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W4255824600.txt	https://medinform.jmir.org/2020/3/e16334/PDF	en		Performance of Computer-Aided Diagnosis in Ultrasonography for Detection of Breast Lesions Less and More Than 2 cm: Prospective Comparative Study (Preprint)	null	2,019	cc-by	5,119	JMIR MEDICAL INFORMATICS Yongping et al Original Paper Performance of Computer-Aided Diagnosis in Ultrasonography for Detection of Breast Lesions Less and More Than 2 cm: Prospective Comparative Study Liang Yongping, MD; Ping Zhou, MD; Zhang Juan, MD; Zhao Yongfeng, MD; Wengang Liu, MD; Yifan Shi, MD The Third Xiangya Hospital, Central South University, Changsha, Hunan, China * these authors contributed equally Corresponding Author: Ping Zhou, MD The Third Xiangya Hospital Central South University 138 Tongzipo Road, Hexiyuelu District Changsha, Hunan, 410013 China Phone: 86 13974809881 Fax: 86 88618403 Email: zhouping1000@hotmail.com Abstract Background: Computer-aided diagnosis (CAD) is used as an aid tool by radiologists on breast lesion diagnosis in ultrasonography. Previous studies demonstrated that CAD can improve the diagnosis performance of radiologists. However, the optimal use of CAD on breast lesions according to size (below or above 2 cm) has not been assessed. Objective: The aim of this study was to compare the performance of different radiologists using CAD to detect breast tumors less and more than 2 cm in size. Methods: We prospectively enrolled 261 consecutive patients (mean age 43 years; age range 17-70 years), including 398 lesions (148 lesions>2 cm, 79 malignant and 69 benign; 250 lesions≤2 cm, 71 malignant and 179 benign) with breast mass as the prominent symptom. One novice radiologist with 1 year of ultrasonography experience and one experienced radiologist with 5 years of ultrasonography experience were each assigned to read the ultrasonography images without CAD, and then again at a second reading while applying the CAD S-Detect. We then compared the diagnostic performance of the readers in the two readings (without and combined with CAD) with breast imaging. The McNemar test for paired data was used for statistical analysis. Results: For the novice reader, the area under the receiver operating characteristic curve (AUC) improved from 0.74 (95% CI 0.67-0.82) from the without-CAD mode to 0.88 (95% CI 0.83-0.93; P<.001) at the combined-CAD mode in lesions≤2 cm. For the experienced reader, the AUC improved from 0.84 (95% CI 0.77-0.90) to 0.90 (95% CI 0.86-0.94; P=.002). In lesions>2 cm, the AUC moderately decreased from 0.81 to 0.80 (novice reader) and from 0.90 to 0.82 (experienced reader). The sensitivity of the novice and experienced reader in lesions≤2 cm improved from 61.97% and 73.23% at the without-CAD mode to 90.14% and 97.18% (both P<.001) at the combined-CAD mode, respectively. Conclusions: S-Detect is a feasible diagnostic tool that can improve the sensitivity for both novice and experienced readers, while also improving the negative predictive value and AUC for lesions≤2 cm, demonstrating important application value in the clinical diagnosis of breast cancer. Trial Registration: Chinese Clinical Trial Registry ChiCTR1800019649; http://www.chictr.org.cn/showprojen.aspx?proj=33094 (JMIR Med Inform 2020;8(3):e16334) doi: 10.2196/16334 KEYWORDS ultrasonography; breast neoplasm; breast imaging reporting and data system (BI-RADS); breast neoplasms diagnosis; cancer screening; computer diagnostic aid https://medinform.jmir.org/2020/3/e16334 XSL• FO RenderX JMIR Med Inform 2020 \| vol. 8 \| iss. 3 \| e16334 \| p. 1 (page number not for citation purposes) JMIR MEDICAL INFORMATICS Introduction Breast cancer is one of the most common cancers in women, and the second leading cause of cancer-related mortality worldwide [1,2]. Early diagnosis of breast cancer can increase the treatment options and survival rate of patients [3], in which breast ultrasound plays an important role in detecting breast cancer. Operator experience-dependence remains the main limitation in ultrasound-based diagnosis [4,5]. S-detect is a recently developed computer-assisted diagnosis (CAD) system for breast cancer, which is based on the Breast Imaging Reporting and Data System (BI-RADS) lexicon and classification [6]. Many studies have reported that S-detect has potential to become a novel diagnostic tool for radiologists [7-10]. However, no study has evaluated the diagnosis performance of CAD in breast lesions with respect to size (less and more than 2 cm). Therefore, the purpose of this study was to compare the performance of detecting breast cancer using CAD between radiologists with different levels of experience for lesions greater and less than 2 cm in size. Methods Patient Selection We prospectively enrolled 261 patients who presented with a total of 398 lesions from November 2018 to May 2019. All patients underwent ultrasound before surgery. The mean age of the examined patients was 43.11 (SD 12.55) years (range 17-70 years). The diameter of lesions ranged from 0.26 to 9.50 cm, with a mean diameter of 1.92 (SD 1.26) cm. All 398 lesions were examined after surgery to confirm their pathological type. This prospective study was approved by the Institutional Review Board of Third Xiangya Hospital. Informed consent was obtained from all patients. The inclusion criteria were follows: patients aged 17-70 years with breast tumor requiring surgery. The exclusion criteria were a history of neoadjuvant chemotherapy or endocrine therapy before surgery, lesions punctured by core-needle biopsy or Mammotome System, breast equipped with a prosthesis, lesions unclear as displayed by ultrasound, and patients unwilling to take part in the study. Ultrasound Image Acquisition All images were obtained with an RS80A ultrasound system (Samsung Medison Co Ltd, Seoul, Korea) with a 5-13–MHz bandwidth linear transducer. All ultrasound examinations were performed by an independent radiologist with 3 years of experience. Typical images of the tumor in longitudinal and transverse planes were stored in the ultrasound system. Computer-Assisted Diagnostic System Our CAD system (S-Detect) extracts features using an integration of artificial neural network classifiers internally installed in the ultrasound equipment (RS80A). The sensitivity of the instrument can be adjusted, with greater sensitivity yielding a higher potential rate of false-positive findings. We chose the default setting. To test the reproducibility of CAD marks with the same image, we randomly selected 20 of 398 https://medinform.jmir.org/2020/3/e16334 XSL• FO RenderX Yongping et al (5.0%) examinations, which were sent through the CAD system three times, and the results showed that the markings were consistent in all images. In S-Detect, the cursor on the center of the lesion was identified, and a region of interest was drawn along the border of the mass automatically by the ultrasound system. The ultrasound features of the lesion were analyzed according to the BI-RADS lexicon, and the final assessment classifications were automatically performed by the ultrasound system. If the borderline was considered inaccurate in any area of the tumor, it was manually edited to achieve the optimum fitness. In the S-Detect system, the final assessment classification was divided into “possibly benign” or “possibly malignant.” Diagnostic Criteria According to the fifth version of BI-RADS, the radiologists classified the lesion from BI-RADS category 3 to BI-RADS category 5. BI-RADS category 4 was further subdivided into category 4A, 4B, and 4C. Category 3 is considered probably benign (<2% likelihood of malignancy) and categories 4A, 4B, 4C range from low to high suspicion (2-10%, 10-50%, 50-95% likelihood of malignancy, respectively). Category 5 indicates a high malignancy rate (>95% likelihood of malignancy). The malignant signs in breast ultrasound imaging included irregular shape, antiparallel orientation, noncircumscribed margin, microcalcification, acoustic halo, posterior shadowing, and abnormalities of the surrounding tissue. No definitive malignant sign is assigned to category 3; one, two, and three malignant signs are assigned to category 4A, 4B, and 4C, respectively; and more than four malignant signs is assigned to category 5. Accordingly, category 3 and 4A lesions were regarded as benign, and category 4B, 4C, and 5 lesions were regarded as malignant [11,12]. For assessments of the combination of ultrasound and the CAD system, we took longitudinal and transverse planes of the tumor for CAD. If one plane indicated “possibly malignant,” it was considered a positive outcome, and the BI-RADS category diagnosis was increased by one level (ie, 3 to 4A, 4A to 4B, 4B to 4C, 4C to 5). If both planes indicated “possibly benign,” it was considered a negative outcome, and the BI-RADS category diagnosis was decreased by one level (ie, 5 to 4C, 4C to 4B, 4B to 4A, 4A to 3) [13]. Readers, Reading Modes, and Training Two readers were involved in the study: a novice reader with 1 year of ultrasound experience and an experienced reader with 5 years of ultrasound experience. Both readers were trained on the reading procedures with 20 ultrasound images that were not part of the study set, 10 of which were read in without-CAD mode. The other 10 images were assessed in combined-CAD mode, in which the readers first read the ultrasound images without CAD and then combined the indications of CAD marks to make the final decision. Both readers reviewed every examination at each reading mode independently and were blinded to any information about the patients, including age, manifestation of symptoms, and previous radiology reports. The readers were asked to read for at least 2 JMIR Med Inform 2020 \| vol. 8 \| iss. 3 \| e16334 \| p. 2 (page number not for citation purposes) JMIR MEDICAL INFORMATICS Yongping et al hours a day to simulate the typical process of batch reading in such examinations. Statistical Analysis Statistical evaluation was performed using SPSS software (SPSS for Windows 19.0, SPSS Inc, Chicago, IL, USA). Taking the pathology results as the gold standard, we analyzed the diagnostic sensitivity, specificity, and area under the receiving operating characteristic curve values (AUCs) in without-CAD mode and combined-CAD mode [14]. The combined-CAD mode and without-CAD mode diagnostic parameters were compared using the McNemar test (sensitivity, specificity, positive predictive value [PPV], negative predictive value [NPV], accuracy) for match-paired data. We used the Hanley and McNeil method to analyze the differences between pairs of AUCs. For all statistical tests, P<.05 was considered to indicate statistical significance. Results Basic Characteristics of Lesions Patient and lesion characteristics on the basis of lesion size are summarized in Table 1. Of the 398 breast lesions in the 261 patients included in this study, 250 (62.8%) were ≤2 cm and 148 (37.2%) were >2 cm. The mean sizes for all lesions, malignant lesions, and benign lesions at ultrasound were similar and close to 2 cm, with benign lesions being the smallest (1.73 cm) and malignant lesions being the largest (2.22 cm). Table 1. Characteristics of patients and lesions. Characteristic All lesions (n=398) Lesions≤2 cm (n=250) Lesions>2 cm (n=148) Mean (SD) 43.10 (12.57) 43.62 (11.875) 42.22 (13.66) Median (range) 45 (17-70) 45.0 (17-70) 44.5 (17-70) Mean (SD) 1.92 (1.26) 1.1629 (0.42) 3.1876 (1.19) Median (range) 1.6 (0.26-9.5) 1.1 (0.26-2.0) 2.8 (2.1-9.5) Mean (SD) 2.22 (1.08) 1.331 (0.42) 3.02 (0.82) Median (range) 2.11 (0.26-6.2) 1.30 (0.26-2.0) 2.8 (2.1-6.2) Mean (SD) 1.73 (1.33) 1.10 (0.40) 3.38 (1.50) Median (range) 1.3 (0.4-9.5) 1.0 (0.4-2.0) 2.9 (2.1-9.5) Total 150 (37.7) 71 (47.3) 79 (52.7) Intraductal carcinoma in situ 5 (3.3) 5 (7.0) 0 (0.0) Invasive lobular carcinoma 11 (7.3) 10 (14.1) 1 (1.3) Mucinous adenocarcinoma 2 (1.3) 2 (2.8) 0 (0.0) Medullary carcinoma 2 (1.3) 1 (1.4) 1 (1.3) Invasive ductal carcinoma 130 (86.7) 53 (74.6) 77 (97.5) Total 248 (62.3) 179 (72.2) 69 (27.8) Intraductal papilloma 29 (11.7) 29 (16.2) 0 (0.0) Granulomatous mastitis 5 (2.0) 1 (0.6) 4 (5.8) Fibroma 171 (69.0) 110 (61.5) 61 (88.4) Hyperplasia-induced lesions 42 (16.9) 38 (21.2) 4 (5.8) Scar tissue 1 (0.4) 1 (0.6) 0 (0.0) Patient age (years) Size of all lesions (cm) Size of malignant lesions (cm) Size of benign lesions (cm) Histologic type of malignant lesions, n (%) Histological type of benign lesions, n (%) Reader Performance In all lesions, the AUCs of the reading improved at combined-CAD mode compared to those of the without-CAD mode for both the novice and experienced reader (Table 2, Figure 1). For the novice reader, the improvement in AUCs was https://medinform.jmir.org/2020/3/e16334 XSL• FO RenderX significant between the without-CAD and combined-CAD modes (Z=4.90, P<.001), whereas there was no significant difference in AUCs between modes for the experienced reader (Z=1.06, P=.29). JMIR Med Inform 2020 \| vol. 8 \| iss. 3 \| e16334 \| p. 3 (page number not for citation purposes) JMIR MEDICAL INFORMATICS In subgroup analysis, for lesions≤2 cm, the AUCs of the reading improved significantly in combined-CAD mode for both the novice and experienced readers. However, in lesions>2 cm, there were no significant differences in AUCs between two reading modes for both the novice and experienced readers (Table 2). When a BI-RADS category 4A threshold was used, the sensitivity and NPV improved at the combined-CAD mode compared with that at the without-CAD mode for both the novice reader and experienced reader in all lesions and subgroup analyses (Table 2). However, in lesions≤2 cm, there were no https://medinform.jmir.org/2020/3/e16334 XSL• FO RenderX Yongping et al significant differences between without-CAD and combined-CAD modes for the novice reader with respect to specificity, PPV, and accuracy. By contrast, significant differences were observed for the experienced reader in specificity and PPV, whereas there was no significant difference in accuracy. In lesions>2 cm, there was a significant decrease in specificity and a significant increase in NPV between without-CAD and combined-CAD modes for both readers, and there was a significant decrease in PPV for only the experienced reader. There was a moderate reduction in accuracy between the without-CAD and combined-CAD modes for both readers, and in PPV for the novice reader (Table 2). JMIR Med Inform 2020 \| vol. 8 \| iss. 3 \| e16334 \| p. 4 (page number not for citation purposes) JMIR MEDICAL INFORMATICS Yongping et al Table 2. Diagnostic performance of the readers in two reading modes with a Breast Imaging Reporting and Data System Category 4A threshold. Lesions Novice Reader Expert Reader Without CADa Combined with CAD + – + + 44 22 – 27 157 P value Without CAD Combined with CAD – + – + – 64 23 52 9 69 33 7 156 19 170 2 146 P value Lesions≤2 cm Pathology Sensitivityb 61.97 90.14 <.001 73.24 97.18 <.001 Specificityb 87.71 87.15 .83 94.97 81.56 .004 PPVc 66.66 73.56 .22 85.25 67.65 .005 NPVd 85.33 95.71 .008 89.95 98.65 .005 Accuracy 80.40 88.00 .12 88.8 86 .52 AUCe (95% CI) 0.74 (0.67-0.82) 0.88 (0.83-0.93) <.001 0.84 (0.77-0.90) 0.90 (0.86-0.94) .002 + 61 11 79 28 67 4 79 23 – 18 58 0 41 12 65 0 46 Lesions>2 cm Pathology Sensitivity 77.22 100 <.001 86.67 100 <.001 Specificity 84.06 59.42 <.001 96.72 66.66 <.001 PPV 84.72 73.83 .05 96.30 77.45 <.001 NPV 76.32 100 <.001 88.06 100 <.001 Accuracy 80.41 81.08 .86 91.74 84.46 .13 AUC (95% CI) 0.81 (0.73-0.88) 0.80 (0.72-0.87) .81 0.90 (0.84-0.95) 0.83 (0.76-0.91) .03 + 105 33 143 51 119 13 148 52 – 45 215 7 197 31 235 2 96 Sensitivity 70 95.33 <.001 79.33 98.66 <.001 Specificity 86.69 79.43 .13 94.75 79.03 .001 PPV 76.08 73.71 .74 90.15 74.00 .003 NPV 82.69 96.57 .001 88.34 98.98 .002 Accuracy 80.40 85.43 .35 88.94 86.43 .52 AUC (95% CI) 0.78 (0.73-0.83) 0.87 (0.84-0.91) <.001 0.87 (0.83-0.91) 0.89 (0.85-0.92) .29 All lesions Pathology a CAD: computer-aided diagnosis. b Breast Imaging Reporting and Data System assessment categories 4B, 4C, and 5 were considered positive for cancer for the calculation of sensitivity and specificity. c PPV: positive predictive value. d NPV: negative predictive value. e AUC: area under the receiver operating characteristic curve. https://medinform.jmir.org/2020/3/e16334 XSL• FO RenderX JMIR Med Inform 2020 \| vol. 8 \| iss. 3 \| e16334 \| p. 5 (page number not for citation purposes) JMIR MEDICAL INFORMATICS Yongping et al Figure 1. Comparison of receiver operating characteristic curves in lesions <2 cm, lesions >2 cm, and lesions in all size. Management of Diagnostic Feature Decision Change At the combined-CAD mode, the management of diagnostic feature decision changes compared with the without-CAD mode was 164/398 (41.2%) of all ultrasound images for the two readers. For the novice reader, with CAD support, 38/150 (25.3%) of the malignant lesions (20 lesions≤2 cm, 18 lesions>2 cm) were correctly upgraded from category 4A to 4B, whereas none (0%) of the malignant lesions was incorrectly changed from 4B to 4A. In addition, 18/248 (7.3%) benign lesions (12 lesions≤2 cm, 6 lesions>2 cm) were correctly downgraded from category 4B to 4A; however, 36/248 (14.5%) benign lesions (11 lesions≤2 cm, 24 lesions>2 cm) were incorrectly changed from category 4A to 4B. For the experienced reader, 30/150 (20.0%) malignant lesions (18 lesions≤2 cm, 12 lesions>2 cm) were correctly changed from category 4A to 4B, and only 1 of 79 (1%) malignant lesions (≤2 cm) was incorrectly changed from category 4B to 4A. In addition, 4/248 (1.6%) benign lesions (3 lesions≤2 cm, 1 lesion>2 cm) were correctly downgraded from category 4B to 4A, whereas 38/248 (15.3%) benign lesions (3 lesions≤2 cm, 20 lesions>2 cm) were incorrectly changed from category 4A to 4B. Discussion Principal Findings CAD systems have been recently applied to improve diagnostic performance in breast ultrasonography. S-Detect is a CAD system based on a neural network learning algorithm [7], which applies a novel feature extraction technique and vector machine classifier that categorizes breast masses into benign or malignant depending on the suggested feature based on the BI-RADS lexicon [15]. Choi et al [10] recently reported that both experienced and inexperienced readers had significantly higher specificity and AUCs in reading ultrasounds in combination with S-Detect, and the inexperienced reader also showed significant improvement in sensitivity. However, the diagnosis of breast lesions of different sizes is one of the most difficult challenges in clinical practice [16,17]. Radiologists with different levels of experience typically perform breast ultrasound, and thus the usefulness of S-Detect may be different according to experience. For example, radiologists with less https://medinform.jmir.org/2020/3/e16334 XSL• FO RenderX experience may have a greater benefit in using S-Detect for the diagnosis of small breast lesions. In our study, when combining ultrasound reading with S-Detect, both the experienced and novice readers showed significantly higher sensitivity and NPV compared to those obtained without S-Detect, which is in line with the findings of the previous studies for CAD systems mentioned above. In addition, 38/150 (25.3%) and 30/150 (20.0%) breast cancers initially assessed as category 4A by the novice and experienced readers were categorized as probably malignant by S-Detect, regardless of size. Combining the results of S-Detect led to significant improvements in AUCs for both readers in lesions<2 cm. However, in lesions>2 cm, the combination of S-Detect did not confer improvements in accuracy and AUC for either reader. Our results suggest that S-Detect could be used as an additional tool with breast ultrasound regardless of the experience of the reader, and may help to reduce the misdiagnosis ratio of early-stage breast cancer. Although the sensitivity, NPV, and AUCs were improved, there was no significant improvement in the accuracy of the readers when using S-Detect compared to that obtained by the ultrasound reading alone. This may be due to the fact that both readers already showed high AUC values with ultrasound alone, and therefore there was minimal room for improvement. Strengths and Prospects Our results showed that readers with less experience may benefit more by using S-Detect in detection of smaller breast lesions. Several studies have reported the application of different types of CAD to breast ultrasound [6,18,19]. Overall, these studies showed that the CAD systems promoted the diagnostic performance of breast ultrasound, especially specificity and accuracy. Shen et al [18] argued that CAD systems could be helpful in evaluating fuzzy category 4 lesions. Wang et al [19] suggested that combining CAD with ultrasound was more helpful for inexperienced radiologists than for experienced radiologists owing to greater improvement in the diagnostic performances observed in the inexperienced group. In our study, the sensitivity, NPV, and AUCs of both readers were improved, supporting the idea that S-Detect can reliably provide a second view that can be referred to by readers. High sensitivity is a remarkable superiority of S-Detect, and similar results were reported in some previous studies [20,21]. Compared to these JMIR Med Inform 2020 \| vol. 8 \| iss. 3 \| e16334 \| p. 6 (page number not for citation purposes) JMIR MEDICAL INFORMATICS previous studies, there was a relatively smaller proportion of benign lesions in our study and the mean size of lesions in our study was larger. In addition, all patients had a breast mass as the prominent symptom, which may explain the different results. Moreover, since S-Detect provides the final assessment in a dichotomized form of possibly benign and possibly malignant, this factor may have also affected the accuracy of readers in the combined-CAD mode. This result is encouraging for clinical breast cancer screening, as breast cancer is a highly aggressive disease with multiple pathological subtypes, including those associated with higher rates of metastases and poorer survival rates [22]. Thus, it is important to detect cancer early to reduce the mortality rate [23]. In addition, S-Detect is a user-friendly and concise program that is integrated in an ultrasound machine to enable obtaining a terse result for radiologists immediately during real-time ultrasonography, which can easily be applied to routine work. However, it is not recommended to apply CAD alone or use it as a replacement for a radiologist in the diagnosis of breast lesions, especially for tumors>2 cm, which is consistent with the results of Kim et al [13]. As one example from this study, a fibroadenoma lesion with a size of 2.94×1.76 cm (Figure 2A) that showed an unclear margin and a large lobulated shape was misdiagnosed as malignant by S-Detect, and was inversely excluded by the radiologist after combining the results with information on the patient’s history. In another example, a lesion of invasive ductal carcinoma with a size of 3.09×1.36 cm (Figure 2B) showing a clear border and microcalcification was classified Yongping et al as BI-RADS category 4B by conventional ultrasound, whereas S-Detect diagnosed this lesion as benign. Further investigation along with technical progress are anticipated to lead to the development of a more sophisticated algorithm using the multiple-planes assessment BI-RADS ultrasonographic categories. Likewise, ultrasound scanning is a real-time and multi-angled imaging method, which can observe the lesion from different planes to collect the imaging features such as the internal situation, relation of the lesion with surrounding tissues, and the blood supply model, along with patient history and other available information. Therefore, more image data and clinical information can be obtained with ultrasound than with CAD. Consequently, in lesions≤2 cm, the combination of S-Detect and ultrasound allows for the weaknesses of each method to be counteracted by the strengths of the other, which could assist both novice and experienced readers in making a more accurate final diagnosis. As one example from this study, an invasive ductal carcinoma lesion with a size of 1.75×1.56 cm (Figure 3A) that showed an unclear margin, irregular shape, and microcalcification was correctly diagnosed as malignant by S-Detect and was classified as BI-RADS category 4C by both readers. In another example, a lesion of fibroadenoma with a size of 1.58×1.10 cm (Figure 3B) showing a clear border and regular shape was classified as BI-RADS category 3 by conventional ultrasound and was correctly diagnosed as benign by S-Detect. Figure 2. A malignant and benign lesion > 2 cm misdiagnosed by S-Detect. https://medinform.jmir.org/2020/3/e16334 XSL• FO RenderX JMIR Med Inform 2020 \| vol. 8 \| iss. 3 \| e16334 \| p. 7 (page number not for citation purposes) JMIR MEDICAL INFORMATICS Yongping et al Figure 3. A malignant and benign lesion < 2 cm diagnosed correctly by S-Detect. Limitations There are several limitations of this study. First, a relatively small number of cases were included (N=261). Second, the presentation of calcifications was not included in the analysis, owing to the limited ability of S-Detect to detect microcalcifications [24]. Third, some small nodules (around 1 cm) classified as BI-RADS category 3 that were not surgically removed were excluded from the study, which may have affected the results. Fourth, only two representative still images of breast masses stored for analysis were chosen during image analysis by the radiologists and S-Detect, which may have caused variability in selecting images of representative planes. Fifth, the criterion of the size of lesions for comparison was set to 2 cm; thus, further studies using other stratifications with a larger number of samples may be warranted. Sixth, both readers had relatively minimal experience as breast imagers. In China, the specialty of breast imaging is somewhat new, and the staff in this field tend to be younger compared with staff of other imaging specialties. Therefore, these factors may have had a slight influence on our results. Conclusion In conclusion, S-Detect is a clinically feasible diagnostic tool that can improve the sensitivity of breast ultrasonography, in addition to improving the NPV and AUC for lesions≤2 cm, with important application value in the clinical diagnosis of breast cancer. Acknowledgments This study was supported by National Natural Science Foundation of China (81871367). Conflicts of Interest None declared. References 1. 2. 3. 4. 5. Siegel RL, Miller KD, Jemal A. Cancer Statistics, 2017. CA Cancer J Clin 2017 Jan;67(1):7-30. [doi: 10.3322/caac.21387] [Medline: 28055103] Chen W, Zheng R, Baade PD, Zhang S, Zeng H, Bray F, et al. Cancer statistics in China, 2015. CA Cancer J Clin 2016;66(2):115-132. [doi: 10.3322/caac.21338] [Medline: 26808342] Brinkley D, Haybrittle JL. The curability of breast cancer. Lancet 1975 Jul 19;2(7925):95-97. [doi: 10.1016/s0140-6736(75)90003-3] [Medline: 49738] Takahashi R, Kajikawa Y. Computer-aided diagnosis: A survey with bibliometric analysis. 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[doi: 10.1016/j.ejrad.2018.01.002] [Medline: 29362145] Abbreviations AUC: area under the receiving operating characteristic curve BI-RADS: Breast Imaging Reporting and Data System CAD: computer-assisted diagnosis NPV: negative predictive value PPV: positive predictive value https://medinform.jmir.org/2020/3/e16334 XSL• FO RenderX JMIR Med Inform 2020 \| vol. 8 \| iss. 3 \| e16334 \| p. 9 (page number not for citation purposes) JMIR MEDICAL INFORMATICS Yongping et al Edited by G Eysenbach; submitted 20.09.19; peer-reviewed by YK Akpak, D Di Stasio; comments to author 16.10.19; revised version received 18.11.19; accepted 26.01.20; published 02.03.20 Please cite as: Yongping L, Zhou P, Juan Z, Yongfeng Z, Liu W, Shi Y Performance of Computer-Aided Diagnosis in Ultrasonography for Detection of Breast Lesions Less and More Than 2 cm: Prospective Comparative Study JMIR Med Inform 2020;8(3):e16334 URL: https://medinform.jmir.org/2020/3/e16334 doi: 10.2196/16334 PMID: 32130149 ©Liang Yongping, Ping Zhou, Zhang Juan, Zhao Yongfeng, Wengang Liu, Yifan Shi. Originally published in JMIR Medical Informatics (http://medinform.jmir.org), 02.03.2020. This is an open-access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in JMIR Medical Informatics, is properly cited. The complete bibliographic information, a link to the original publication on http://medinform.jmir.org/, as well as this copyright and license information must be included. https://medinform.jmir.org/2020/3/e16334 XSL• FO RenderX JMIR Med Inform 2020 \| vol. 8 \| iss. 3 \| e16334 \| p. 10 (page number not for citation purposes)
https://openalex.org/W4310207722	https://link.springer.com/content/pdf/10.1007/s00025-022-01790-6.pdf	English	null	Non-periodic solutions of the Goła̧b–Schinzel type functional equation	Results in mathematics	2,022	cc-by	8,179	Results Math (2023) 78:28 c⃝2022 The Author(s) 1422-6383/23/010001-15 published online November 29, 2022 https://doi.org/10.1007/s00025-022-01790-6 Results Math (2023) 78:28 c⃝2022 The Author(s) 1422-6383/23/010001-15 published online November 29, 2022 https://doi.org/10.1007/s00025-022-01790-6 Results in Mathematics Mathematics Subject Classiﬁcation. 39B12, 39B22, 39B52. Mathematics Subject Classiﬁcation. 39B12, 39B22, 39B52. Keywords. Gol¸ab-Schinzel functional equation, semigroup-valued solu- tions, non-periodic solutions. Non-periodic solutions of the Gol¸ab–Schinzel type functional equation Jacek Chudziak Abstract. We determine the solutions of the Gol¸ab-Schinzel type func- tional equation in the class of non-periodic functions. Applying this re- sult we give a positive answer to the problem raised by E. Jablo´nska (Aequationes Math 87:125–133, 2014). 1. Introduction The paper is inspired by an open problem, posed by Jablo´nska [16], concerning the solutions of the Gol¸ab–Schinzel type functional equation f(x + g(x)y) = f(x)f(y) for x, y ∈R (1) (1) where f, g : R →R are unknown functions. Equation (1) is a generalization of the Gol¸ab–Schinzel type equations f(x + f(x)y) = f(x)f(y) (2) (2) and f(x + f(x)ny) = f(x)f(y), (3) f(x + f(x)ny) = f(x)f(y), ( ) ( ) (3) where n is a ﬁxed positive integer. Equations (2)-(3) play an important role in determination of substructures of algebraic structures (see e.g. [1,2,4,5,12,13] and [18]). Furthermore, the conditional versions of (2) are strictly related to some problems arising in meteorology and ﬂuid mechanics (cf. [17]). In a more general setting, where an unknown function f maps a real linear space into R, solutions of (2) and (3) have been investigated by several authors under various where n is a ﬁxed positive integer. Equations (2)-(3) play an important role in determination of substructures of algebraic structures (see e.g. [1,2,4,5,12,13] and [18]). Furthermore, the conditional versions of (2) are strictly related to some problems arising in meteorology and ﬂuid mechanics (cf. [17]). In a more general setting, where an unknown function f maps a real linear space into R, solutions of (2) and (3) have been investigated by several authors under various 28 Page 2 of 15 J. Chudziak Results Math regularity assumptions. More details concerning properties of the solutions of these equations as well as their applications can be found in a survey paper [6]. In order to formulate the aforementioned problem, recall the main result proved in [16]. Theorem 1.1. Assume that f, g : R →R, f is locally bounded above at each point, f(R) \ {−1, 0, 1} ̸= ∅and g(R) \ {0, 1} ̸= ∅. If the pair (f, g) satisﬁes equation (1) then there exist a c ∈R\{0}, an inﬁnite subgroup G of the group (R \ {0}, ·) and a non-constant multiplicative function φ : G →R such that g(x) = cx + 1 whenever cx + 1 ∈G, 0 otherwise (4) g(x) = cx + 1 whenever cx + 1 ∈G, 0 otherwise (4) and f(x) = φ(cx + 1) whenever cx + 1 ∈G, 0 otherwise. (5) (5) It has been noted in [16] that from Theorem 1.1 one can directly derive the following result. 1. Introduction It has been noted in [16] that from Theorem 1.1 one can directly derive the following result. Corollary 1.2. Assume that a function f : R →R is locally bounded above at each point and f(R)\{−1, 0, 1} ̸= ∅. If f satisﬁes equation (2) then there exist a c ∈R \ {0} and an inﬁnite subgroup G of the group (R \ {0}, ·) such that f(x) = cx + 1 whenever cx + 1 ∈G, 0 otherwise. The same form of solutions of equation (2) was established in [19] under the assumption that an unknown function is not microperiodic. This fact inspired E. Jablo´nska to raise the following question: does Theorem 1.1 hold, if we replace the assumption that f is locally bounded above at each point of the line by the assumption that f is not periodic? In this paper we give a positive answer to this question. In fact, we determine the form of non-periodic solutions of a signiﬁcantly more general functional equation, namely f(x + g(x)y) = f(x) ◦f(y) for x, y ∈X, (6) (6) where X is a real linear space, (S, ◦) is a commutative semigroup, f : R →S and g : R →R are unknown functions. In the case X = R equation (6) has been introduced and studied in [7]. The solutions of (6) under some regularity assumptions have been determined in [8]. The following particular case of (6) f(x + g(x)y) = f(x)f(y) for x, y ∈X, (7) (7) f(x + g(x)y) = f(x)f(y) for x, y ∈X, and some its conditional versions have been investigated under various reg- ularity assumptions in [9–11] and [14,15]. It is remarkable that solutions of (6) and their applications to invariance under binomial thinning have been recently considered in [3]. Vol. 78 (2023) Vol. 78 (2023) Vol. 78 (2023) Page 3 of 15 28 2. Results In what follows, X is a non-zero real linear space and (S, ◦) is a commuta- tive semigroup. Furthermore, E(S) denotes the set of all idempotents of the semigroup (S, ◦), that is E(S) := {s ∈S : s ◦s = s}. Moreover, for a function g : X →R, we set Ag := {x ∈X : g(x) ̸= 0}. E(S) := {s ∈S : s ◦s = s}. Moreover, for a function g : X →R, we set Ag := {x ∈X : g(x) ̸= 0}. Remark 1. Let f : X →S and g : X →R. If the pair (f, g) satisﬁes equation (6) then Remark 1. Let f : X →S and g : X →R. If the pair (f, g) satisﬁes equation (6) then f(0) = f(0 + g(0) · 0) = f(0) ◦f(0), f(0) = f(0 + g(0) · 0) = f(0) ◦f(0), that is, f(0) ∈E(S). Furthermore, applying (6) with y = x ∈X \ Ag, we get f(x) ∈E(S) for x ∈X \ Ag. (8) that is, f(0) ∈E(S). Furthermore, applying (6) with y = x ∈X \ Ag, we get f(x) ∈E(S) for x ∈X \ Ag. (8) (8) The following three auxiliary results will play a crucial role in our con- siderations. Lemma 2.1. Assume that f : X →S, g : X →R and the pair (f, g) satisﬁes equation (6). If Ag ̸= X then there exists a z ∈E(S) such that Lemma 2.1. Assume that f : X →S, g : X →R and the pair (f, g) satisﬁes equation (6). If Ag ̸= X then there exists a z ∈E(S) such that f( ) f ∈X \ A (9) f(x) = z for x ∈X \ Ag f(x) = z for x ∈X \ Ag (9) (9) and z ◦f(y) = f(y) ◦z = z for y ∈X. (10) (10) Proof. Assume that Ag ̸= X and ﬁx an x0 ∈X \ Ag. Since the semigroup (S, ◦) is commutative, setting in (6) x = x0, we obtain (10) with z := f(x0). In view of (8), we have z ∈E(S). Moreover, applying (6) and (10), for every x ∈X \ Ag, we get Proof. Assume that Ag ̸= X and ﬁx an x0 ∈X \ Ag. 2. Results □ Lemma 2.3. Assume that a pair (f, g), where f : X →S and g : X →R, satisﬁes equation (6). If f is non-periodic then Lemma 2.3. Assume that a pair (f, g), where f : X →S and g : X →R, satisﬁes equation (6). If f is non-periodic then Lemma 2.3. Assume that a pair (f, g), where f : X →S and g : X →R, satisﬁes equation (6). If f is non-periodic then x + g(x)y −g(y)x −y = 0 for x, y ∈Ag. (12) (12) Proof. In view of (6), for every x, y, z ∈X, we get Proof. In view of (6), for every x, y, z ∈X, we get f(x + g(x)y + g(x)g(y)z) = f(x + g(x)(y + g(y)z)) = f(x) ◦f(y + g(y)z) = f(x) ◦(f(y) ◦f(z)) = (f(x) ◦f(y)) ◦f(z) = (f(y) ◦f(x)) ◦f(z) = f(y) ◦(f(x) ◦f(z)) = f(y) ◦f(x + g(x)z) = f(y + g(y)(x + g(x)z)) f( + ( ) + ( ) ( ) ) f(x + g(x)y + g(x)g(y)z) = f(x + g(x)(y + g(y)z)) = f(x) ◦f(y + g(y)z) = f(x) ◦(f(y) ◦f(z)) = (f(x) ◦f(y)) ◦f(z) = (f(y) ◦f(x)) ◦f(z) = f(y) ◦(f(x) ◦f(z)) f( ) f( + ( ) ) f( + ( )( + ( ) )) = f(y) ◦f(x + g(x)z) = f(y + g(y)(x + g(x)z)) = f(y + g(y)x + g(y)g(x)z), that is that is f(x + g(x)y + g(x)g(y)z) = f(y + g(y)x + g(x)g(y)z). Replacing in this equality z by z−g(y)x−y g(x)g(y) , for every x, y ∈Ag and z ∈X, we obtain f(x + g(x)y + g(x)g(y)z) = f(y + g(y)x + g(x)g(y)z). Replacing in this equality z by z−g(y)x−y g(x)g(y) , for every x, y ∈Ag and z ∈X, we obtain f(z + x + g(x)y −g(y)x −y) = f(z). non-periodic, then (12) holds. □ f(z + x + g(x)y −g(y)x −y) = f(z). f(z + x + g(x)y −g(y)x −y) = f(z). □ Therefore, if f is non-periodic, then (12) holds. Therefore, if f is non-periodic, then (12) holds. 2. Results Since the semigroup (S, ◦) is commutative, setting in (6) x = x0, we obtain (10) with z := f(x0). In view of (8), we have z ∈E(S). Moreover, applying (6) and (10), for every x ∈X \ Ag, we get f(x) = f(x + g(x)x0) = f(x) ◦f(x0) = f(x) ◦z = z, f(x) = f(x + g(x)x0) = f(x) ◦f(x0) = f(x) ◦z = z, 9) holds. □ □ Lemma 2.2. Assume that a pair (f, g), where f : X →S and g : X →R, satisﬁes equation (6). If f is non-constant then: Lemma 2.2. Assume that a pair (f, g), where f : X →S and g : X →R satisﬁes equation (6). If f is non-constant then: g (ii) for every x, y ∈X, it holds: x + g(x)y ∈Ag if and only if x ∈Ag and y ∈Ag. (11) (11) x + g(x)y ∈Ag if and only if x ∈Ag and y ∈Ag. (11) Proof. Assume that f is non-constant. Suppose that 0 ̸∈Ag. Then Ag ̸= X and so, according to Lemma 2.1, there exists a z ∈E(S) such that (9)–(10) hold. Hence, in view of (6), for every x ∈X, we get f(x) = f(x + g(x) · 0) = f(x) ◦f(0) = f(x) ◦z = z. Since f is non-constant, this yields a contradiction and proves (i). J. Chudziak 28 Page 4 of 15 28 Page 4 of 15 Results Math In order to prove (ii) note that, applying (6), for every x, y, z ∈X, we obtain f(x + g(x)y + g(x + g(x)y)z) = f(x + g(x)y) ◦f(z) = (f(x) ◦f(y)) ◦f(z) = f(x + g(x)y) ◦f(z) = (f(x) ◦f(y)) ◦f(z) = f(x) ◦(f(y) ◦f(z)) = f(x) ◦f(y + g(y)z) = f(x + g(x)(y + g(y)z)) ( ( ) ( ) ( ) ) = f(x) ◦(f(y) ◦f(z)) = f(x) ◦f(y + g(y)z) = f(x + g(x)(y + g(y)z)) = f(x + g(x)y + g(x)g(y)z). = f(x + g(x)y + g(x)g(y)z). Hence, as f is non-constant, for every x, y ∈X, we get g(x + g(x)y) = 0 if and only if g(x)g(y) = 0, g(x + g(x)y) = 0 if and only if g(x)g(y) = 0, g(x + g(x)y) = 0 if and only if g(x)g(y) = 0, which implies (11). □ g( g( )y) y g( )g(y) , which implies (11). □ which implies (11). 2. Results In what follows we call a mapping φ deﬁned on a subset G of R periodic provided there exists a t0 ∈R \ {0} such that t −t0, t + t0 ∈G for t ∈G (13) t −t0, t + t0 ∈G for t ∈G (13) and and φ(t −t0) = φ(t + t0) = φ(t) for t ∈G. (14) (14) The next theorem is the main result of the paper. Theorem 2.4. Assume that f : X →S and g : X →R. If the pair (f, g) sat- isﬁes equation (6) and f is non-periodic then one of the following possibilities holds: Vol. 78 (2023) Non-periodic solutions of the Gol¸ab–Schinzel type Page 5 of 15 28 ol. 78 (2023) Non-periodic solutions of the Gol¸ab–Schinzel type Page 5 of 15 28 Vol. 78 (2023) Non-periodic solutions of the Gol¸ab–Schinzel type Page 5 of 15 28 Vol. 2. Results 78 (2023) (a) there exist an a ∈R \ {0} and s, z ∈E(S) such that s ̸= z, s ◦z = z, (a) there exist an a ∈R \ {0} and s, z ∈E(S) such that s ̸= z, s ◦z = z, g(x) = a for x = 0, 0 for x ̸= 0 (15) g(x) = a for x = 0, 0 for x ̸= 0 (15) (15) and and f(x) = s for x = 0, z for x ̸= 0; (16) (16) (b) g = 1 and f is an injective homomorphism of the group (X, +) into (S, ◦); (c) there exist a non-trivial proper subgroup A of the group (X, +), an injec- tive homomorphism ψ : A →S and a z ∈E(S) such that (c) there exist a non-trivial proper subgroup A of the group (X, +), an injec- tive homomorphism ψ : A →S and a z ∈E(S) such that ψ(x) ◦z = z for x ∈A, (17) g(x) = 1 for x ∈A, 0 otherwise (18) (18) and and f(x) = ψ(x) for x ∈A, z otherwise; (19) (19) (d) there exist an x0 ∈X \ {0}, a non-trivial subgroup G of the group (R \ {0}, ·), a non-periodic homomorphism φ : G →S and a z ∈E(S) such that φ(t) ̸= z for t ∈G, (20) φ(t) ◦z = z for t ∈G, (21) g(x) = 1 −t whenever x = tx0 and 1 −t ∈G, 0 otherwise (22) (22) and and f(x) = φ(1 −t) whenever x = tx0 and 1 −t ∈G, z otherwise. (23) (23) Conversely, in any case (a)-(d) the pair (f, g) satisﬁes equation (6) and f is non-periodic. Proof. Assume that the pair (f, g) satisﬁes equation (6) and f is non-periodic. Then f is non-constant and so, according to Lemma 2.2 (i), we have 0 ∈Ag. We shall divide our considerations into the following three cases: 1. Ag = {0}; 1. Ag = {0}; 1. Ag = {0}; g { } 2. Ag \ {0} ̸= ∅and g(x) = 1 for x ∈Ag; g \ { } ̸ g( ) g 3. Ag \ {0} ̸= ∅and g(y0) ̸= 1 for some y0 ∈Ag. g \ { } ( ) g 3. Ag \ {0} ̸= ∅and g(y0) ̸= 1 for some y0 ∈Ag. Case 1. 2. Results Taking a := g(0), s := f(0) and applying Remark 1 and Lemma 2.1, we obtain that a ̸= 0, s ∈E(S) and there exists a z ∈E(S) such that (15)–(16) hold. Moreover, setting in (6) x = 0 and y ∈X \ {0}, in view of (15)–(16), we get s ◦z = z. As f is non-constant, we have also s ̸= z. Thus, (a) holds. J. Chudziak 28 Page 6 of 15 Results Math Case 2. If Ag = X then g = 1 and so, in view of (6), f is a homomorphism of the group (X, +) into (S, ◦). Furthermore, if f(y1) = f(y2) for some y1, y2 ∈ X, then for every x ∈X, we obtain f(x + y1 −y2) = f(x) ◦f(y1) ◦f(−y2) = f(x) ◦f(y2) ◦f(−y2) = f(x) ◦f(0) = f(x). Since f is non-periodic, this implies that y1 = y2 and proves the injectivity of f. Thus, (b) holds. Assume that A := Ag ̸= X. Then 0 ∈A, g is of the form (18) and, in Since f is non-periodic, this implies that y1 = y2 and proves the injectivity of f. Thus, (b) holds. Since f is non-periodic, this implies that y1 = y2 and proves the injectivity of f. Thus, (b) holds. f ( ) Assume that A := Ag ̸= X. Then 0 ∈A, g is of the form (18) and, in view of (11), we get x + y = x + g(x)y ∈A for x, y ∈A. Furthermore, since x + g(x)(−x) = 0 ∈A for x ∈A, applying (11) again, we conclude that −x ∈A for x ∈A. Hence, A is a subgroup of the group (X, +). Moreover, as A \ {0} ̸= ∅and A ̸= X, the subgroup is non-trivial and proper. applying (11) again, we conclude that −x ∈A for x ∈A. Hence, A is a subgroup of the group (X, +). Moreover, as A \ {0} ̸= ∅and A ̸= X, the subgroup is non-trivial and proper. Let ψ : A →S be given by ψ(x) = f(x) for x ∈A. Then, making use of Lemma 2.1, we obtain that there exists a z ∈E(S) such that (17) and (19) hold. Furthermore, taking into account (6) and (18), we get ψ(x + y) = f(x + g(x)y) = f(x) ◦f(y) = ψ(x) ◦ψ(y) for x, y ∈A. 2. Results ψ(x + y) = f(x + g(x)y) = f(x) ◦f(y) = ψ(x) ◦ψ(y) for x, y ∈A. Thus, ψ is a homomorphism. In order to show that ψ is injective, suppose that ψ(y1) = ψ(y2) for some y1, y2 ∈A. Since A is a subgroup of the group (X, +), we have y1 −y2 ∈A. Hence, according to (11) and (18), we obtain Thus, ψ is a homomorphism. In order to show that ψ is injective, suppose that ψ(y1) = ψ(y2) for some y1, y2 ∈A. Since A is a subgroup of the group (X, +), we have y1 −y2 ∈A. Hence, according to (11) and (18), we obtain x + y1 −y2 ∈A if and only if x ∈A. x + y1 −y2 ∈A if and only if x ∈A. Therefore, in view of (19), we get f(x + y1 −y2) = z = f(x) for x ∈X \ A Therefore, in view of (19), we get f(x + y1 −y2) = z = f(x) for x ∈X \ A and and f(x + y1 −y2) = ψ(x + y1 −y2) = ψ(x) ◦ψ(y1) ◦ψ(−y2) = ψ(x) ◦ψ(y2) ◦ψ(−y2) = ψ(x) ◦ψ(0) = ψ(x) = f(x) for x ∈A. As f is non-periodic, this implies that y1 = y2. Hence, ψ is injective and so, (c) is valid. As f is non-periodic, this implies that y1 = y2. Hence, ψ is injective and so, (c) is valid. 3. According to Lemma 2.3, we get (1 −g(y0))x −(1 −g(x))y0 = 0 for x ∈Ag. Case 3. According to Lemma 2.3, we get Case 3. According to Lemma 2.3, we get (1 −g(y0))x −(1 −g(x))y0 = 0 for x ∈Ag. Hence x = 1 −g(x) 1 −g(y0)y0 for x ∈Ag. (24) (24) ol. 78 (2023) Non-periodic solutions of the Gol¸ab–Schinzel type Page 7 of 15 28 Vol. 78 (2023) Page 7 of 15 28 Since Ag \ {0} ̸= ∅, this implies that y0 ̸= 0. Thus, replacing in (24) x by ty0, we obtain Since Ag \ {0} ̸= ∅, this implies that y0 ̸= 0. Thus, replacing in (24) x by ty0, we obtain g(ty0) = 1 −(1 −g(y0))t whenever ty0 ∈Ag. g(ty0) = 1 −(1 −g(y0))t whenever ty0 ∈Ag. n particular, we have g(ty0) = 1 −(1 −g(y0))t whenever ty0 ∈Ag. 2. Results In particular, we have In particular, we have g t 1 −g(y0)y0 = 1 −t whenever t 1 −g(y0)y0 ∈Ag. Hence, putting x0 := 1 1 −g(y0)y0, (25) (25) we get x0 ̸= 0 and we get x0 ̸= 0 and g(tx0) = 1 −t whenever tx0 ∈Ag. (26) Moreover, in view of (24)–(25), we have Ag ⊆Span{x0} := {tx0 : t ∈R}. (27) (26) Moreover, in view of (24)–(25), we have (27) Therefore, taking G := {1 −t : tx0 ∈Ag}, (28) (28) we conclude that g is of the form (22). Since {0, y0} ⊆Ag, it follows from (25) and (28) that {1, g(y0)} ⊆G ̸= ∅. (29) (29) Note that, in view of (26), we have x0 ̸∈Ag. Thus, taking into account (28), we get G ⊆R \ {0}. We show that G is a non-trivial subgroup of the group (R \ {0}, ·) and φ : G →S, given by φ(t) = f((1 −t)x0) for t ∈G, (30) (30) is a homomorphism of G into (S, ◦). To this end, ﬁx ω, ξ ∈G. Then ω = 1 −s and ξ = 1 −t with some s, t ∈R such that sx0 ∈Ag and tx0 ∈Ag. Hence, applying Lemma 2.2(ii), in view of (26), we obtain (s + (1 −s)t)x0 = sx0 + g(sx0)tx0 ∈Ag. Thus, ωξ = (1 −s)(1 −t) = 1 −(s + (1 −s)t) ∈G. Furthermore, using (26) again, we get Furthermore, using (26) again, we get sx0 + g(sx0) − s 1 −sx0 = sx0 + (1 −s) − s 1 −sx0 = 0 ∈Ag. Hence, according to Lemma 2.2 (ii), we have − s 1−sx0 ∈Ag and so ω−1 = 1 1 −s = 1 − − s 1 −s ∈G. J. Chudziak 28 Page 8 of 15 Results Math Results Math In this way we have proved that G is a subgroup of the group (R\{0}, ·). Note also that, in view of (29), G is non-trivial. Furthermore, making use of (6), (26) and (30), we get φ(ωξ) = φ((1 −s)(1 −t)) = f((1 −(1 −s)(1 −t))x0) = f(sx0 + (1 −s)tx0) = f(sx0 + g(sx0)tx0) = f(sx0) ◦f(tx0) = φ(1 −s) ◦φ(1 −t) = φ(ω) ◦φ(ξ). Therefore, φ is a homomorphism of G into (S, ◦). 2. Results Moreover, taking into account (27)–(28) and (30), in view of Lemma 2.1, we obtain that there exists a z ∈ E(S) such that (21) and (23) hold. Note that, if φ(tz) = z for some tz ∈G then, making use of (21), we get φ(t) = φ t tz tz = φ t tz ◦φ(tz) = φ t tz ◦z = z for t ∈G. Since f is a non-constant function, this contradicts (23). Thus, (20) holds. Suppose that φ is periodic. We claim that f(x −t0x0) = f(x + t0x0) = f(x) for x ∈X. (31) (31) To this end, ﬁx an x ∈X. If x ̸∈Span{x0} then x −t0x0 ̸∈Span{x0} and x + t0x0 ̸∈Span{x0}. Hence, in view of (23), we get f(x −t0x0) = f(x + t0x0) = f(x) = z. (32) (32) If x = tx0 for some t ∈R with 1 −t ̸∈G then, by (13), we have 1 −t −t0 ̸∈G and 1−t+t0 ̸∈G. Thus, applying (23) again, we obtain (32). Finally, if x = tx0 for some t ∈R with 1 −t ∈G then, in view of (13), we get 1 −t + t0 ∈G and 1 −t −t0 ∈G. Hence, making use of (14) and (23), we conclude that If x = tx0 for some t ∈R with 1 −t ̸∈G then, by (13), we have 1 −t −t0 ̸∈G and 1−t+t0 ̸∈G. Thus, applying (23) again, we obtain (32). Finally, if x = tx0 for some t ∈R with 1 −t ∈G then, in view of (13), we get 1 −t + t0 ∈G and 1 −t −t0 ∈G. Hence, making use of (14) and (23), we conclude that f(x −t0x0) = f((t −t0)x0) = φ(1 −t + t0) = φ(1 −t) = f(tx0) = f(x) f(x −t0x0) = f((t −t0)x0) = φ(1 −t + t0) = φ(1 −t) = f(tx0) = f(x) and f(x + t0x0) = f((t + t0)x0) = φ(1 −t −t0) = φ(1 −t) = f(tx0) = f(x). f(x + t0x0) = f((t + t0)x0) = φ(1 −t −t0) = φ(1 −t) = f(tx0) = f(x). f(x + t0x0) = f((t + t0)x0) = φ(1 −t −t0) = φ(1 −t) = f(tx0) = f(x). In this way we have proved (31). Since f is non-periodic and t0x0 ̸= 0, this yields a contradiction. 2. Results Therefore, φ is non-periodic and so, (d) is valid. In this way we have proved (31). Since f is non-periodic and t0x0 ̸= 0, this yields a contradiction. Therefore, φ is non-periodic and so, (d) is valid. yields a contradiction. Therefore, φ is non-periodic and so, (d) is valid. A standard computations show that, if one of the possibilities (a)–(d) holds, then the pair (f, g) satisﬁes equation (6). Furthermore, it is obvious that in the cases (a)–(b) f is non-periodic. In the case (c), for every x ∈A, we have ψ(x) = ψ(x + 0) = ψ(x) ◦ψ(0). Hence, as ψ is injective, taking into account (17), we get ψ(0) ̸= z. Thus, applying the injectivity of ψ again, in view of (19), we obtain f(x) ̸= ψ(0) = f(0) for x ∈X \ {0}. This implies that f is non-periodic. ( ) \ { } Consider the case (d). Suppose that f is periodic and ﬁx its period y ∈ X \ {0}. Then, taking a t ∈G and using (23) and then (20), we get f((1 −t)x0 −y) = f((1 −t)x0 + y) = f((1 −t)x0) = φ(t) ̸= z. (33 (33) Hence, applying (23) again, we conclude that Hence, applying (23) again, we conclude that (1 −t)x0 −y = s1x0 (34) (1 −t)x0 −y = s1x0 (34) Vol. 78 (2023) Non-periodic solutions of the Gol¸ab–Schinzel type Page 9 of 15 28 Vol. 78 (2023) Non-periodic solutions of the Gol¸ab–Schinzel type Page 9 of 15 28 Vol. 78 (2023) and (1 −t)x0 + y = s2x0 (35) (1 −t)x0 + y = s2x0 (1 −t)x0 + y = s2x0 (35) with s1, s2 ∈R such that 1 −si ∈G for i ∈{1, 2}. Thus, setting t0 := s2−s1 2 , we get with s1, s2 ∈R such that 1 −si ∈G for i ∈{1, 2}. Thus, setting t0 := s2−s1 2 , we get y = t0x0. (36) (36) y = t0x0. (36 y = t0x0. Since x0 ̸= 0, from (34)-(36) we derive that t + t0 = 1 −s1 ∈G and t −t0 = 1 −s2 ∈G. Thus, taking into account (23), (33) and (36), we obtain Since x0 ̸= 0, from (34)-(36) we derive that t + t0 = 1 −s1 ∈G and t −t0 = 1 −s2 ∈G. 2. Results Thus, taking into account (23), (33) and (36), we obtain φ(t) = f((1 −t)x0 + y) = f((1 −t + t0)x0) = φ(t −t0). φ(t) = f((1 −t)x0 + y) = f((1 −t + t0)x0) = φ(t −t0). and φ(t) = f((1 −t)x0 −y) = f((1 −t −t0)x0) = φ(t + t0). In this way we have proved that (13)-(14) hold. Moreover, as y ̸= 0, it follows from (36) that t0 ̸= 0. Therefore, t0 is a period of φ, which yields a contradiction and shows that f is non-periodic. □ In this way we have proved that (13)-(14) hold. Moreover, as y ̸= 0, it follows from (36) that t0 ̸= 0. Therefore, t0 is a period of φ, which yields a contradiction and shows that f is non-periodic. □ Corollary 2.5. Assume that f : X →R and g : X →R. 2. Results If the pair (f, g) sat- isﬁes equation (7) and f is non-periodic then one of the following possibilities holds: (i) there exists an a ∈R \ {0} such that g is of the form (15) and f(x) = 1 for x = 0, 0 for x ̸= 0; (37) (37) (ii) g = 1 and there exist an injective additive function a : X →R such that ii) g = 1 and there exist an injective additive function a : X →R such that f(x) = ea(x) for x ∈X; f(x) = ea(x) for x ∈X; (38) (38) (iii) there exist a non-trivial proper subgroup A of the group (X, +), a non- trivial subgroup A0 of A and an injective additive function a : A →R such that (iii) there exist a non-trivial proper subgroup A of the group (X, +), a non- trivial subgroup A0 of A and an injective additive function a : A →R such that x + y ∈A0 for x, y ∈A \ A0, (39) he form (18) and x + y ∈A0 for x, y ∈A \ A0, (39) (39) \ ( ) g is of the form (18) and g is of the form (18) and f(x) = ⎧ ⎨ ⎩ ea(x) for x ∈A0, −ea(x) for x ∈A \ A0, 0 otherwise; (40) (40) (iv) there exist an x0 ∈X \ {0}, a non-trivial subgroup G of the group (R \ {0}, ·) and a non-periodic multiplicative function φ : G →R \ {0} such that g is of the form (22) and (iv) there exist an x0 ∈X \ {0}, a non-trivial subgroup G of the group (R \ {0}, ·) and a non-periodic multiplicative function φ : G →R \ {0} such that g is of the form (22) and f(x) = φ(1 −t) whenever x = tx0 and 1 −t ∈G, 0 otherwise. (41) (41) Conversely, if one of the possibilities (i)–(iv) holds, then the pair (f, g) satisﬁes equation (7) and f is non-periodic. J. Chudziak 28 Page 10 of 15 Results Math Results Math Proof. Assume that the pair (f, g) satisﬁes equation (7) and f is non-periodic. Then, applying Theorem 2.4, with (S, ◦) being the multiplicative semigroup of real numbers, we conclude that one of the possibilities (a)-(d) holds. The converse is a direct consequence of Theorem 2.4. 2. Results Note that in this case, we have E(S) = {0, 1} and so (s, z) = (1, 0) is the only pair of elements of E(S) such that s ̸= z and s ◦z = z. Hence, (a) leads to (i). In the case (b), we have f(x) ̸= 0 for x ∈X. In fact, if f(y) = 0 for some y ∈X, then f(x) = f(x −y + y) = f(x −y)f(y) = 0 for x ∈X, which contradicts the injectivity of f. Hence, which contradicts the injectivity of f. Hence, f(x) = f x 2 + x 2 = f x 2 2 > 0 for x ∈X. Thus, deﬁning a function a : X →R by a(x) = ln f(x) for x ∈X, we conclude that a is injective and additive. Moreover, f is of the form (38) and so (ii) is valid. Thus, deﬁning a function a : X →R by a(x) = ln f(x) for x ∈X, we conclude that a is injective and additive. Moreover, f is of the form (38) and so (ii) is valid. Assume that (c) holds. Then, (17) becomes z(1 −ψ(t)) = 0 for t ∈A. Hence, as A is a non-trivial subgroup of the group (X, +) and ψ is injective, we have z = 0. Moreover, arguing as in the previous case, one can show that ψ does not vanish. Thus, ψ(0) = ψ(0)2 > 0, that is 0 ∈A0 := {x ∈A : ψ(x) > 0}. Furthermore, ψ(x −y) = ψ(x −y)ψ(y) ψ(y) = ψ(x) ψ(y) > 0 for x, y ∈A0 and ψ(x + y) = ψ(x)ψ(y) > 0 for x, y ∈A \ A0. Hence, A0 is a subgroup of A and (39) holds. Note also that, as A is a non- trivial subgroup of the group (X, +), in view of (39), A0 is non-trivial. Let a : A →R be given by a(x) = ln \|ψ(x)\| for x ∈A. Then a is an additive function and ψ(x) = ea(x) for x ∈A0, −ea(x) for x ∈A \ A0. (42) (42) Therefore, as z = 0, taking into account (19), we conclude that f is of the form (40). In order to show that a is injective, suppose that a(x0) = 0 for some x0 ∈A. Then a(2x0) = 0. The converse is a direct consequence of Theorem 2.4. 2. Results Moreover, as A0 is a subgroup of A, in view of (39), we have 2x0 ∈A0. Thus, applying (42), we get ψ(2x0) = ψ(0) = 1. Since ψ is injective this implies that 2x0 = 0 and so x0 = 0. Hence, a is injective. In this way we have proved that (iii) holds. Therefore, as z = 0, taking into account (19), we conclude that f is of the form (40). In order to show that a is injective, suppose that a(x0) = 0 for some x0 ∈A. Then a(2x0) = 0. Moreover, as A0 is a subgroup of A, in view of (39), we have 2x0 ∈A0. Thus, applying (42), we get ψ(2x0) = ψ(0) = 1. Since ψ is injective this implies that 2x0 = 0 and so x0 = 0. Hence, a is injective. In this way we have proved that (iii) holds. In the case (d) from (20)–(21) we derive that z ̸= 1. Since z ∈E(S) = {0, 1}, this means that z = 0. Therefore, applying (20) and (23), we obtain that φ : G →R \ {0} and f is of the form (41), respectively. Hence, (iv) is valid. □ The converse is a direct consequence of Theorem 2.4 Page 11 of 15 28 Vol. 78 (2023) Non-periodic solutions of the Gol¸ab–Schinzel type Vol. 78 (2023) Page 11 of 15 28 Vol. 78 (2023) Non-periodic solutions of the Gol¸ab–Schinzel type Remark 2. It follows from (13) that, if G ⊆R\{0}, G ̸= ∅and φ : G →R\{0} is a periodic function, then none of its periods belongs to G. Furthermore, as one can easily check, any multiplicative function, in particular a constant function φ = 1, deﬁned on (0, ∞), Q \ {0} or R \ {0} is non-periodic. On the other hand, the function φ = 1 deﬁned on the following subgroup of the group (R \ {0}, ·) , (R \ {0}, ·) G := m n : m and n are odd integers , is multiplicative and periodic. In fact, t0 = 2 is a period of φ. is multiplicative and periodic. In fact, t0 = 2 is a period of φ. The existence of a non-constant periodic multiplicative function deﬁned on a (proper) subgroup of (R \ {0}, ·) remains an open problem. 2. Results The following two examples show that, for any real linear space, equation (7) possesses several solutions with periodic f. Example 1. Assume that A is a non-trivial subgroup of the group (X, +) and f : X →R is of the form f(x) = 1 for x ∈A, 0 for x ∈X \ A. Then every element of A \ {0} is a period of f and, as one can easily check, the pair (f, g), where g = f, satisﬁes equation (7). Example 2. Assume that the dimension of X is greater than 1. Let L : X →R be a non-zero linear functional. Then ker L is a non-trivial linear subspace of X and every element of ker L \ {0} is a period of L. Thus, a function f : X →R, given by f(x) = L(x) + 1 for x ∈X, f(x) = L(x) + 1 for x ∈X, is periodic. Moreover, as a standard computation shows, a pair (f, g), where g = f, satisﬁes equation (7). Corollary 2.6. Assume that f : R →S and g : R →R. If the pair (f, g) satisﬁes equation Corollary 2.6. Assume that f : R →S and g : R →R. 2. Results If the pair (f, g) satisﬁes equation f(x + g(x)y) = f(x) ◦f(y) for x, y ∈R, (43) (43) and f is non-periodic then one of the following possibilities is valid: and f is non-periodic then one of the following possibilities is valid: and f is non-periodic then one of the following possibilities is valid: (i) there exist an a ∈R \ {0} and s, z ∈E(S) such that s ̸= z, s ◦z = z and (15)–(16) hold; (i) there exist an a ∈R \ {0} and s, z ∈E(S) such that s ̸= z, s ◦z = z and (15)–(16) hold; (ii) g = 1 and f is an injective homomorphism of the additive group of real numbers into (S, ◦); (ii) g = 1 and f is an injective homomorphism of the additive group of real numbers into (S, ◦); ( ) (iii) there exist a non-trivial proper subgroup A of the group (R, +), an in- jective homomorphism ψ : A →S and a z ∈E(S) such that (17)–(19) hold; (iii) there exist a non-trivial proper subgroup A of the group (R, +), an in- jective homomorphism ψ : A →S and a z ∈E(S) such that (17)–(19) hold; hudziak Results Math J. Chudziak 28 Page 12 of 15 Results Math (iv) there exist a c ∈R\{0}, a non-trivial subgroup G of the group (R\{0}, ·), a non-periodic homomorphism φ : G →S and a z ∈E(S) such that (20)– (21) hold, g is of the form (4) and (iv) there exist a c ∈R\{0}, a non-trivial subgroup G of the group (R\{0}, ·), a non-periodic homomorphism φ : G →S and a z ∈E(S) such that (20)– (21) hold, g is of the form (4) and (iv) there exist a c ∈R\{0}, a non-trivial subgroup G of the group (R\{0}, ·), a non-periodic homomorphism φ : G →S and a z ∈E(S) such that (20)– (21) hold, g is of the form (4) and f(x) = φ(cx + 1) whenever cx + 1 ∈G, z otherwise. (44) (44) Conversely, if one of the possibilities (i)–(iv) holds, then the pair (f, g) satisﬁes equation (43) and f is non-periodic. Proof. Assume that the pair (f, g) satisﬁes equation (43) and f is non-periodic. Then, applying Theorem 2.4 (with X = R), we obtain that one of the pos- sibilities (a)–(d) holds. □ The converse implication follows from Theorem 2.4. Corollary 2.7. Let f, g : R →R. If the pair (f, g) satisﬁes equation (1) and f is non-periodic then one of the following possibilities holds: (i) there exist an a ∈R\{0} such that g and f are of the forms (15) and (2.5), respectively; (ii) g = 1 and there exist an injective additive function a : R →R such tha (ii) g = 1 and there exist an injective additive function a : R →R such that f(x) = ea(x) for x ∈R; (iii) there exist a non-trivial proper subgroup A of the additive group of real numbers, a non-trivial subgroup A0 of A and an injective additive function a : A →R such that (39) holds, g is of the form (18) and f is of the form (40); (iii) there exist a non-trivial proper subgroup A of the additive group of real numbers, a non-trivial subgroup A0 of A and an injective additive function a : A →R such that (39) holds, g is of the form (18) and f is of the form (40); (iii) there exist a non-trivial proper subgroup A of the additive group of real numbers, a non-trivial subgroup A0 of A and an injective additive function a : A →R such that (39) holds, g is of the form (18) and f is of the form (40); (iv) there exist a c ∈R\{0}, a non-trivial subgroup G of the group (R\{0}, ·) and a non-periodic multiplicative function φ : G →R \ {0} such that g and f are of the forms (4) and (5), respectively. (iv) there exist a c ∈R\{0}, a non-trivial subgroup G of the group (R\{0}, ·) and a non-periodic multiplicative function φ : G →R \ {0} such that g and f are of the forms (4) and (5), respectively. Conversely, if one of the possibilities (i)-(iv) holds, then the pair (f, g) satisﬁes equation (1) and f is non-periodic. Conversely, if one of the possibilities (i)-(iv) holds, then the pair (f, g) satisﬁes equation (1) and f is non-periodic. Proof. Assume that the pair (f, g) satisﬁes equation (1) and f is non-periodic. Then, applying Theorem 2.4 with X = R and (S, ◦) being a multiplicative semigroup of real numbers, we get that one of the possibilities (a)-(d) holds. 2. Results Obviously, (a), (b) and (c) imply (i), (ii) and (iii), respectively. Furthermore, as x = x x0 x0 for x ∈R, if (d) is valid, then applying (22) and (23), for every x ∈R, we obtain g(x) = 1 −x x0 whenever 1 −x x0 ∈G and f(x) = φ 1 −x x0 whenever 1 −x x0 ∈G, respectively. Thus, taking c := −1 x0 , we conclude that g and f are of the forms (4) and (44), respectively. Hence, (iv) holds. respectively. Thus, taking c := −1 x0 , we conclude that g and f are of the forms (4) and (44), respectively. Hence, (iv) holds. such that g and f are of the forms (4) and (5), respectively. Proof. If (A) is valid then, applying Corollary 2.7, we obtain that there exist a c ∈R \ {0}, a non-trivial subgroup G of the group (R \ {0}, ·) and a non- periodic multiplicative function φ : G →R \ {0} such that g and f are of the forms (4) and (5), respectively. Furthermore, as f(R) \ {−1, 0, 1} ̸= ∅, in view of (5), we get (45). Taking into account Corollary 2.7, one can easily get the inverse impli- cation. □ Remark 3. It follows from (45) that a subgroup G is inﬁnite. Acknowledgements The author is grateful to the referee for a number of valuable suggestions. Funding The author declare that no funds, grants or other support were re- ceived during the preparation of this manuscript. □ Let f, g : R →R. The following two statements are equivalent: (A) the pair (f, g) satisﬁes equation (1), f(R)\{−1, 0, 1} ̸= ∅, g(R)\{0, 1} ̸= ∅ and f is non-periodic; (A) the pair (f, g) satisﬁes equation (1), f(R)\{−1, 0, 1} ̸= ∅, g(R)\{0, 1} ̸= and f is non-periodic; (B) there exist a c ∈R \ {0}, a subgroup G of the group (R \ {0}, ·) and a non-periodic multiplicative function φ : G →R \ {0}, with φ(G) \ {−1, 1} ̸= ∅, (45) (45) such that g and f are of the forms (4) and (5), respectively. □ Repeating the arguments from the proof of Corollary 2.5, we obtain that (a), Proof. Assume that the pair (f, g) satisﬁes equation (1) and f is non-periodic. Then, applying Theorem 2.4 with X = R and (S, ◦) being a multiplicative semigroup of real numbers, we get that one of the possibilities (a)-(d) holds. Repeating the arguments from the proof of Corollary 2.5, we obtain that (a), Vol. 78 (2023) Non-periodic solutions of the Gol¸ab–Schinzel type Page 13 of 15 28 Vol. 78 (2023) (b) and (c) imply (i), (ii) and (iii), respectively. Moreover, if (d) holds, then g and f are given by (22) and (41), respectively, where x0 ∈R \ {0}, G is a non-trivial subgroup of the group (R \ {0}, ·) and φ : G →R \ {0} is a non- periodic multiplicative function. Hence, taking c := −1 x0 and arguing as in the proof of Corollary 2.6, we conclude that g and f are of the forms (4) and (5), respectively. Therefore, (iv) is valid. (b) and (c) imply (i), (ii) and (iii), respectively. Moreover, if (d) holds, then g and f are given by (22) and (41), respectively, where x0 ∈R \ {0}, G is a non-trivial subgroup of the group (R \ {0}, ·) and φ : G →R \ {0} is a non- periodic multiplicative function. Hence, taking c := −1 x0 and arguing as in the proof of Corollary 2.6, we conclude that g and f are of the forms (4) and (5), respectively. Therefore, (iv) is valid. (b) and (c) imply (i), (ii) and (iii), respectively. Moreover, if (d) holds, then g and f are given by (22) and (41), respectively, where x0 ∈R \ {0}, G is a non-trivial subgroup of the group (R \ {0}, ·) and φ : G →R \ {0} is a non- periodic multiplicative function. Hence, taking c := −1 x0 and arguing as in the proof of Corollary 2.6, we conclude that g and f are of the forms (4) and (5), respectively. Therefore, (iv) is valid. □ The converse is a consequence of Theorem 2.4. □ The converse is a consequence of Theorem 2.4. We conclude the paper with the result providing a positive answer to the question raised in [16]. Corollary 2.8. Let f, g : R →R. The following two statements are equivalent ary 2.8. J. Chudziak Results Math Results Math 28 Open Access. 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II. 211, 117–136 (2002) [18] Luneburg, H., Plaumann, P.: Die Funktionalgleichung von Gol¸ab und Schinzel in Galoisfeldern. Arch. References Math. (Basel) 28, 55–59 (1977) [19] Wolod´zko, S.: Solution g´en´erale de l’equation fonctionnelle f[x + yf(x)] = f(x)f(y). Aequationes Math. 2, 12–29 (1968) Jacek Chudziak Institute of Mathematics University of Rzesz´ow Pigonia 1 35-310 Rzesz´ow Poland e-mail: jchudziak@ur.edu.pl Jacek Chudziak Jacek Chudziak Institute of Mathematics Institute of Mathematics University of Rzesz´ow e-mail: jchudziak@ur.edu.pl Received: February 16, 2022. Accepted: November 3, 2022. Publisher’s Note Springer Nature remains neutral with regard to jurisdic- tional claims in published maps and institutional aﬃliations.
https://openalex.org/W4307370122	https://www.mdpi.com/2073-4409/11/21/3362/pdf?version=1666699780	English	null	Diabetic Macular Edema: Current Understanding, Molecular Mechanisms and Therapeutic Implications	Cells	2,022	cc-by	22,011	cells cells Citation: Zhang, J.; Zhang, J.; Zhang, C.; Zhang, J.; Gu, L.; Luo, D.; Qiu, Q. Diabetic Macular Edema: Current Understanding, Molecular Mechanisms and Therapeutic Implications. Cells 2022, 11, 3362. https://doi.org/10.3390/ cells11213362 Academic Editor: Shikun He Received: 31 August 2022 Accepted: 20 October 2022 Published: 25 October 2022 Citation: Zhang, J.; Zhang, J.; Zhang, C.; Zhang, J.; Gu, L.; Luo, D.; Qiu, Q. Diabetic Macular Edema: Current Understanding, Molecular Mechanisms and Therapeutic Implications. Cells 2022, 11, 3362. https://doi.org/10.3390/ cells11213362 Keywords: diabetic retinopathy; diabetic macular edema; blood-retinal barrier breakdown; drainage dysfunction; inﬂammation; anti-VEGF; proteomics; metabolomics Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Jingfa Zhang 1,2,3,4,5,,† , Jingxiang Zhang 6,†, Chaoyang Zhang 1,2,3,4,5, Jingting Zhang 1,2,3,4,5, Limin Gu 7, Dawei Luo 1,2,3,4,5 and Qinghua Qiu 1,2,3,4,5, Jingfa Zhang 1,2,3,4,5,,† , Jingxiang Zhang 6,†, Chaoyang Zhang 1,2,3,4,5, Jingting Zhang 1,2,3,4,5, Limin Gu 7, Dawei Luo 1,2,3,4,5 and Qinghua Qiu 1,2,3,4,5, Jingfa Zhang 1,2,3,4,5,,† , Jingxiang Zhang 6,†, Chaoyang Zhang 1,2,3,4,5, Jingting Zhang 1,2,3,4,5, Limin Gu 7, Dawei Luo 1,2,3,4,5 and Qinghua Qiu 1,2,3,4,5, 1 Department of Ophthalmology, Shanghai General Hospital (Shanghai First People’s Hospital), School of Medicine, Shanghai Jiao Tong University, 100 Haining Road, Hongkou District, Shanghai 200080, China 2 National Clinical Research Center for Eye Diseases, Shanghai 200080, China 3 Shanghai Key Laboratory of Ocular Fundus Diseases, Shanghai 200080, China 4 Shanghai Engineering Center for Visual Science and Photomedicine, Shanghai 200080, China 5 Shanghai Engineering Center for Precise Diagnosis and Treatment of Eye Diseases, Shanghai 200080, China 6 Nursing Department, People’s Hospital of Huangdao District, Qingdao 266400, China 7 Department of Ophthalmology, Shanghai Aier Eye Hospital, Shanghai 200336, China * Correspondence: jingfa.zhang@shgh.cn (J.Z.); qinghuaqiu@163.com (Q.Q.) † These authors contributed equally to this work. Abstract: Diabetic retinopathy (DR), with increasing incidence, is the major cause of vision loss and blindness worldwide in working-age adults. Diabetic macular edema (DME) remains the main cause of vision impairment in diabetic patients, with its pathogenesis still not completely elucidated. Vascular endothelial growth factor (VEGF) plays a pivotal role in the pathogenesis of DR and DME. Currently, intravitreal injection of anti-VEGF agents remains as the ﬁrst-line therapy in DME treatment due to the superior anatomic and functional outcomes. However, some patients do not respond satisfactorily to anti-VEGF injections. More than 30% patients still exist with persistent DME even after regular intravitreal injection for at least 4 injections within 24 weeks, suggesting other pathogenic factors, beyond VEGF, might contribute to the pathogenesis of DME. Recent advances showed nearly all the retinal cells are involved in DR and DME, including breakdown of blood-retinal barrier (BRB), drainage dysfunction of Müller glia and retinal pigment epithelium (RPE), involvement of inﬂammation, oxidative stress, and neurodegeneration, all complicating the pathogenesis of DME. The profound understanding of the changes in proteomics and metabolomics helps improve the elucidation of the pathogenesis of DR and DME and leads to the identiﬁcation of novel targets, biomarkers and potential therapeutic strategies for DME treatment. The present review aimed to summarize the current understanding of DME, the involved molecular mechanisms, and the changes in proteomics and metabolomics, thus to propose the potential therapeutic recommendations for personalized treatment of DME. 1. Overview of Diabetic Retinopathy (DR) and Diabetic Macular Edema (DME) Diabetes mellitus is a chronic, metabolic disease, which is characterized by a prolonged period of hyperglycemia. According to the reports of International Diabetes Federation (IDF) Diabetes Atlas 10th edition (https://diabetesatlas.org, accessed on 21 October 2022), the prevalence of diabetes has continued to increase globally, and diabetes still remains a signiﬁcant challenge to the health and well-being of people. As estimated in IDF Diabetes Atlas 10th edition, there were 537 million adults (20–79 years) with diabetes in 2021. This number is estimated to rise to 643 million by 2030 and 783 million by 2045. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Given the global pandemic of diabetes, diabetes causes serious damage to many organs, including the heart, blood vessels, eyes, and kidneys, which remains a major cause https://www.mdpi.com/journal/cells Cells 2022, 11, 3362. https://doi.org/10.3390/cells11213362 Cells 2022, 11, 3362 2 of 29 of heart attacks, stroke, blindness, and kidney failure (https://www.who.int, accessed on 21 October 2022). As one of the common complications of diabetes, diabetic retinopathy (DR) remains a major cause of visual impairment and blindness in working-age adults. Despite the advances in optimal control of systemic risk factors, i.e., hyperglycemia, hypertension and hyperglycemia, and the application of anti-vascular endothelial growth factor (VEGF) agents (Anti-VEGF), the prevalence of DR remains high in diabetic patients. Among diabetic patients, about one third of patients suffered from DR, which increases markedly after the age of 60 years due to the longer duration of diabetes [1]. A comprehensive systematic review and meta-analysis showed that the estimated pooled prevalence of DR was 28.41% (95% CI: 25.98 to 30.84), 25.93% (95% CI: 23.54 to 28.31), and 28.95% (95% CI: 26.57 to 31.32) in the diabetic population in general, in female, and in male, respectively; with no inter-gender difference for DR prevalence [1]. g p Diabetic macular edema (DME) represents the major cause of vision impairment in diabetic patients with an increasing prevalence worldwide [2]. DME is considered as the retinal thickening, which involves or approaches the fovea due to abnormal accumulation of ﬂuid in the macula under diabetic condition. In diabetic retina, ﬂuid accumulation in the macular area leads to increased central retinal/macular thickness, resulting in DME. 1. Overview of Diabetic Retinopathy (DR) and Diabetic Macular Edema (DME) The prevalence of DME varies widely, ranging from 4.2% to 14.3% in people with type 1 diabetes mellitus (T1DM) and 1.4% to 5.57% in people with type 2 diabetes mellitus (T2DM) [3,4]. Based on the ﬁndings of optical coherence tomography (OCT)-diagnosed DME, a systematic review showed the analysis of the pooled prevalence of DME was 5.47% (95% CI: 3.66–7.62%) overall worldwide, 5.81% (95% CI: 0.07–18.51%) in low-to-middle- income countries, and 5.14% (95% CI: 3.44–7.15%) in high-income countries, respectively [5]. The high prevalence of DR and DME not only seriously affects people’s life quality, but also lays a heavy economic burden on health care budgets. 2. OCT-Based DME Classiﬁcation and Evaluation Nowadays, OCT is widely used for the early detection of DME. Based on OCT ﬁnd- ings, several types of DME were classiﬁed, i.e., diffuse edema (sponge-like diffuse retinal thickening), cystoid edema (retinal thickening with intraretinal cystoid change), macular edema with serous retinal detachment (macular thickening with subretinal ﬂuid (SRF)), and mixed edema with at least two different edema types [6]. Other types of DME detected with OCT were also reported and proposed, including diffuse edema, cystoid edema, and cystoid degeneration, combined with different morphological features such as serous mac- ular detachment, vitreomacular interface abnormalities, hard exudates, and photoreceptor status [7]. Arf S, et al. pointed that serous macular detachment should not be classiﬁed as a type of DME, which was an accompanying morphological change [7]. The author also indicated that cystoid macular degeneration should be differentiated from cystoid macular edema, because cystoid macular degeneration was correlated with the poor out- comes of the retinal function and morphology [7]. Based on OCT classiﬁcation, cystoid macular edema is considered as intraretinal cystoid-like, hypo-reﬂective spaces with highly reﬂective septa separating the cystoid-like spaces in OCT B-scan, while cystoid macular degeneration is accepted when the horizontal diameter of the cystoid spaces larger than 600 µm (≥600 µm) [7], even though there is no consensus on its deﬁnition. In a retrospective study, DME was classiﬁed based on the localization and area of the ﬂuid using en face images of swept-source OCT [8]. The retina was segmented into two segments, i.e., Segment 1, mainly comprising the inner nuclear layer (INL) and outer plexi- form layer (OPL), including Henle’s ﬁber layer (HFL); and Segment 2, mainly comprising the outer nuclear layer (ONL). Fujiwara et al. reported that the DME patients with diffuse ﬂuid at Segment 2 demonstrated signiﬁcantly poorer visual acuity, higher disruption rates of ellipsoid zone (EZ), and greater central subﬁeld thickness (CSFT) than those without ﬂuid at Segment 2 [8]. The study indicated the impact of the localization and extent of the ﬂuid on the visual outcome, and also suggested other biomarkers besides CSFT should be considered for evaluating the outcome of DME [8]. Cells 2022, 11, 3362 3 of 29 DME development and treatment outcome can also be well evaluated with OCT. In one study, 205 eyes from 129 diabetic patients underwent OCT angiography (OCTA) examination and were followed-up for at least 2 years [9]. 3. The Pathogenesis of DME DME is due to an imbalance between ﬂuid entry, ﬂuid exit and retinal hydraulic conductivity, leading to the accumulation of intraretinal ﬂuid (IRF) or SRF [2]. IRF is the ﬂuid accumulates in retinal parenchyma, mainly in the extracellular spaces of INL, OPL and ONL, while SRF is the ﬂuid accumulation in subretinal space right underneath the neurosensory retina and above retinal pigment epithelium (RPE). Based on the Starling equation, in normal retina, the balance of inﬂux and efﬂux of the ﬂuid in retina is maintained by the blood-retinal barrier (BRB) integrity and the active drainage function of Müller glia and RPE [2,11]. The intact BRB and the active drainage function of both Müller glia and RPE maintain the retina under a relative dehydrated condition and normal function [2]. Under physiologic conditions, Müller glia removes the ﬂuid from the retinal interstitial tissue to the blood vessels or vitreous, while RPE removes the SRF to the choroid by active transport [12,13]. However, the pathogenesis of DME is really complex, and it has still not been fully elucidated yet. Among the multiple, intricate mechanisms (Figure 1), DME develops mainly due to the two major underlying mechanisms, i.e., BRB breakdown, increasing ﬂuid inﬂux into retina parenchyma, and the decrease in drainage functions by Müller glia and RPE, resulting in the reduced ﬂuid efﬂux out of retina [14,15]. Moreover, inﬂammation also plays a contributory role in BRB breakdown (Figure 1), resulting in DME [2,16–18]. The pathogenesis of DME starts with retinal hypoxia, resulting in the hyperpermeabil- ity of retinal capillary and increased intravascular pressure due to dysfunction of vascular autoregulation. The capillary hyperpermeability is mainly attributed to hypoxia-induced upregulation of VEGF, which plays a major role in the pathogenesis of DME. Currently, intravitreal anti-VEGF injection has become the ﬁrst-line therapy in DME treatment, due to its many clinical beneﬁts including functional and morphological improvements, and even halted or reversed DR progression. However, some patients respond unsatisfactorily to anti-VEGF injections. Based on the ﬁndings of Diabetic Retinopathy Clinical Research (DRCR) Retina Network Protocol T, there are 31.6~65.6% patients with DME who still exist with persistent edema even after regular intravitreal injection for at least 4 injections within 24 weeks [19], suggesting other pathogenic factors, beyond VEGF, might contribute to the pathogenesis of DME. 2. OCT-Based DME Classiﬁcation and Evaluation The ﬁndings showed that some OCTA metrics could predict the progression of DR, which included foveal avascular zone area, vessel density, and fractal dimension of deep capillary plexus; whereas vessel density of superﬁcial capillary plexus predicted the development of DME [9]. Toto L, et al. used wideﬁeld optical coherence tomography angiography (WFOCTA) to evaluate the changes in retinal capillary nonperfusion areas and retinal capillary vessel density in DME patients treated with an intravitreal dexamethasone implant [10]. The results showed that a reduction in retinal capillary nonperfusion areas after intravitreal injection of dexamethasone implant, suggesting the reperfusion of retinal capillaries might be drug- related in the early period [10]. 3.1. Two Forms of Edema in DME: Intracellular Edema and Extracellular Edema 3.1. Two Forms of Edema in DME: Intracellular Edema and Extracellular Edema In addition to the anatomic location of macular ﬂuid accumulation as detected with OCT (SRF and IRF), there are two types of edema, i.e., intracellular edema and extracellular edema [17]. The ﬂuid accumulation in the intracellular space is considered as intracel- lular swelling or cytotoxic edema, while ﬂuid accumulation in the extracellular space is considered as extracellular edema or vasogenic edema [17]. In DME patients, both intracellular edema and extracellular edema can occur [2,17]. Diabetic intracellular edema can result from intracellular accumulation of sorbitol, lactate, and phosphates secondary to hyperglycemia-induced metabolic abnormalities [17]. Intracellular edema in the form of DME is the consequence of metabolic disturbances, which can be detected in RPE cells, Müller glia, and retinal neurons [20,21]. With the development of DME, the intracellular edema of the involved cells results in neuronal toxicity, contributing to vision loss and extracellular fluid volume increase [2]. Extracellular edema is mainly due to BRB breakdown, which leads to increased fluid accumulation and the decreased clearance of accumulated fluid with diabetes progression. Thus, in DME patients, intracellular edema, extracellular edema and even mixed edema can occur with the development of diabetes. 3. The Pathogenesis of DME p g Moreover, it remains largely unknown whether or not these anti-VEGF agents can restore the drainage functions of Müller glia and RPE to facilitate the absorbance and transport of excess ﬂuid out of retina. Alongside the VEGF pathway, DME formation is also attributed to other factors including leakage of microaneurysms, tractional effects by epiretinal membranes or posterior vitreous cortex, and inﬂammation from both retina and vitreous pool [2,17,18]. Cells 2022, 11, 3362 4 of 29 Figure 1. The proposed diagram for the pathogenesis and treatments of DR and DME. Anti-VEGF, anti-vascular endothelial growth factor; Ang-2, angiopoietin 2; DME, diabetic macular edema; DR, diabetic retinopathy; IL-6, interleukin 6; IL-6R, interleukin 6 receptor; NADPH, the reduced form of nicotinamide adenine dinucleotide phosphate; NSAIDs, nonsteroidal anti-inﬂammatory drugs; Tie2, tyrosine kinase with immunoglobulin-like and epidermal growth factor-like domains 2; TNF-α, tumor necrosis factor α; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor. Figure 1. The proposed diagram for the pathogenesis and treatments of DR and DME. Anti-VEGF, anti-vascular endothelial growth factor; Ang-2, angiopoietin 2; DME, diabetic macular edema; DR, diabetic retinopathy; IL-6, interleukin 6; IL-6R, interleukin 6 receptor; NADPH, the reduced form of nicotinamide adenine dinucleotide phosphate; NSAIDs, nonsteroidal anti-inﬂammatory drugs; Tie2, tyrosine kinase with immunoglobulin-like and epidermal growth factor-like domains 2; TNF-α, tumor necrosis factor α; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor. 3.2. Drainage Dysfunction of Müller Cells Leading to Intracellular Edema Müller glia, the major macroglia unique to the retina, transport and remove excess ﬂuid from retinal parenchyma into the vitreous and retinal vessels [2,22,23]. Müller cells have many aquaporins, ion channels, transmembrane proteins, and enzymes. The numerous functions of Müller glia include drainage of extracellular ﬂuid into the retinal vessels or vitreous body, regulation of retinal blood ﬂow, maintenance of retinal pH by ionic homeostasis, their great conductance for potassium, glutamate recycling due to neuronal transmission and maintenance of glucose metabolism. Cells 2022, 11, 3362 5 of 29 5 of 29 In DR, the metabolism of Müller cells is disturbed, which compromises the drainage function of Müller cells and leads to intracellular accumulation of ﬂuid and inadequate discharge of the ﬂuid into retinal blood vessels or vitreous. Consequently, the intracellular swelling or cytotoxic edema of Müller cells leads to rupture of cell membranes as well as increased liquid accumulation in the extracellular space, presenting intraretinal cystoid abnormal spaces visible as focal defects in the deep capillary plexus and hypo-reﬂective cystoid spaces in B-scan of OCTA [2]. y p Müller cells maintain the homeostasis of ion and water in the retina via inward rectifying potassium channel 4.1 (Kir4.1) and aquaporin 4 (AQP4) [12,24]. The proteins Kir4.1 and AQP4 are anchored by Dystrophin 71 (Dp71) on Müller cellular membranes [25,26]. The downregulation or redistribution of Kir4.1 and AQP4 results in the intracellular edema of Müller cells, which was reported in many retinal diseases, including retinal vein occlusion, ischemia-reperfusion injury and DR [27–29]. In 3-month rat experimental DR, Kir4.1 was absent in the inner limiting membrane (ILM) and perivascular areas [30]. In our previous study using 3-month diabetic rat retinas, the expression of Kir4.1 and AQP4 were reduced [29]. The decreased expression and the altered distribution of Kir4.1 and AQP4 may be a molecular marker of the drainage dysfunction of Müller cells, leading to Müller intracellular edema. According to the published method [31], we observed the swollen apical processes of Müller glia as ribbon-like transparent gaps in ONL in semithin section of retina, indicating intracellular edema of Müller glia in streptozotocin-induced diabetic rat [29]. In our previous clinical observation in diabetic patients with DME, there was a strong positive correlation between INL thickness, where somas of Müller cells are located, and the CSFT, indicating that the Müller intracellular edema might contribute to DME development [32]. 3.2. Drainage Dysfunction of Müller Cells Leading to Intracellular Edema The Müller glia intracellular edema was also observed and reported in patients with DR when examined with OCTA, as indicated by the hypo-reﬂective cystoid edema spaces in the deep capillary network in both b-scan and en face of OCTA [2,29,33]. p p y However, there is no direct evidence showing intracellular edema of RPE cells, which merits further study with high resolution multi-modal imaging to validate the intracellular edema in vivo both experimentally and clinically. With the advancement of the imaging technologies, metabolic imaging is promising to evaluate the function of RPE cells, which measures the intracellular levels of the metabolites non-invasively and real-timely [34]. Bianchetti G, et al. investigated the effect of docosahexaenoic acid (DHA) on the redox homeostasis in the human retinal pigment epithelial cell line (ARPE-19) under high-glucose conditions, using both metabolic imaging and molecular biology [34]. The metabolic imag- ing, using two-photon microscopy, showed that DHA treatment could increase intracellular nicotinamide adenine dinucleotide plus hydrogen (NADH) to upregulate the production of reductive species in high-glucose + DHA group compared with high-glucose treated cells [34]. Moreover, artiﬁcial intelligence-based metabolic imaging analysis will largely facilitate the evaluation of the metabolic changes in retinal cells, including RPE, both in vivo and in vitro [35,36]. 3.3. BRB Breakdown Leading to Increased Fluid Leakage into Retina The BRB protects retinal neuronal functions via regulating the protein, ion, and water to maintain an adequate microenvironment in retina. The BRB is consisted of both inner BRB and outer BRB. Abnormal ﬂuid accumulation in the retina is mainly attributed to the BRB breakdown. Following BRB breakdown, the equilibrium is lost between hydro- static and oncotic pressure gradients across the BRB leading to further progression of the macular edema [37,38]. In DR, BRB breakdown was observed both experimentally and clinically [2,37,39,40]. The loss of protective function of BRB is mainly due to the impair- ment of cells comprising the BRB and cell-cell junctions. Three BRB pathologies in DR are critical in the development of DME, i.e., pericyte loss, impaired cell-cell junctions, and capillary basement membrane thickening [41]. In DR, cell-cell junctions between the endothelial cells are lost, resulting in the increased leakage of plasma, lipid, and even red blood cells [42,43], manifesting clinically as edema, hard exudates and intraretinal Cells 2022, 11, 3362 6 of 29 hemorrhages. Bianchetti G, et al. analyzed and compared the alteration of erythrocyte membrane ﬂuidity by calculating the generalized polarization (GP), representative for membrane ﬂuidity of red blood cells, in T1DM patients with or without DR, and found that erythrocyte membranes of DR patients were more ﬂuid than those of T1DM without DR, indicating alteration of erythrocyte membrane ﬂuidity could represent a biomarker for DR development in T1DM patients [44]. The changes in GP in DR might be related to the early stages of diabetic microangiopathy, including capillary occlusion, microaneurysm formation, and retinal ischemia, resulting in BRB breakdown. g Hyperglycemia-induced upregulation of many cytokines, chemokines, and enzymes, such as angiopoietins, interleukins (ILs), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and matric metalloproteinases (MMPs), play a synergic role leading to the breakdown of BRB [41,45]. The predominant molecular mechanisms leading to BRB breakdown and subsequent DME include activated VEGF downstream pathways and inﬂammation leading to the increased production of many cytokines and growth factors [46,47], playing a causative role in BRB breakdown through multiple signaling pathways [46]. For example, tumor necrosis factor α (TNF-α) increases the expression of intercellular adhesion molecule 1 (ICAM-1), promoting leukocyte adhesion and leukostasis in early DR. TNF-α inhibition might provide a therapeutic target to prevent BRB break- down, retinal leukostasis, and apoptosis in DR [46,48]. 3.3. BRB Breakdown Leading to Increased Fluid Leakage into Retina Interleukin-1β (IL-1β) stimulates reactive oxygen species (ROS) production and accelerates the apoptosis of retinal cap- illary endothelial cells through activating the nuclear factor kappa B (NF-κB) pathway under diabetic conditions. [49,50]. Furthermore, the activated kallikrein-kinin system in DR also contributes to retinal edema, hemorrhage and neovascularization through activa- tion of bradykinin B1 and B2 receptors, resulting in vasodilation, vascular permeability, inﬂammation, and leukostasis [51]. Alongside inner BRB breakdown, outer BRB breakdown can also participate the patho- genesis of DME. In experimental DR, the degeneration of RPE and the breakdown of outer BRB were observed [39,40]. In early diabetic rats, the majority leakage of ﬂuorescein isoth- iocyanate (FITC)-dextran was examined in the ONL, indicating the outer BRB breakdown and RPE dysfunction. The breakdown of outer BRB was due to the decreased protein levels of zonula occludens-1 (ZO-1) and occludin in the RPE-Bruch’s membrane-choriocapillaris complex, which was caused by the activated hypoxia inducible factor 1α (HIF-1α) and c-Jun N-terminal kinase (JNK) pathways [39]. One previous study reported that high-glucose induced upregulation of claudin-1, which decreased apical-basolateral permeability in ARPE-19 cell line; however, the transepithelial permeability of ARPE-19 cells was increased in high glucose treated ARPE-19 cells [52]. In hyperglycemia cultured bovine RPE cells, Na+-K+-ATPase function was impaired, which could be restored by the aldose reductase inhibitor [53]. Therefore, it appears that hyperglycemia impairs the transport of water from subretinal space to the choriocapillaris, contributing to DME development. RPE degen- eration, as evidence with electron microscopy in diabetic animal models demonstrating shrunken nuclei, reduced endoplasmic reticulum, infolding of cell membranes, altered melanosomes and reduced number of RPE cells [54], further aggregating the breakdown of outer BRB. OCT characteristics of the outer retina in DME have proven that ellipsoid zone (EZ) disruption occurs subsequent to the disruption of the external limiting membrane (ELM) [55]. Since the ELM, the apical microvilli of Müller cells, functions as the third retinal barrier, ELM disruption may result in photoreceptor and RPE damage. In fact, decreased RPE thickness was observed in DME patients with non-proliferative DR (NPDR) or prolif- erative DR (PDR) [56]. Thus, both inner and outer BRB breakdown in DR contributes the ﬂuid accumulation in macular region, leading to DME formation. 3.4. Inﬂammatory Effect Contributing to DME Formation 3.4.1. Inﬂammation-Related Factors Are Upregulated in DME 3.4.1. Inﬂammation-Related Factors Are Upregulated in DME Leukocyte adhesion and the subsequent leukostasis is the early inﬂammatory response in DME. In DR, endothelial cells upregulated ICAM-1 expression, leading to increased leukocyte adhesion, resulting in retinal vascular leakage [60]. In diabetic animals, neu- trophils increased the expression of integrin such as CD18, and exhibited higher integrin- mediated adhesion, whereas, antibodies against CD18 or ICAM-1, and knockout of these genes inhibited the leukostasis and decreased BRB breakdown [61]. In DR, chronic hy- perglycemia increases expression of chemokines, including MCP-1/CCL2, that increase and mediate leukocyte adhesion, leukostasis, and inﬁltration of monocytes into the retinal parenchyma [16]. The inﬁltrated monocytes differentiate into activated macrophages and produce many cytokines and inﬂammation-related factors, including VEGF, IL-6, TNF-α and angiopoietin-2 (Ang-2) [41]. These cytokines and mediators disrupt the cell-cell junc- tions, resulting in BRB breakdown. BRB breakdown further aggregates the over-production of the inﬂammation-related factors. The inﬂammation-related factors contributing to BRB breakdown include but are not limited to VEGF, TNF-α, IL-1α, IL-1β, IL-6, IL-8, IL-10, ICAM-1, MCP-1, placental growth factor (PlGF), hepatocyte growth factor (HGF), insulin- like growth factor-1 (IGF-1), histamine and complement factors, which were reported to be upregulated in the vitreous or aqueous humor of DME patients [18,60], indicating the signiﬁcant contribution of inﬂammation-related factors to the pathogenesis of DME. 3.4. Inﬂammatory Effect Contributing to DME Formation DR is considered as a disease of chronic microinﬂammation [57,58]. Accumulating evidence showed that immunological and inﬂammatory mechanisms play a prominent role in the pathogenesis of DR and DME [2,18]. In DR, the microinﬂammation, a low-grade Cells 2022, 11, 3362 7 of 29 7 of 29 background inﬂammation, is maintained by cytokines such as IL-6, IL-8 and monocyte chemoattractant protein-1 (MCP-1, also known as the C-C motif chemokine ligand 2 (CCL2)) [18,59]. IL-6 alters the function of the astrocytes, leading to disruption of the inner BRB. IL-8 and MCP-1 act on neutrophils and monocytes, promoting inﬁltration of these cells into the retina. Alongside inﬂammation-related factors, inﬂammatory cells, such as leukocytes and microglia, also play pivotal roles in DME. 3.4.2. Inﬂammatory Cells Are Activated in DME In addition to inﬂammation-related factors, inﬂammatory cells, such as activated microglia, also contribute to the pathogenesis of DME and DR [62]. In diabetic eyes, hyperglycemia-induced activation of retinal microglia and inﬁltration of immune cells, including macrophages, lymphocytes, and neutrophils, have been reported [63–65]. In the retina, resident microglia, regarded as the immunological guard, are sensitive to the changes in the retinal microenvironment and respond quickly to various insults [66,67]. Microglia are activated early in streptozotocin-induced diabetic rat retinas [68]. Activated microglia, with a more amoeboid phenotype with increased motility, migrate from inner retina to the outer retina in experimental DR. The activated microglia, releasing the inﬂam- matory factors and phagocytosing the apoptotic neurons, contribute to the anatomical and functional abnormalities in diabetic retina [69]. The phenomena of microglia activation in diabetic retina has been documented by our recent work [70,71]. In the diabetic rat, microglia became activated with increased cell proliferation, close contact with the retinal capillaries, especially the deep capillary plexus, and enhanced migration from inner to outer retina, even to the subretinal space and RPE layer [70]. Our observation showed that the activated microglia penetrated the basement membrane of the capillaries and phagocytosed endothelial cells, leading to BRB breakdown and the formation of acellular capillaries [70]. This enhanced phagocytosis by microglia was associated with decreased Src/Akt/Coﬁlin pathway signaling [70]. Alongside direct interaction, activated microglia mediate the death of retinal ganglion cells (RGCs) non-cell-autonomously by releasing TNF-α [72]. In experimental DR, activated microglia synthesize inﬂammatory factors, cytokines, proteases, nitrous oxide, and ROS, accompanying with neuronal death in DR [66,69]. In a proof-of-concept ﬁnding, the increased inﬂammatory factors and intracellular ROS could be prevented in experimental DR by intravitreal injection of fractalkine (FKN), an inhibitor of microglia activation [71]. Graeber et al. observed the number of holes in the RPE layer was increased in diabetic mice Cells 2022, 11, 3362 8 of 29 8 of 29 model [73], which facilitated the migration of the inﬂammatory cells. Activated microglia recruit macrophages and other inﬂammatory cells into the retina by increasing the release of CCL2/MCP-1 [16,74]. In patients with DR, activated microglia, increased in number and becoming hyper- trophy, clustered around the retinal vessels, microaneurysms, intraretinal hemorrhages, and cotton-wool spots, etc. [75]. Activated microglia even migrate to the outer retina and subretinal space in some retinas with cystoid macular edema [75]. 3.4.2. Inﬂammatory Cells Are Activated in DME Recently, hyperreﬂective foci (HRF) viewed with OCT or OCTA in the diabetic retina have been considered as a biomarker of active inﬂammatory cells, especially microglia and/or macrophages. One previous study demonstrated a positive correlation between the level of aqueous CD14, the cytokine released by microglia and macrophages, and the increased HRF number in DME patients, indicating that inﬂammatory cells, such as microglia, participate in the pathogenesis of DME [62]. The above ﬁndings indicate that DR is an inﬂammatory disease, characterized by in- creased proinﬂammatory mediators and activated cellular inﬂammatory processes resulting in BRB breakdown and DME formation [60,61,76–79]. . Diabetic Retinal Neurodegeneration (DRN) Aggregating the Functional Outcome in DME 3.5. Diabetic Retinal Neurodegeneration (DRN) Aggregating the Functional Outcome in DME DRN involves degenerative alterations in retinal neurons and glia, including RGCs, photoreceptors, amacrine cells, bipolar cells and glial cells. The characteristics of DRN include increasing neural cell apoptosis, reduced retinal function, and reactive gliosis. DRN might be driven by accumulating glutamate, inﬂammation, oxidative stress, and altered balance of neurotrophic factors in retina. It should be noted that DRN and diabetic retinal microangiopathy are distinct but interdependent components of DR [80–83]. g p y p p Neurodegeneration, characterized by an increase in neuronal apoptosis, could be detected in the early stage of DR [84]. In diabetic retina, a high apoptotic rate of RGCs was reported [84,85]. In diabetic donors, RGCs showed cytoplasmic immunoreactivity for caspase-3, Fas, and Bax [86]. In ONL, increasing apoptosis of photoreceptors was also observed between 4 and 24 weeks after diabetes onset in diabetic rats [87,88]. In the eyes of patients with NPDR, the immunohistochemistry showed the association of upregulated expressions of Bax, caspase-9 and -3 with neuronal cell death [89]. p p In a prospective longitudinal study including 45 T1DM patients with no or minimal DR, OCT showed the thickness of NFL, ganglion cell layer (GCL), and inner plexiform layer (IPL) decreased progressively over time [90]. In this study, after adjustment for diverse factors, an obvious, progressive loss of NFL (0.25 µm/year) both parafoveally and perifoveally and loss of the GCL+IPL (0.29 µm/y) parafoveally was detected in patients with T1DM [90]. In a separate observation comparing 5 donor eyes with diabetes mellitus and no or minimal DR (diabetic group) to 5 age-matched donor eyes without diabetes mellitus (control group), the thickness of NFL was obviously decreased (17.3 µm) in diabetic group than that in control group (30.4 µm), while the retinal vascular density remained relatively unchanged between the two groups [90]. The above observations suggested that diabetes could directly affect the neuroretina in addition to retinal capillaries. The neuroprotection should not be ignored during the management of DME. 3.6. Proteomics and Metabolomics Leading to Deep Understanding and Targeted Treatments for DME 3.6. Proteomics and Metabolomics Leading to Deep Understanding and Targeted Treatments for DME 3.6. Proteomics and Metabolomics Leading to Deep Understanding and Targeted Treatments for DME In DR and DME, chronic hyperglycemia alters retinal homeostatic mechanisms, result- ing in the changes in the proteomic and metabolomic microenvironment, which is crucial to retinal cell function. With the rapid advancement in methodology, numerous studies on proteomics and metabolomics have been conducted on samples from DR and DME patients and shed a light in the pathogenesis of DR and DME, advancing and expanding our understanding of the pathogenesis of DR and DME. The changes in proteomics in DR and DME have expanded our recognition of this blinding disease using proteomic approaches, e.g., two-dimensional difference gel elec- Cells 2022, 11, 3362 9 of 29 trophoresis (2D-DIGE) coupled with mass spectrometry (MS), sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) coupled with MS [91], liquid chromatog- raphy coupled with tandem MS (LC-MS/MS) [92], and bead-based multiplex immunoas- says [93], and etc. With the advancing methods detecting the proteomics in vitreous humor of DR patients, several proteins, such as complement component C3, ICAM-1, IL-6, serum amyloid A protein (SAA), amyloid-β A4 protein, kininogen-1, metalloproteinase inhibitor 1, and VEGF, etc., have been identiﬁed as potential biomarkers for different stages of DR [94]. Thus, a thorough understanding of the proteomic changes could provide new insight into elucidation of the pathogenesis as well as biomarker for DR and DME, which would lead to the development of potential treatments for DR and DME. p p Using the proteomics technologies, the potential biomarker for DR could be discovered. The vitreous samples from pre-proliferative DR associated with DME (DME group) and without DME (non-DME group) were analyzed for the changes in proteomics. The results showed that a total of 14 proteins (DME group) and 15 proteins (non-DME group) were differentially expressed. Further analysis showed six proteins were upregulated, i.e., pigment epithelium-derived factor (PEDF), apolipoprotein A (ApoA)-1 (ApoA-1), ApoA-4, thyroid hormone receptor- interacting protein-11 (Trip-11), plasma retinol-binding protein (PRBP), and vitamin D binding protein (VDBP) in DME group; while apolipoprotein H (Apo H) was expressed only in non-DME group [95]. The differential changes in proteomics indicate the biomarker for DME group and also demonstrated the involvement of these molecules in the pathogenesis of DME. 3.6. Proteomics and Metabolomics Leading to Deep Understanding and Targeted Treatments for DME A recent study using LC-MS/MS analysis to detect the proteomics in aqueous humor in 73 eyes of DME patients with NPDR or PDR showed that about 308 signiﬁcantly changed proteins between NPDR group and PDR group [96]. Compared with NPDR group, in PDR group, the upregulated proteins are involved in the immune system and/or negative regulation of the cell cycle, while the downregulated proteins are related with the VEGF receptor (VEGFR) pathway and/or metabolism [96]. Further analysis showed that, compared with the NPDR and non-diabetic groups, the immune-associated protein cystatin C (CST3), downregulated in the PDR group, might be served as a novel target in DME treatment [96]. Metabolomics, the qualitative and quantitative assessment of the metabolites in body ﬂuids [97], is a promising branch of omics to detect the changes in metabolites and the underlying mechanisms in various diseases, including DR and DME [98]. Metabolomics could provide the novel metabolic biomarkers and potential pathways in DR and DME, facilitating the elucidation of the mechanisms, and proposing new therapeutic strategies for DR and DME. One current review reported that several metabolites, such as L-glutamine, L-lactic acid, pyruvic acid, acetic acid, L-glutamic acid, D-glucose, L-alanine, L-threonine, citrulline, L-lysine, and succinic acid, were potential biomarkers of DR [99], which involved new pathogenic pathways in DR. Alongside the ocular samples, the metabolites in serum and metabolic pathways were detected and compared between different stages of DR in patients with T2DM [100]. The results showed that the pathways including arginine biosynthesis metabolism, linoleic acid metabolism, glutamate metabolism, D-glutamine and D-glutamate metabolism, etc., were dysregulated in DR patients of the Asian popula- tion [100]. The metabolic signatures, such as upregulated levels of glutamate, aspartate, glutamine, N-acetyl-L-glutamate, and N-acetyl-L-aspartate, and downregulated levels of dihomo-gamma-linolenate, docosahexaenoic, and eicosatetraenoic, can be employed to differentiate PDR from NPDR in the Asian population [100]. Thus, with the advancements in biotechnologies of proteomics and metabolomics, the key new pathways, biomarkers, and establishing therapeutic targets would be in the practice in the management of DR and DME, further enhancing our understanding of this disease. 4.1. Control of Systemic Risk Factors 4.1. Control of Systemic Risk Factors Since DME remains a common complication of DR caused by diabetes, control of systemic risk factors including tight control of hyperglycemia, hyperlipidemia and hyper- tension should be considered as the fundamental strategy for the prevention and treatment of DR and DME. The Diabetes Control and Complications Trial (DCCT) showed that in- tensive glycemic control in T1DM reduced the risk of developing retinopathy by 76% and also reduced the risk of proliferative disease and the need for laser treatment [101]. For patients with T2DM, the UK Prospective Diabetes Study (UKPDS) showed that intensive glycemic control can reduce the need of laser photocoagulation treatment and decrease the risk of progression to blindness [102]. The UKPDS analyzed the effect of intensive control of blood pressure with captopril or atenolol on microvascular complications in 1148 hypertensive patients with T2DM [103]. The results showed that tight control of blood pressure decreased the risk of complications related to diabetes, progression of DR, and deterioration in visual acuity. The Action to Control Cardiovascular Risk in Diabetes (AC- CORD) Eye Study showed a 40% reduction in relative risk of retinopathy progression with the addition of fenoﬁbrate to simvastatin to control blood lipids [104]. Thus, the intensive control of hyperglycemia, hypertension and hyperlipidemia seems to reduce the rate of progression of DR, which will also beneﬁt the treatment outcome of DME. Furthermore, erythrocyte membrane ﬂuidity, might be served as a novel biomarker to predict the risk of developing complications in T1DM patients supplementing hemoglobin A1c in long-term T1DM management [44]. 4.2. Laser Therapy The efﬁcacy and safety of focal laser for treating DME was validated by the Early Treat- ment of Diabetic Retinopathy Study (ETDRS) in the 1980s [105]. Today, the focal/grid laser is an alternative in eyes with DME, mostly for non-center involved DME (Non-CI-DME). The subthreshold micropulse laser has been accepted as a potential and promising treatment in some cases for DME [106], due to its safe, non-scarring alternative procedure [107–110]. Subthreshold micropulse laser therapy is known to improve RPE function, modulate the activation of heat-shock proteins and normalize cytokine expression [111], and it seems to result in the normalization of retinal neuroinﬂammatory metabolic pathways [112]. Both the infrared subthreshold micropulse laser (810-nm wavelength) and yellow subthreshold micropulse laser (577-nm wavelength) were shown to be effective for DME treatment with good safety [113,114]. In a prospective study, the morphological changes in the retina and choroid, and the function of macula were evaluated and compared in patients with center- involved DME (CI-DME), who were treated with yellow (577-nm) or infrared (810-nm) subthreshold micropulse laser [115]. The data showed that both treatments are safe based on the morphological and functional evaluations in mild CI-DME [115]. 4. Therapeutic Strategies for DME Since the pathogenesis of DME involves different mechanisms with multiple factors and pathways participation, the treatment of DME should be the multimodality therapies, Cells 2022, 11, 3362 10 of 29 10 of 29 comprising the systemic control of the risk factors, regulating the potential targets, anti- inﬂammation, anti-oxidative stress, neuroprotection, laser and subthreshold micropulse laser therapy, photobiomodulation, as well as the vitrectomy, etc. (Figure 1). 4.4.2. OPT-302 OPT-302 (Opthea; Victoria, Australia) is a soluble form of VEGF receptor 3 (VEGFR-3) consisting of the extracellular domains 1–3 of human VEGFR-3 and the Fc fragment of human IgG1. OPT-302 blocks the activity of the proteins VEGF-C and VEGF-D [134], which may serve a complementary therapeutic role in VEGF-mediated DR pathogenesis, and overcome the limitation of the current anti-VEGF drugs that only target VEGF-A. Intravitreal OPT-302 was safe and well tolerated, and the combined treatment with OPT- 302 may enhance the efﬁcacy in neovascular suppression in nAMD [134]. A multicenter phase 1b/2a trial has evaluated OPT-302 in combination with aﬂibercept for refractory DME [135]. Combo-therapy using OPT-302 and aﬂibercept or conbercept may target all the VEGF family members, which might be effective in patients with retinal vascular diseases and is worth trying. 4.4.1. Abicipar Pegol Abicipar pegol (AGN-150998, Allergan plc/Molecular Partners) belongs to a family of the designed ankyrin repeat proteins (DARPin). Abicipar pegol binds VEGF-A with high afﬁnity [126]. Compared with ranibizumab, abicipar pegol improved its pharmacokinetic properties, i.e., lower molecular weight (34 vs. 48 kDa), higher target binding afﬁnity (2 vs. 46 pM) and longer ocular half-life (≥13 vs. 7 days in the aqueous humor) [127–129]. In phase I/II, open-label, multicenter dose-escalation trial for DME, prolonged edema reduction and visual improvement was achieved in several patients, however, ocular inﬂammation was a major concern [128]. Phase III clinical trials, SEQUOIA (NCT02462486) and CEDAR (NCT02462928), showed non-inferior result in visual acuity improvement compared with monthly ranibizumab injection in patients with neovascular age-related macular degeneration (nAMD) [130,131]. However, it failed to gain FDA approval due to signiﬁcant intraocular inﬂammation and an unfavorable risk-beneﬁt ratio [130,132]. Further studies are needed for evaluation of the efﬁcacy and safety of abicipar pegol in DR treatment before its approval [133]. 4.4. Emerging Therapeutic Strategies Targeting VEGF/VEGFR System and the Accessory Proteins The current trend for anti-VEGF development is toward either smaller molecular weight targeting VEGF-A (e.g., Beovu and abicipar), fusion proteins targeting VEGF-A in combination with other factors (e.g., faricimab), targeting other VEGF family members (OPT-302), reducing the cost of burden (developing biosimilars), or improving treatment durability (KSI-301, port delivery system, gene therapy), and etc. 4.3. Intravitreal Injection of Anti-VEGF Agents Since many cytokines and various pathways are implicated in the pathogenic process of DME, treating targets become the fundamental strategy for DME treatment [116]. In the pathogenesis of DR and DME, VEGF is upregulated and plays a pivotal role leading to BRB breakdown, macular edema, and neovascularization [117], and the severity of leakage in DME correlates with the level of VEGF [118]. Targeting VEGF (anti-VEGF) treatment has demonstrated signiﬁcant beneﬁts for patients with DME, which has become the ﬁrst- line treatment for DME [116], supplanting focal photocoagulation. Currently, there are several anti-VEGF agents which are commercially available for DME, including Lucentis (ranibizumab), Eylea (aﬂibercept), Lumitin (conbercept), Beovu (brolucizumab), and off- label Avastin (bevacizumab) [119–124], differing in molecular weight and structure, binding Cells 2022, 11, 3362 11 of 29 11 of 29 afﬁnity, targeted VEGF isoforms, and concentration, etc. For instance, brolucizumab treat- ment improved visual function and retinal morphology signiﬁcantly in DME patients with a favorable beneﬁt/risk proﬁle [125]. Anti-VEGF drugs maintain BRB integrity by antagonizing VEGF-A and/or PlGF. However, some patients respond unsatisfactorily to anti-VEGF therapy. There are 31.6~65.6% patients with DME who still exist with persistent edema even after regular intravitreal injection for at least 4 injections within 24 weeks [19]. Furthermore, many DME patients need repeated injections with high cost due to multiple injections, and some patients even respond incompletely or are unresponsive to anti-VEGF treatment (non-responders). These limitations and suboptimal responses to anti-VEGF treat- ment indicate other pathogenic factors, beyond VEGF, might contribute to the pathogenesis of DME, which prompted the researchers to develop novel therapeutic approaches. 4.4.6. High-Dose of Anti-VEGF Agents An intravitreal injection of high-dose anti-VEGF agents might prolong the intravitreal injection intervals and improve drug efﬁcacy. Using rabbits, Kim et al. showed that a two-fold increase in retinal half-life and prolonged effective concentration of ranibizumab in retina when administered a 10-fold dose of ranibizumab with good safety in rabbit eyes [149]. Currently, phase 3 clinical trials are underway in DME (PHOTON; NCT04429503) and nAMD (PULSAR; NCT04423718). 4.4.5. Port Delivery System (PDS) with Ranibizumab Currently, the delivery of anti-VEGF drugs is largely dependent on repeated intrav- itreal injections. PDS allows continuous release of ranibizumab, and minimizes the need for frequent injections [146]. Sustained and controlled release is achieved by the porous metal element allowing passive diffusion of drugs from PDS to the vitreous [147]. ARCH- WAY (NCT03677934) randomized Phase 3 trial of PDS with ranibizumab showed that PDS with ranibizumab met its primary objective, demonstrating equivalent efﬁcacy of monthly ranibizumab injection [148]. Phase 3 clinical trials for DR (PAVILION; NCT04503551), and DME (PAGODA; NCT04108156) are currently in progress. 4.4.3. Anti-VEGF Biosimilars The anti-VEGF medications have been available for more than a decade and their patent expiration dates are coming. For example, ranibizumab’s patent expired in June 2020 in the United States (2022 in the European Union) and aﬂibercept’s patent will expire in 2023 in the United States (2025 in the European Union) [136]. With the expiry of these Cells 2022, 11, 3362 12 of 29 12 of 29 patents, the transition to biosimilars can have a signiﬁcant impact worldwide due to the favorable cost-effectiveness [137]. patents, the transition to biosimilars can have a signiﬁcant impact worldwide due to the favorable cost-effectiveness [137]. Many bevacizumab biosimilars were approved for cancer treatment. However, due to a cheaper alternative to ranibizumab, the off-label use of bevacizumab is still increasing in ophthalmology [137]. Currently, there are several anti-VEGF biosimilars to ranibizumab and aﬂibercept in the development stage or acquiring approval [137]. For example, Razumab® (Intas Pharmaceutical Ltd., Ahmedabad, GJ, India) is the ﬁrst biosimilar to ranibizumab approved for ophthalmic use in India by the drug controller general of India for nAMD, myopic choroidal neovascularization, DME, and retinal vein occlusion-macular edema (RVO-ME) [137–140]. In 2021, the US FDA approved Byooviz (ranibizumab-nuna) as the ﬁrst biosimilar to Lucentis (ranibizumab injection) for the treatment of several eye diseases, including nAMD, RVO-ME, and CNV. As for aﬂibercept biosimilars, ABP-938 (Amgen, Thousand Oaks, CA, USA) is under phase 3 trial, which is scheduled to be completed by July 2023 (NCT04270747). Other biosimilars to aﬂibercept (MYL-1710P, ABP- 938, and CHS-2020, USA; FYB203, Germany; SB15, South Korea) underwent the clinical trials [141]. 4.4.4. KSI-301 KSI-301 (KODIAK sciences, Palo Alto, CA, USA) comprises a speciﬁc anti-VEGF IgG1 antibody and an inert immune effector, covalently linked to a high molecular weight phos- phorycholine biopolymer (950 kDa). Intravitreal injection of KSI-301 showed prolonged intravitreal half-life (about 6 months) due to slow diffusion and decreased elimination in the eye [142,143]. Clinical trials (GLEAM Study and GLIMMER study) are underway. The patients are randomized into two groups receiving either intravitreal KSI-301 or aﬂiber- cept [144]. Phase 2b/3 clinical trial failed to meet the primary endpoint of visual acuity gains in nAMD patients treated with KSI-301 compared to aﬂibercept [145]. 4.4.9. Targeting Neuropilin-1 Vesencumab is a human IgG1 monoclonal antibody against neuropilin-1 (NRP-1), with potential anti-angiogenic and anti-neoplastic activities. Vesencumab speciﬁcally targets and binds to NRP-1, preventing the subsequent coupling of NRP-1 to VEGFR-2, thereby decreasing VEGF-mediated signaling. When combined with other anti-VEGF therapies, vesencumab may enhance their anti-angiogenic effect [166]. Vesencumab is currently undergoing clinical study for cancer patients [167]. 4.4.8. Targeting VEGFRs The inhibition of VEGFRs is one of the promising strategies for treatment of VEGF- driven neovascular diseases [156–160]. Targeting VEGFRs has been extensively studied in clinical oncology. There are several approaches to inhibiting VEGFR signaling, i.e., VEGFR antibodies, VEGFR allosteric inhibitors, and inhibition of the intracellular tyrosine kinase of VEGFR by tyrosine kinase inhibitors (TKIs). Ramucirumab (Cyramza®), a fully humanized anti-VEGFR-2 monoclonal antibody, was approved for the treatment of cancer patients who experience disease progression during chemotherapy [158]. Its ophthalmic use is to treat retinal vascular diseases, including DME, which deserves further exploration. p GB-102 (GrayBug Vision; Redwood City, CA, USA), sunitinib maleate and a TKI with activity against both VEGF-A and PDGF, is encapsulated within bioerodible polymer nanoparticles degrading slowly over time [161]. Single GB-102 treatment can last up to 6 months with comparable visual acuity and CSFT outcomes [162,163]. Phase 1/2a study (ADAGIO) reported that the majority of nAMD patients were maintained at 3 months (88%) and 6 months (68%) with a single dose of GB-102 [164]. Among the emerging therapies, GB- 102, with longer duration between treatments, would impact signiﬁcantly on the patient’s life with less frequent follow-up and less expenditure [164]. X-82 (Tyrogenex) is an oral anti-PDGF and VEGF-A inhibitor. In a Phase 1 study (NCT02348359) for nAMD, 29% patients (10 of 35) did not complete the 24-week endpoint, with 6 (17%) withdrawing due to adverse events, including diarrhea, nausea, fatigue, and transaminase elevation [165]. Phase 2 APEX study (NCT02348359) is underway, which compares X-82 (Tyrogenex) with as-needed aﬂibercept injections to aﬂibercept monotherapy. PAN-90806 (PanOptica; Mount Arlington, NJ, USA), a TKI eyedrop, was shown to inhibit VEGF signaling with topical once daily dosing. According to a phase 1/2 study (NCT03479372), PAN-90806 showed favorable safety and effectiveness as monotherapy. However, it may be applicable for certain patients and further studies are needed [162]. 4.4.7. Gene Therapy to Deliver Anti-VEGF Agents 4.4.7. Gene Therapy to Deliver Anti-VEGF Agents Given the burden of repeated anti-VEGF treatments, gene therapy can achieve long- term expression of anti-VEGF proteins to suppress of VEGF in retinal vascular diseases. Several gene therapy drugs, including RGX-314, ADVM-022 and rAAV-sFlt1, are currently under clinical evaluation. RGX-314 is an adeno-associated virus 8 (AAV8) vector encoding ranibizumab. The Phase II ALTITUDE trial is studying the patients with DR but without DME, who are treated with a single dose of RGX-314, delivered in suprachoroidal space [150]. Positive 3-month interim data from cohort 1, treated with a single injection at a dose of 2.5 × 1011 Cells 2022, 11, 3362 13 of 29 13 of 29 genomic copies per eye, showed that treatment was well-tolerated and 33% of patients had a ≥2-step improvement from baseline [150]. genomic copies per eye, showed that treatment was well-tolerated and 33% of patients had a ≥2-step improvement from baseline [150]. ADVM-022, an AAV2-7m8 vector encoding aﬂibercept, is optimized for intravitreal delivery. Prolonged expression and efﬁcacy of ADVM-022 was evaluated in a laser-induced CNV model in non-human primates with promising outcomes [151]. Clinical trials for nAMD (NCT04645212; NCT03748784) and DME (NCT04418427) are currently underway, evaluating safety and efﬁcacy following a single intravitreal injection of ADVM-022. rAAV-sFlt1, a recombinant AAV2 vector expressing soluble VEGF receptor 1, works as a decoy receptor for VEGF. A pre-clinical study showed safety and well-toleration in non-human primates after a single subretinal injection of rAAV-sFlt1 [152]. Although phase I study (NCT01494805) demonstrated the safety in nAMD patients [153], phase IIa clinical trial (NCT01494805) showed no obvious beneﬁt in visual acuity or anatomy [154]. The potential effect of rAAV-sFlt1 on DME deserves further study [155]. 4.5. Anti-Inﬂammatory Therapy Since inﬂammation plays a critical role in DR and DME, suppression of inﬂammation seems to be a reasonable approach for treating DR and DME [57]. Corticosteroids have Cells 2022, 11, 3362 14 of 29 14 of 29 been proven to be beneﬁcial in treating DR and DME due to their anti-inﬂammatory and anti-angiogenic properties [168]. At present, intravitreal preservative-free triamcinolone, the extended-release dexamethasone implant (Ozurdex) and the ﬂuocinolone acetonide implant (Iluvein) are FDA-approved for treating DME. Intravitreal injection of sustainable dexamethasone (Ozurdex, Allergen) was safe and effective in DME treatment, achieving visual improvement, reducing edema, and decreasing the inﬂammatory cytokines, such as VEGF, MCP-1, and IL-6. A MEAD study (NCT00168337 and NCT00168389) evaluated the safety and efﬁcacy of Ozurdex (0.7 mg and 0.35 mg) and demonstrated both doses of the Ozurdex implant met the primary objective for visual improvement with acceptable safety proﬁle [169]. A FAME study evaluated long-term effects, including efﬁcacy and safety, of intravitreal inserts of ﬂuocinolone acetonide (Iluvien, Alimera Sciences, Alpharetta, GA, USA), releasing 0.2 µg/d (low dose) or 0.5 µg/d (high dose) for DME treatment. Iluvein inserts provided substantial visual beneﬁt for up to 3 years [170], especially for those who are unresponsive to other therapy, such as anti-VEGF treatment [171]. However, due to con- cerns regarding the intraocular pressure elevation and cataract formation, corticosteroids are used as the second-line therapy for DME. been proven to be beneﬁcial in treating DR and DME due to their anti-inﬂammatory and anti-angiogenic properties [168]. At present, intravitreal preservative-free triamcinolone, the extended-release dexamethasone implant (Ozurdex) and the ﬂuocinolone acetonide implant (Iluvein) are FDA-approved for treating DME. Intravitreal injection of sustainable dexamethasone (Ozurdex, Allergen) was safe and effective in DME treatment, achieving visual improvement, reducing edema, and decreasing the inﬂammatory cytokines, such as VEGF, MCP-1, and IL-6. A MEAD study (NCT00168337 and NCT00168389) evaluated the safety and efﬁcacy of Ozurdex (0.7 mg and 0.35 mg) and demonstrated both doses of the Ozurdex implant met the primary objective for visual improvement with acceptable safety proﬁle [169]. A FAME study evaluated long-term effects, including efﬁcacy and safety, of intravitreal inserts of ﬂuocinolone acetonide (Iluvien, Alimera Sciences, Alpharetta, GA, USA), releasing 0.2 µg/d (low dose) or 0.5 µg/d (high dose) for DME treatment. Iluvein inserts provided substantial visual beneﬁt for up to 3 years [170], especially for those who are unresponsive to other therapy, such as anti-VEGF treatment [171]. 4.5.3. TAK-779 TAK-779, a dual CCR2/CCR5 inhibitor, signiﬁcantly reduced retinal vascular perme- ability in diabetic mice [181]. TAK-779 also decreased inﬁltration of macrophage/microglia, reduced the expressions of ICAM-1 and stromal cell-derived factor 1 (SDF-1), and restored zonula occludens-1 (ZO-1) in diabetic mouse retina [181]. Targeting CCR2/CCR5 might provide a novel strategy for DME management. 4.5.2. Diﬂuprednate and Dexamethasone-Cyclodextrin 4.5.2. Diﬂuprednate and Dexamethasone-Cyclodextrin Diﬂuprednate (diﬂuprednisolone butyrate acetate, DFBA) is an anti-inﬂammatory steroid, effective in the treatment of anterior uveitis, postoperative ocular inﬂammation, and pain [175,176]. Diﬂuprednate ophthalmic emulsion 0.05% (Durezol (TM), Sirion Thera- peutics Inc., Tampa, FL, USA) effectively reduces refractory DME post-vitrectomy [177], and diffuse DME without surgical intervention [178]. Topical dexamethasone-cyclodextrin eye drops were safe, improved visual acuity and decreased central macular thickness in DME patients [179]. In a randomized, controlled trial, topical dexamethasone-cyclodextrin nanoparticle eye drops (1.5%) signiﬁcantly improved the vision and decreased macular thickness in DME patients [180]. 4.5. Anti-Inﬂammatory Therapy However, due to con- cerns regarding the intraocular pressure elevation and cataract formation, corticosteroids are used as the second-line therapy for DME. py Based on the inﬂammatory theory of DME formation, the ongoing translational re- search targeting inﬂammatory cells and factors is shedding new light on the management of DME beyond anti-VEGF therapy. Anti-inﬂammation treatment can be roughly clas- siﬁed into several categories, i.e., regulation/inhibition of inﬂammatory cells (such as minocycline, dextromethorphan), targeting various inﬂammatory mediators (such as corti- costeroids, TAK-779, TNF-α inhibitor, IL-6/IL-6R inhibitor, non-steroid anti-inﬂammatory drugs (NSAIDs)), and alternative delivery route (Suprachoroidal injection, oral and subcu- taneous injection), and etc. 4.5.1. Minocycline and Dextromethorphan Minocycline, besides its antimicrobial activity, has anti-inﬂammatory, anti-oxidant, anti-apoptotic, neuroprotective, and immunomodulatory effects [172]. In phase I/II clinical trial (ClinicalTrials.gov number, NCT01120899), oral minocycline treatment improved visual function and reduced central macular edema [173]. Dextromethorphan was effective in decreasing vascular leakage in 5 DME patients in phase I/II clinical trial, in which oral dextromethorphan was administered 60 mg twice daily for 6 months as monotherapy [174]. 4.5.5. Targeting TNF-α TNF-α is an inﬂammatory cytokine that promotes the upregulation of adhesion molecule expression, leukocyte recruitment and monocyte attraction. TNF-α was increased in the aqueous and vitreous of diabetic patients compared to control subjects [190–192]. The targeting TNF-α might provide an option for treating DR and DME. Currently, there are monoclonal anti-TNF-α full IgG1 antibodies (inﬂiximab, adalimumab, and golimumab), PEGylated Fab’ fragment of anti-TNF-α antibody (certolizumab pegol) and extracellular domain of TNF receptor 2/IgG1-Fc fusion protein (etanercept), effective for the treatment of rheumatoid arthritis [193]. In fact, a clinical study with inﬂiximab achieved functional and anatomical improvement in DME patients, highlighting the pathogenic role of TNF-α in DR [194]. 4.5.4. Targeting Integrin Integrins are involved in many biological processes and play a critical role in the pathogenesis of many diseases. Some integrins are associated with vitreolysis, angiogenesis, Cells 2022, 11, 3362 15 of 29 15 of 29 and ocular surface diseases [182]. Anti-β2-integrin or anti-ICAM-1 decreased leukocyte adhesion, the death of endothelial cells, and BRB breakdown [183–185]. Therefore, targeting integrins, independent of anti-VEGF therapies, has the potential to prevent vision loss. g p p p p Risuteganib (Luminate, ALG-1001, Allegro Ophthalmics, LLC, San Juan Capistrano, CA, USA) is an engineered arginyl-glycyl-aspartic acid (RGD) class synthetic peptide targeting integrin. RGD peptide treatment suppressed retinal neovascularization and released cellular adhesion to induce posterior vitreous detachment [186,187]. Risuteganib has potential as a therapy for DR and DME [182,188]. SB-267268 (GlaxoSmithKline) is a small molecule inhibitor of αvβ3 and αvβ5 integrins [189]. In an animal model of retinopathy of prematurity (ROP), SB-267268 decreased the mRNA expressions of VEGF and VEGFR2, and reduced the pathological angiogenesis by 50% [189]. 4.5.6. Targeting IL-6/IL-6R IL-6/IL-6R exerts an important role in initiating the breakdown of BRB in DR [195,196], due to the disrupting of the barrier function and increasing vascular leakage via the downregulation of tight junction proteins [197]. IL-6 signaling occurs through its membrane- bound receptor IL-6R (classical signaling) or through the soluble IL-6R (sIL-6R, trans- signaling) [198,199]. Anti-IL-6 and anti-IL-6R strategies target both classical and trans- signaling pathways to block IL-6 signaling. Several therapeutic strategies targeting IL-6 signaling pathways are in progress [200], including anti-IL-6 antibodies (e.g., siltuximab and sirukumab), anti-IL-6R antibodies (e.g., tocilizumab and vobarilizumab), and IL-6 trans-signaling selective inhibitor (olamkicept). Tocilizumab is effective in the treatment of various autoimmune and inﬂammatory diseases, including rheumatoid arthritis, with a favorable outcome [201]. Thus, blocking IL-6 and IL-6R may be potential approaches for treating DR. 4.5.9. Suprachoroidal Injection of Steroid The suprachoroidal space (SCS) has become an applicable route to deliver drugs to the back of the eye via suprachoroidal injection [213]. When delivered to the suprachoroidal space, the drug can target both the retina and the choroid, overcoming multiple ocular tissue barriers and achieving the efﬁcacy at low dose [142,214]. The phase 2 TYBEE clinical trial enrolled 71 eyes with treatment-naïve DME [215], with 36 eyes received SCS injection of triamcinolone acetonide (TA) (CLS-TA, 4 mg/100 µL) and aﬂibercept (2 mg/0.05 mL) at baseline and week 12 (active group) and 35 eyes which were treated with aﬂibercept (control group). At 24 weeks from baseline, the visual acuity gain was similar between two groups, with mild anatomic improvement and less treatment burden in the active group [215]. 4.6. Targeting Ang-2/Tyrosine Kinase with Immunoglobulin-like and Epidermal Growth Factor-like Domains 2 (Tie2) System The angiopoietin (Ang)/Tie2 pathway is involved in many retinal vascular diseases. Angiopoietin-1 (Ang-1) and Ang-2 ligands compete for the Tie2 receptor. Tie2 is a tyrosine kinase receptor in vascular endothelial cells and maintains vascular stability. Tie2 activation by Ang-1 increases the survival, adhesion, and cell junction integrity of endothelial cells, while Ang-2 interferes with the Ang-1/Tie2 axis, resulting in vascular instability. Vascular endothelial-protein tyrosine phosphatase (VE-PTP) is an endothelial cell-speciﬁc phos- phatase, which forms a complex with Tie2 and dephosphorylates Tie2, against the actions of Ang-1 [216]. In DR, there is an increased production of Ang-2, competitively binding Tie2 to reduce Ties phosphorylation, whereas VE-PTP directly decreases Tie2 phosphorylation. Tie2 inactivation destabilizes the vasculature, resulting in pericyte dropout, reduction in endothelial cell viability, decreased endothelial cell anchor and cell junction integrity. Thus, activating the Tie2 signaling pathway, by the inhibition of Ang-2 or VE-PTP, should be a therapeutic strategy for retinal vascular diseases. 4.5.8. Non-Steroid Anti-Inﬂammatory Drugs (NSAIDs) 4.5.8. Non-Steroid Anti-Inﬂammatory Drugs (NSAIDs) NSAIDs inhibit the cyclooxygenase (COX) enzyme that is an essential mediator through the regulation of prostaglandin dependent pathways [207]. Bromfenac mainly inhibits the activity of COX-2 [208], and nepafenac, a prodrug, acts on COX-1 and COX-2 through its active metabolite amfenac [209]. NSAIDs were reported effective in DME with various and heterogeneous results. In a pilot study, topical bromfenac signiﬁcantly reduced central macular thickness in patients with DME, however, without obvious effect on visual acuity [210]. The safety and efﬁcacy of topical nepafenac 0.1% were tested in 6 eyes of 5 patients with DME, which showed that topical nepafenac treatment improved vision and decreased retinal thickness [211]. Postoperative topical nepafenac was shown to be effective for prophylaxis of macular edema in diabetic patients underwent phacoemulsiﬁcation and intraocular lens implantation [212]. Further investigations on whether topical NSAIDs could serve as an alternative or adjunctive treatment to intravitreal anti-VEGF therapy are required. 4.5.7. Vascular Adhesion Protein-1 (VAP-1) Inhibitor VAP-1, known as amine oxidase copper-containing 3 (AOC3) and semicarbazide- sensitive amine oxidase, is a membrane-bound adhesion protein facilitating leukocyte adhesion and transmigration to the inﬂammatory site [202]. Previous studies showed that the level of soluble VAP-1 was higher in the vitreous of PDR patients than in that of non- diabetic patients [203]. In diabetic rats, the leukocyte transmigration rate was reduced by UV-002 (a speciﬁc inhibitor of VAP-1) [202]. In diabetic animals, VAP-1 inhibition improved retinal function and structure as evidenced by electroretinogram and histopathological studies [204]. Thus, VAP-1 could be an underlying target for DR treatment [205]. A phase 2 study (VIDI study, NCT02302079) tested the effect of ASP8232, a speciﬁc VAP-1 inhibitor, on CI-DME [206]. The primary data showed that ASP8232 nearly inhibited the activity of plasma VAP-1, while had no effect on CSFT in patients with CI-DME. The clinical application of VAP-1 inhibition still requires further study. Cells 2022, 11, 3362 16 of 29 4.6.3. Targeting VE-PTP ARP-1536 (Aerpio Therapeutics, Cincinnati, OH, USA) is a monoclonal antibody tar- geting VE-PTP. ARP-1536 is intravitreally administered, currently undergoing preclinical studies [161]. AKB-9778 (Aerpio Therapeutics, Cincinnati, OH, USA) is a small molecule antagonist of VE-PTP, increasing Tie2 phosphorylation. AKB-9778 is administered by subcu- taneous injection. In preclinical studies, AKB-9778 reduced vascular leakage and ocular neo- vascularization, with synergistic effect when combined with VEGF inhibition [221]. AKB- 9778 reduced macular edema more effectively when combined with monthly ranibizumab in a phase 2 study of DME [222]. 4.6.1. Targeting Ang-2 Nesvacumab (Regeneron, Tarrytown, NY, USA) is a fully human IgG1 monoclonal antibody selectively binding Ang-2. In phase 2 studies of nAMD and DME, nesvacumab co-formulated with aﬂibercept failed to show beneﬁcial effects over aﬂibercept in visual gains improvement [217]. AXT107 (Asclepix Therapeutics, Baltimore, MD, USA) is a peptide derived from the non-collagenous domain of collagen IV [217]. AXT107 modiﬁes Ang-2 and promotes its conversion into the Tie2 agonist, and AXT107 also inhibits the signaling of VEGFR-2 and other receptor tyrosine kinases [217]. In the presence of AXT107 and Ang-2, α5β1 integrin is disrupted, promoting Tie2 clustering and converting Ang-2 into a Tie2 agonist [218]. Currently, AXT107 is in the preclinical phase of study [217]. 17 of 29 17 of 29 Cells 2022, 11, 3362 4.7. Neuroprotection in DME Management Since DR is also a neurovegetative disease, neuroprotection should be considered in the management of DR and DME. The neuroprotective agents include but not limited to erythropoietin (EPO), Cibinetide (known as ARA 290 and helix B surface peptide (HBSP)), somatostatin and brimonidine. EPO’s protective mechanisms comprise anti-apoptosis and neuroprotection via activat- ing the ERK and AKT pathways [40,223], neurotrophic effect and anti-reactive gliosis [224], anti-VEGF via inhibition of HIF-1α [225], anti-inﬂammatory effect by decreasing inﬂam- matory factors from Müller glia [226], increase in the expression of zinc transporter 8 (ZnT8) [227], downregulation of glutamate [228], and maintenance of VE-cadherin expres- sion via inhibiting VEGF/VEGFR-2/Src pathway. In addition, EPO is able to improve the integrity of the inner BRB [229], and maintain outer BRB integrity through downregulation of HIF-1α and c-Jun N-terminal kinase (JNK) signaling, and upregulation of ZO-1 and occludin expressions in RPE cells [39]. Recently, we found that EPO protects the inner BRB by inhibiting microglia phagocytosis via Src/Akt/coﬁlin signaling in experimental DR [70]. A clinical cohort study showed that intravitreal EPO could improve the visual acuity and reduce macular edema in refractory DME patients [230], demonstrating its potential usage in treatment of DME. Cibinetide is a synthetic 11 amino acid peptide, derived from EPO, having anti- apoptotic, anti-permeability and anti-inﬂammatory functions, with no erythropoietic function [231–233]. Both somatostatin and brimonidine were tested in diabetic patients, however, no neuroprotective effect was found for both drugs to achieve the primary end- point [234]. However, the topical administration of somatostatin and brimonidine appears to be useful in preventing the worsening of preexisting retinal dysfunction [234]. Topical treatment with either somatostatin or brimonidine was observed to cause retinal arteriolar and venous dilation in patients with T2DM and early DR [235]. 4.6.2. Bispeciﬁc Drug Faricimab (faricimab-svoa; Vabysmo™), known as RG7716, (Roche, Basel, Switzerland and Genentech, South San Francisco, CA, USA), is a bispeciﬁc antibody binding both VEGF- A and Ang-2. Phase 3 trials for DME (YOSEMITE NCT03622580 and RHINE NCT03622593) showed robust vision gains and anatomical improvements in patients treated with faricimab and the personalized treatment interval was extended to 16 weeks [219]. In 2022, faricimab received its ﬁrst approvals in the USA for the treatment of nAMD or DME [220]. 4.9. Combo Therapy and Other Strategies Based on the severity of the DME, combo therapy can be proposed, such as ranibizumab + OPT + 302, aﬂibercept/conbercept + OPT + 302 or anti-VEGF + anti-inﬂammatory treat- ment. Moreover, other approaches are also attempted to treat DME, including targeted laser photocoagulation for non-perfusion area, micropulse laser for macular microaneurysms, photobiomodulation to enhance RPE function, vitrectomy to relieve the abnormalities of vitreoretinal interface and clear vitreous body, and etc. The growing achievements of translational research will lead to future treatments for DME with better efﬁcacy, longer duration, and greater cost-effectiveness. 4.8. Antioxidative Therapy Oxidative stress, resulting from the metabolic abnormalities, is regarded as a pivotal contributor to the pathogenesis of DR [236]. The reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) system is as a key enzymatic source of oxidative stress [236,237]. Nox-derived ROS contributes to retinal damage through inducing the expressions of pro-angiogenic and pro-inﬂammatory cytokines, including VEGF-A, EPO, Ang-2 and ICAM-1 [238,239]. The Nox2 gene knockout reduced oxidative stress, attenuated vascular permeability, and reduced leucocyte-endothelial interaction and leukostasis in diabetic mice [237]. Nox4 knockdown with small interfering RNA Cells 2022, 11, 3362 18 of 29 18 of 29 signiﬁcantly decreased retinal vascular permeability, indicating the causal role of Nox4 in BRB breakdown [240]. Thus, the inhibition of Nox would provide a potential strategy for the treatment of DR and DME. Idebenone, a ubiquinone short-chain synthetic analog, is believed to restore mitochon- drial ATP synthesis with antioxidant properties [241]. Punicalagin (2,3-hexahydroxydiphenoyl- gallagyl-D-glucose), a polyphenol extracted from pomegranate (Punica granatum), is a potent antioxidant in several cell types [242]. Previous studies showed that both idebenone and punicalagin could protected RPE from oxidative damage, suggesting their possible roles in DR and DME treatment. Idebenone protected RPE through modulation of the intrinsic mitochondrial pathway of apoptosis [241]. Punicalagin exerted its effect to reduce oxidative stress and decrease the apoptosis via enhancing mitochondrial functions [242]. Other potential antioxidants, such as quercetin, resveratrol, curcumin, lutein, vitamin E, nicanartine, and lipoic acid, etc., are promising against oxidative stress in treatment of DR and DME [236], deserving further exploration. Funding: National Natural Science Foundation of China (82171062, 81970810, 81970811) and Aier Eye Hospital Group Scientiﬁc Research Fund (AF2101D8). References 1. Hashemi, H.; Rezvan, F.; Pakzad, R.; Ansaripour, A.; Heydarian, S.; Yekta, A.; Ostadimoghaddam, H.; Pakbin, M.; Khabazkhoob, M. Global and Regional Prevalence of Diabetic Retinopathy; A Comprehensive Systematic Review and Meta-Analysis. Semin. Ophthalmol. 2022, 37, 291–306. [CrossRef] [PubMed] p aruich, A.; Matet, A.; Moulin, A.; Kowalczuk, L.; Nicolas, M.; Sellam, A.; Rothschild, P.-R.; Omri, S.; Gélizé 2. Daruich, A.; Matet, A.; Moulin, A.; Kowalczuk, L.; Nicolas, M.; Sellam, A.; Rothschild, P.-R.; Omri Mechanisms of Macular Edema: Beyond the Surface. Prog. Retin. Eye Res. 2018, 63, 20–68. [CrossRe 2. Daruich, A.; Matet, A.; Moulin, A.; Kowalczuk, L.; Nicolas, M.; Sellam, A.; Rothschild, P.-R.; Omri, S.; Gélizé, E.; Jonet, L.; et al. Mechanisms of Macular Edema: Beyond the Surface. Prog. Retin. Eye Res. 2018, 63, 20–68. [CrossRef] [PubMed] , ; , ; , ; , ; , ; , ; , ; , ; , ; J , ; Mechanisms of Macular Edema: Beyond the Surface. Prog. Retin. Eye Res. 2018, 63, 20–68. [CrossRef] [PubMed] 3. Thomas, R.L.; Dunstan, F.D.; Luzio, S.D.; Chowdhury, S.R.; North, R.V.; Hale, S.L.; Gibbins, R.L.; Owens, D.R. 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Romero-Aroca, P.; Baget-Bernaldiz, M.; Pareja-Rios, A.; Lopez-Galvez, M.; Navarro-Gil, R.; Verges, R. Pathophysiology: Vasogenic versus Inﬂammatory. J. Diabetes Res. 2016, 2016, 2156273. [CrossRef] 18. Rübsam, A.; Parikh, S.; Fort, P.E. Role of Inﬂammation in Diabetic Retinopathy. Int. J. Mol. Sci. 2018, 18. Rübsam, A.; Parikh, S.; Fort, P.E. 5. Conclusions and Future Directions DR is the leading cause of vision loss and blindness in the working-age adults with increasing incidence. DME is the main cause of vision impairment in DR patients with its pathogenesis still not completely elucidated. Although anti-VEGF therapy becomes the ﬁrst-line treatment for DME, not all patients respond satisfactorily to anti-VEGF in- jections. The limitations and unsatisfactory response indicate other pathogenic factors, beyond VEGF, might be involved in the pathogenesis of DME (Figure 1). The advance- ment of the elucidation of the pathogenesis of DR and DME, including the proteomics and metabolomics studies have greatly revolutionized our recognition of DR and DME, which laid down a solid foundation for uncovering the key pathways, novel biomarkers, and establishing therapeutic targets. Based on the underlying mechanisms of DME, the future direction of DME treatment (Figure 1) should be focused on the emerging anti-VEGF agents, bispeciﬁc antibody targeting VEGF-A and other potential molecule such as Ang-2 (faricimab), targeting VEGF/VEGFR system, enhancing anti-inﬂammatory effect, includ- ing the suprachoroidal route delivery, integrin antagonists, NASIDs, etc. Furthermore, neuroprotection, anti-oxidation, and other approaches, such as targeted laser photocoag- ulation, subthreshold micropulse laser, photobiomodulation, and vitrectomy, should be considered during DME management (Figure 1). Therefore, DME treatment should be tailored treatment based on its full elucidation of the complex mechanism, which leads to the identiﬁcation of new targets and therapeutic strategies for DME treatment. Author Contributions: J.Z. (Jingfa Zhang) and J.Z. (Jingxiang Zhang) reviewed the literature, con- tributed to manuscript draft writing, discussion, and analysis; C.Z., J.Z. (Jingting Zhang), D.L., Q.Q. and L.G. contributed the draft writing, revision and extensive discussion of the manuscript. J.Z. (Jingfa Zhang), L.G., D.L. and Q.Q. contributed funding acquisition. J.Z. (Jingfa Zhang) is guarantor of this work, who has full access to all the data in this study and take responsibility for the integrity and accuracy of the data; All authors have read and agreed to the published version of the manuscript. 19 of 29 19 of 29 Cells 2022, 11, 3362 Institutional Review Board Statement: Not applicable. References PLoS ONE 200 26. 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https://openalex.org/W3039841667	https://www.revistas.usp.br/espinosanos/article/download/171666/161604	Portuguese	null	ENTRE SERVIDÃO E LIBERDADE DE HOMERO SANTIAGO	Cadernos Espinosanos/Cadernos espinosanos	2,020	cc-by	1,687	imagem Detalhe de A arte da pintura, 1666, óleo sobre tela de Johannes Vermeer. Cadernos Espinosanos estudos sobre o século xvii 2020 n. 42 issn 1413-6651 jan-jun estudos sobre o século xvii santiago, h. (2019). Entre servidão e liberdade. São Paulo, Politeia. Cadernos Espinosanos São Paulo n.42 jan-jun 2020 ENTRE SERVIDÃO E LIBERDADE DE HOMERO SANTIAGO resenha de Luis César Oliva Professor, Universidade de São Paulo, São Paulo, Brasil lcoliva@uol.com.br resenha de Luis César Oliva Professor, Universidade de São Paulo, São Paulo, Brasil lcoliva@uol.com.br resenha p. 341 - 346 341 O caminho de um jovem professor e pesquisador depois de seu período de formação (supondo abstratamente que a formação termine com a conclusão das teses de mestrado e doutorado, o que é sabida mente falso) é sempre sinuoso. Depois de anos regido por um projeto de pesquisa e “vigiado” por agências de fomento, ele se vê dividido em múltiplas tarefas didáticas, administrativas e intelectuais (algumas feitas de bom grado, outras nem tanto) que produzem, na sua obra publicada, uma inevitável impressão de dispersão. De certo modo, é esse percurso fragmentado que Homero Santiago nos traz em Entre Servidão e Liber dade (2019). Depois de seu mestrado e doutorado sobre Espinosa, ambos revelando um historiador da filosofia de boa cepa, capaz de abordar temas circunscritos e pouco explorados pela tradição interpretativa, mesclando coerentemente rigorosas análises de texto com eruditas considerações históricas, eis que o autor agora deve dividir-se em escritos dos mais diferentes formatos: artigos especializados e para o público geral, tradu ções, prefácios, resenhas, entrevistas, etc. Ao reunir boa parte deles neste livro, Santiago não esconde a multiplicidade de formas nem a variedade de ocasiões que os motivaram. Porém, se a forma talvez denuncie a dispersão do percurso, o conteúdo a nega, atestando a admirável unida de de pensamento de um filósofo que usou as mais variadas vias de expressão para discutir uma única e fundamental questão: a transição (infelizmente nem sempre de mão única) da servidão à liberdade. Dividido em cinco partes (e uma introdução que mencionare mos mais tarde), o livro se abre com um notável capítulo sobre a supers tição em Espinosa, no qual o autor apresenta uma das mais completas e detalhadas análises já feitas sobre o famoso apêndice da parte I da Ética. Mais do que mostrar a renitência do historiador da filosofia meticuloso, este longo capítulo tem a função de revelar o ponto de vista com o qual Cadernos Espinosanos São Paulo n.42 jan-jun 2020 342 Santiago pretende abordar os diversos assuntos presentes no livro. ENTRE SERVIDÃO E LIBERDADE DE HOMERO SANTIAGO É com lentes espinosanas que o fará, e com atenção específica para o aspecto que talvez afaste Espinosa do outro filósofo que mais marcou o percur so de Santiago e é quase onipresente na etapa final do livro: Antonio Negri. Enquanto Negri, também inspirado em Espinosa, deixa-se levar pelo otimismo dos movimentos multitudinários e dá todo o destaque para a liberdade, Santiago ancora-se na análise espinosana da superstição para dar conta da inegável servidão que nos assola. Ao final do livro, quando Santiago explicitar suas críticas (que não impedem a enorme admiração) a Negri, o leitor perceberá plenamente o quanto era signi ficativo ter no apêndice seu ponto de partida. O mergulho na servidão, porém, não ocorre sem uma visão da praia. Embora as decepções com o sistema de crenças da superstição não apontem, na letra do apêndice, para uma saída, elas abrem ao menos uma possibilidade, que não será explorada neste texto, mas que remete Santiago a outro de Espinosa, o Tratado da Emenda do Intelecto, onde as decepções com os valores da vida comum nos levam à necessidade de começar a filosofar, ou pelo menos, como dirá o segundo capítulo, já que os valores são necessários, tratemos nós de forjá-los em vista da alegria e do benefício à vida. Poder fazê-lo talvez seja o mais difícil, mas precisamos fazê-lo. Se não o fizermos, se nos restringir mos ao mais fácil, o preconceito, a tristeza, a superstição e os seus lugares-tenentes o farão por nós (santiago, 2019, pág. 118). De um lado, esta surpreendente conclusão nos remete à introdução do livro, onde Santiago usa, também surpreendentemente, Pascal para pensar a transição entre servidão e liberdade. É no interior do deter minismo da teologia jansenista da graça que Pascal inventa um sentido para a apologia: é preciso crer que estamos entre os eleitos, assim como resenha p. 341 - 346 343 os homens que nos cercam, por mais ímpios que sejam, enquanto lhes restar um momento de vida. Para Santiago, ao fazê-lo, Pascal está crian do um possível no seio do necessário, e aí se encontra a surpreendente afinidade entre os dois pensadores. De outro lado, o trecho citado nos remete a outro capítulo funda mental, em que o que é apenas intuído na teologia de Pascal ganhará clareza conceitual para aplicar-se a Espinosa. Cadernos Espinosanos São Paulo n.42 jan-jun 2020 ENTRE SERVIDÃO E LIBERDADE DE HOMERO SANTIAGO A partir de uma engenhosa interpretação do papel da ignorância nas definições de contingente e possível, Santiago encontra para este último um lugar fundamental na ética e na política espinosanas: o ponto de vista do possível ignora a causa, mas a ignora sobre tudo porque a considera, ou seja, toma a coisa como tendo causa (...). Com efeito, se possível é aquilo cuja causa é indeterminada, possível é igualmente aquilo cuja causa pode ser determinada; sobre a qual, em suma, pode-se agir, pois o indivíduo se enxerga (correta ou incorretamente) como agente possível de um aconte cimento (santiago, 2019, p. 153). Sem desconsiderar o fato de que, ontologicamente, só há o necessário, Santiago dá ao possível a realidade de uma tarefa cujo cumprimen to é imperioso para o uso da vida. Este capítulo, assim, fecha o bloco mais estritamente espinosano do livro, apresentando tanto a realidade da servidão, quanto a possível porta aberta para a liberdade. Como passar por ela? Eis a pergunta que o restante do livro tentará responder das mais variadas maneiras; nenhuma delas, porém, definitiva. Já chegando à terceira parte do livro, é curioso que o primeiro texto explicitamente dedicado ao pensamento de Negri seja antecedido por um capítulo dedicado a outra notória espinosana, Marilena Chaui. A despeito das diferenças interpretativas dos dois filósofos sobre a obra Cadernos Espinosanos São Paulo n.42 jan-jun 2020 344 de Espinosa, a contiguidade dos dois capítulos acaba destacando algo de comum entre eles, e que remete à influência marxista que ambos compartilham: a importância dada à luta de classes. Olhar para os confli tos no interior da sociedade, e não só para a história do Estado, como se este fosse um ente transcendente e causa de si próprio, é uma tônica de vários textos de Chaui. Em Negri, é a partir da análise das lutas operá rias que se desenvolverá a apropriação particular que o italiano faz do conceito espinosano de multidão, central em sua filosofia (e também na de Santiago, que com ele pensará, dentre outros temas, junho de 2013). Mas a atenção aos conflitos sociais e a recusa espinosana da transcen dência, comuns a ambos, levarão Chaui e Negri a caminhos diversos. ENTRE SERVIDÃO E LIBERDADE DE HOMERO SANTIAGO Para Chaui, nas palavras de Santiago, “se a tirania persiste é porque se enraíza na vida social, dela emergindo como efeito que decorre de uma causa e envolve, de alguma maneira, todo o corpo social” (santiago, 2019, pág. 192). Daí surgirão as reflexões de Chaui sobre Brasil como sociedade autoritária. Seria o lado amargo da multidão? Para Negri, a multidão é o sujeito da práxis coletiva que brota do desejo primordial de libertação, subjacente a todas as carências particulares que aparecem nas lutas sociais. Como explica Santiago: a noção restritiva de classe sai de cena em benefício de uma noção bem mais ampla, que permite pensar a unidade de todos os ex plorados em sua própria diferença, sem recurso à tradicional sub sunção dessas diferenças à identidade do operário industrial, isto é, o operário-massa. Em segundo lugar, a luta de classes passa a ser considerada como possuindo seu motor no desejo. É a articulação dessas duas inovações que, nitidamente, vai nos direcionando para o conceito de multidão, que ao fim e ao cabo se revelará o único capaz de nomear essa nova classe (santiago, 2019, p. 212). resenha p. 341 - 346 345 Aqui decerto não há lado amargo, mas a lente crítica de Santiago não se furtará ao questionamento óbvio (aliás retomando, por outro viés, o problema da passagem da servidão à liberdade): como pode este concei to fazer-se acontecimento? A filosofia de Negri será retomada mais à frente, em detalhe. Antes disso, porém, a quarta parte do livro dará lugar a três preciosos ensaios sobre temas recorrentes em nossa realidade social: a polícia e seu pendor à violenta obediência abstrata; o Estado e seu escopo; e final mente o dinheiro e a liberdade. Todos têm por ponto de partida objetos empíricos particulares, como o colaboracionismo da polícia francesa durante a segunda guerra ou as transformações sociais decorrentes do Bolsa-Família, mas a questão teórica de fundo é a mesma: os conceitos de servidão e liberdade. Ademais, depois da passagem por Chaui e Negri, não poderiam ser mais claras as razões de Santiago para voltar-se para os acontecimentos sociais. As conclusões, porém, são sempre teóricas, e tão surpreendentes quanto (para um leitor atento) coerentes com as bases conceituais estabelecidas nos capítulos anteriores. ENTRE SERVIDÃO E LIBERDADE DE HOMERO SANTIAGO Sem entrar em mais detalhes, inclusive para não entregar todas as voltas e reviravoltas ao leitor, cabe destacar mais uma vez a impor tância deste trabalho nos dias que correm. Útil tanto para especialistas (em Espinosa, Negri, Chaui e até mesmo Pascal e Nietzsche) quanto para o público geral, o livro traz reflexões particularmente vivas quando espasmos autoritários nos ameaçam de longe ou de perto. Embora seja o retrato do percurso intelectual singular de Homero Santiago, Entre Servidão e Liberdade alcança, se não a universalidade, pelo menos a comu nidade dos bens que podem ser partilhados. 46 Cadernos Espinosanos São Paulo n.42 jan-jun 2020 Cadernos Espinosanos São Paulo n.42 jan-jun 2020 346
https://openalex.org/W2083229040	https://europepmc.org/articles/pmc3320120?pdf=render	English	null	The Effect of Motor Learning of Serial Reaction Time Task (SRTT) Through Action Observation on Mu Rhythm and Improvement of Behavior Abilities	Journal of clinical medicine research	2,012	cc-by	3,503	J Clin Med Res • 2012;4(2):114-118 ress Elmer J Clin Med Res • 2012;4(2):114-118 ress Elmer Original Article Original Article J Clin Med Res • 2012;4(2):114-118 Abstract Conclusions: The result suggests that imitation and learning are involved even in the action that requires task understanding in hu mans. Background: The aim of this study was investigate whether an ac tion observation would have an effect on the action that requires task understanding in humans. Methods: Participants who met the criteria for this study (n = 36). To evaluate the performance, reaction time and performance ac curacy, the stimulus scheduling software was employed. For the electroencephalogram, the equipment QEEG-8 was used. Keywords: Accuracy; Mirror neuron system; Mu rhythm; Reaction time; SRTT Results: Concerning the reaction time of the groups of different learning methods a significant difference was found after the learn ing among the three groups. Regarding the accuracy among the groups of different learning methods, significant difference was found in the action performance accuracy among the three groups. The relative mu power during the SRTT implementation was com pared in the CZ, C3 and C4 regions before, during, and after the learning for each group. In the CZ and C4 region, a significant dif ference was found in the action observation group. In the C3 and The Effect of Motor Learning of Serial Reaction Time Task (SRTT) Through Action Observation on Mu Rhythm and Improvement of Behavior Abilities Sang-Yeol Leea, f, Sung-Soo Baeb, Jin-Tae Hanc, Seung-Deuk Byund, Jong-Sung Change C4 region a significant difference was found in the actual practice group. C4 region a significant difference was found in the actual practice group. Articles © The authors \| Journal compilation © J Clin Med Res and Elmer Press™ \| www.jocmr.org doi:10.4021/jocmr727w Introduction Recent studies using the brain image during action obser vation reported that the brain area involved in actual prac tice was activated during action observation [3, 5], and the motor evoked potential of the corresponding area was also elevated [6, 7]. These results mean that action observation can produce the same neuromuscular response with that of actual practice. When the dynamic imaging of finger move ment for SRTT was shown to adults, the brain response that was similar to that by actual practice was found even by the observation through dynamic imaging [3]. This was reported as the result of the action of the brain mirror neuron [3, 8]. The activation of the mirror neuron suggests that action ob servation for a teleological and well-trained action can help to form the appropriate, coordinated patterns of the action and learn the action by activating the brain area involved in the same action [9]. group, 12 subjects in the action observation group, and 12 subjects in the motor imagery group. There was no signifi cant difference in the age, weight and height among the three groups (P > 0.05). All the subjects sat in front the desk on which a com puter is laid. The height of the chair was controlled so that the elbow joint angle could be about 90° when the right up per limb was placed on the desk. The subjects were asked to perform the task as accurately and quickly as possible fol lowing the visuoauditory signals. While performing the task, the subjects performed the consecutive reaction-time tasks with the minimum finger movement, fixing the elbow joint and the wrist joint. The SRTT for this study was to press the correspond ing key on the keyboard with the designated figure when 40 consecutive signals were provided. The 40 signals included “one” to “eight” that were repeated for five times each. The subjects were asked to press the left-arrow key with the in dex finger for “one” and “eight,” the up-arrow key with the ring finger for “two” and “seven,” and the down-arrow key with the middle finger for “four” and “five” (Fig. 1). The forty consecutive signals composed of five-time repetition of eight signals constituted one block which was implemented for one-time practice. Introduction When responding the forty consecu tive signals, the next signal follows in one-second interval regardless of the key-pressing by the subjects in response to the consecutive signals. The tasks with the same order were assigned to the subjects. The visuoauditary signals were prepared and provided in the form of dynamic imaging file including the letters and audio files of “one” to “eight.” The subjects were asked to perform the task according to the signals. All the subjects wore an earphone while performing the task. Perry and Bentin [10] studied the effect of oneness in the objective and grip shape and reported that mu rhythm was repressed the most and the mirror neuron in the mirror neuron was activated the most. This result suggests that there is a high correlation between the mirror neuron activation and the mu rhythm repression that take place during action observation. The mu rhythm, an electroencephalogram, is in the range of alpha wave, but it has difference features from al pha wave. Although alpha wave is relatively regular, the mu rhythm looks like a bow and is often asymmetric and asyn chronous [11]. While the amplitude of alpha wave is 30 - 50 µV, the amplitude of the mu rhythm is lower than that. While alpha wave appears in all the brain areas, mu rhythm appears usually around the central sulcus (CZ, C3 and C4) in general [12]. Many recent studies showed that action observation is involved in motor learning as it greatly affects the mirror neuron activation and mu rhythm repression. Through the one-time repetition performance in the method described above, we measured the reaction time, ac curacy and mu rhythm before learning. The subjects in the actual practice group learned the movement by means of actual practice of three times, while the subjects in the ac tion observation group learned it for three times through the dynamic imaging (visual and auditory) of the performance by the third person who was well-trained . Additionally, the subjects in the motor imagery group learned the movement only by using the visuoauditary file used in SRTT. However, even though many studies and analyses have been carried out on the action observation of the movement with simple objectives, the effect of the performance obser vation of the task where the response to stimulus is required, such as SRTT, on the task accuracy and reaction time has been little studied. Introduction Motor learning is the human ability to maintain flexibility to the environment in learning and performing motor skills [1]. Motor learning is a comprehensive concept that includes the sensory processing procedure, motor control, and mo tor skill learning and it also refers to the ability to perform and memorize learned skill under variety of conditions [2]. It was known that traditional motor learning can be obtained by means of repeated movement. In fact, however, most of the motor functions include both physical and cognitive fac tors. Recently, studies have actively been conducted show ing that motor skill can be obtained through mental practice. In the case of the traditional movement method through di rect practice, motor learning is difficult if there is limitation in physical movement. For example, initial motor learning is impossible for stroke patients in their initial stage because of the limitation in movement. The indirect practice meth ods to supplement such a drawback include mental practice through motor imagery and action observation using the vi sual and auditory senses [3, 4]. Studies are actively carried out about the effect of mental practice by means of motor imagery among the motor learning methods through indirect practice. However, disadvantages such as lowered level of concentration during the learning should be supplemented. For that, action observation using the visual and auditory senses have been actively studied in recent times [3]. Manuscript accepted for publication November 11, 2011 aDepartment of phyical therapy, Kyoungsung University, Republic of Korea bDepartment of Physical Therapy, Daegu University, Gyeongsan, Republic of Korea cDepartment of Physical Therapy, Kyungsung University, Pusan, Republic of Korea dDepartment of Physical Medicine and Rehabilitation, Daegu Fatima Hospital, Daegu, Republic of Korea eDepartment of Physical Therapy, Daegu Haany University fCorresponding author: Sang-Yeol Lee. 314-79, Daeyeon-dong, Nam- gu, Busan, 609-736, Republic of Korea. Email: sjslh486@hanmail.net doi:10.4021/jocmr727w eDepartment of Physical Therapy, Daegu Haany University f Action observation is the method to understand, choose and imitate the form and motion of an action by observing other’s action. [1]. Initially, action observation was the re 114 Articles © The authors \| Journal compilation © J Clin Med Res and Elmer Press™ \| www.jocmr.org J Clin Med Res • 2012;4(2):114-118 Lee et al search theme of social psychology about imitation [2], and it is nowadays highlighted as a method for cognitive inter vention to supplement the limitation of motor imagery [3]. Introduction In this study, we investigate the mu rhythm and the mirror neuron system activation that appears during the action observation, motor imagery, and actual practice of the task that requires understanding of movement, such as SRTT, and studied the effect of the individual motor learning methods on the movement accuracy and reaction time. To evaluate the reaction time and accuracy of SRTT, we employed the stimulus scheduling software (LAXTHA, Ko rea). While all the subjects were performing the task, the per formance reaction time and the performance accuracy were recorded in all the intervals. The electroencephalogram was measured using QEEG- 8 (LXE3208, LAXTHA Inc., Korea). The electroencepha logram sampling rate of the measured subjects was 256 Hz, filtered in the range of 0.5 – 50 Hz, and the data was saved in a computer by the 12-bit AD conversion. The electroen cephalogram was measured in three regions on the head sur Articles © The authors \| Journal compilation © J Clin Med Res and Elmer Press™ \| www.jocmr.org Methods This study was conducted with a total of 36 healthy adults at the age of 20’s including 12 subjects in the actual practice 115 Articles © The authors \| Journal compilation © J Clin Med Res and Elmer Press™ \| www.jocmr.org J Clin Med Res • 2012;4(2):114-118 Effects of Action Observations Figure 1. Method of reaction. Figure 1. Method of reaction. Results face using the monopoly system. The electrode was attached using the international 10 - 20 system. To measure the mu rhythm that appears during the motor performance of hand, action observation and motor imagery in this study, three channels, Cz, C3 and C4, were attached, and the reference electrode and ground electrode were attached to the styloid process on the right and left sides. Articles © The authors \| Journal compilation © J Clin Med Res and Elmer Press™ \| www.jocmr.org Behaviour change after practice With respect to the reaction time among the groups of dif ferent learning methods, no significant difference was found in the reaction time among the group before the learning (P > 0.05), where the reaction time after the learning showed significant differences among the three groups (P < 0.05). The post-hoc test to examine the reaction time difference among the groups after the learning, the reaction time was significantly longer in the motor imagery group (Table 1) (P < 0.05). Regarding the action performance accuracy among the groups of different learning methods, no significant dif ference was found in the action performance accuracy among the group before the learning (P > 0.05), where the action performance accuracy after the learning showed significant differences among the three groups (P < 0.05). The post-hoc test to examine the action performance accuracy difference among the groups after the learning, the performance ac curacy was significantly lower in the motor imagery group In this study, to quantify the generation of mu rhythm in the electroencephalogram which was known to be closely related to the mirror neuron activity, the ratio of mu rhythm to the total wave was calculated and referred as relative mu power. Mu rhythm(μ)/(θ wave + α wave + β wave + γ wave) = (7 - 11 Hz)/( 4 - 50 Hz) To investigate the change in the behavior in each group before and after the learning, one-way ANOVA and an inde pendent t-test were performed. Repeated ANOVA was used to compare the relative mu power before, during and after the learning in each group. This study was approved by the Institutional Review Board (IRB) and the approval number is DFH09OT055. Table 1. Comparison of Reaction Time and Accuracy mean ± SD; RT: Reaction time; AC: Accuracy; AO: Action observation; AP: Actually practice; MI: Motor imagery;P < 0.05. Table 1. Behaviour change after practice Comparison of Reaction Time and Accuracy AO (n = 12) AP (n = 12) MI (n = 12) F P RT (msec) Pre 0.69 ± 0.04 0.69 ± 0.54 0.67 ± 0.58 0.69 0.50 Post 0.61 ± 0.03 0.61 ± 0.05 0.65 ± 0.05 3.47 0.04 AC (%) Pre 68.61 ± 12.06 65.46 ± 12.26 72.96 ± 14.51 1.00 0.37 Post 86.95 ± 5.74 89.16 ± 8.60 80.18 ± 10.47 3.63 0.03* Articles © The authors \| Journal compilation © J Clin Med Res and Elmer Press™ \| www.jocmr.org 116 Articles © The authors \| Journal compilation © J Clin Med Res and Elmer Press™ \| www.jocmr.org 116 J Clin Med Res • 2012;4(2):114-118 Lee et al Table 2. Comparison of Relative Mu Power Within Intervention Period on Each Group at CZ, C3 and C4 Area P < 0 05 Table 2. Comparison of Relative Mu Power Within Intervention Period on Each Group at CZ, C3 and C4 Area Area Group Pre Mid Post F P CZ AO 0.21 ± 0.06 0.18 ± 0.04 0.13 ± 0.05 12.39 0.00 AP 0.22 ± 0.08 0.18 ± 0.04 0.13 ± 0.05 2.71 0.08 MI 0.24 ± 0.06 0.23 ± 0.07 0.22 ± 0.06 0.353 0.70 C3 AO 0.18 ± 0.05 0.17 ± 0.04 0.14 ± 0.04 2.15 0.14 AP 0.21 ± 0.07 0.18 ± 0.03 0.16 ± 0.061 9.48 0.00* MI 0.21 ± 0.03 0.22 ± 0.04 0.20 ± 0.06 0.56 0.57 C4 AO 0.20 ± 0.02 0.16 ± 0.04 0.13 ± 0.04 20.55 0.00* AP 0.21 ± 0.08 0.17 ± 0.04 0.15 ± 0.04 11.71 0.00* MI 0.24 ± 0.05 0.22 ± 0.07 0.22 ± 0.03 1.35 0.27 omparison of Relative Mu Power Within Intervention Period on Each Group at CZ, C3 and C4 (Table 1) (P < 0.05). (Table 1) (P < 0.05). (Table 1) (P < 0.05). limited by time and space, the learning ability is not maxi mized in the cases when understanding of the task perfor mance is insufficient and the ability, control and concentra tion to remind the task performance scene are lacking [13]. However, the motor learning through action observation, which has drawn attention of many researchers recently, is known as the method that helps to better remind the task performance scene specifically than the learning through motor imagery and supplement control and concentration [3]. Behaviour change after practice In this study, to exclude the effect of audiovisual stimu lation, all the audiovisual stimulations were provided also to the motor imagery group except the action by the third person. Mu rhythm change after practice The relative mu power was compared for the SRTT per formance before, during and after the learning in the CZ, C3 and C4 regions in each group. The result showed that there were significant differences in the action observation group and the actual practice group in the CZ region (P < 0.05), while not significant difference was found in the mo tor imagery group. In the C3 region, a significant reduction was found only in the actual practice group, while a little decreasing tendency was found in the action observation group. In the C4 region, a significant difference was found in the action observation group and the actual practice group (P < 0.05), while no significant difference was found in the motor imagery group (Table 2). In this study, we measured the mu rhythm of 7 - 11Hz waveform that is characterized by its repression during the mirror neuron activation in learning process to analyze the learning process status and the degree of concentration. The result showed that the mu rhythm became repressed as the learning proceeded in the CZ, C3 and C4 regions in the ac tual practice group and the action observation group. This result indicates that accurate learning took place as the mir ror neuron was activated in the action observation group and the actual practice group. Articles © The authors \| Journal compilation © J Clin Med Res and Elmer Press™ \| www.jocmr.org Discussion Various methods are applied for motor learning and each method has unique characteristics. Learning through actual practice, which is direct motor learning, is effective, but limited by time and space. Although the learning by means of motor imagery, one of the motor learning methods, is less The accuracy and reaction time support the result. The accuracy and reaction time were measured in this study for each of the learning methods before, during and after 117 Articles © The authors \| Journal compilation © J Clin Med Res and Elmer Press™ \| www.jocmr.org J Clin Med Res • 2012;4(2):114-118 Effects of Action Observations Lippincott Williams and Wilkins. 2007. the learning. The result showed that the accuracy was sig nificantly increased after the learning in the actual practice group and the action observation group, and the reaction time was significantly reduced in those groups. This result might have been caused by the repression of the mu rhythm which was known to play the identical functional role with that of the mirror neuron. In the conventional studies where the mu rhythm was observed when observing the movement of a person and a robot, significant repression was found in the C3 and C4 regions, which indicated that the first-order movement region can be activated when observing an objec tive-oriented movement [10, 13]. In our study also, the result similar to that of previous studies was found. The result sug gests that the learning mechanism that involves understand ing and imitating of task by mirror neuron system activation takes place not only in the observation of simple actions but also in the performance of task that requires action respond ing to stimulus such as SRTT. 3. Lee SY, Lee MH, Bae SS, Lee KS, Gong WT. The ef fects of action observation and motor imagery of SRTT in mirror neuron activation. Journal of The Korean Soci ety of Physical Medicine. 2010; 5(3): 395-404. 4. Stenekes MW, Geertzen JH, Nicolai JP, De Jong BM, Mulder T. Effects of motor imagery on hand function during immobilization after flexor tendon repair. Arch Phys Med Rehabil. 2009;90(4):553-559. 5. Filimon F, Nelson JD, Hagler DJ, Sereno MI. Human cortical representations for reaching: mirror neurons for execution, observation, and imagery. Neuroimage. 2007;37(4):1315-1328. 6. Fadiga L, Craighero L, Olivier E. Human motor cortex excitability during the perception of others’ action. Curr Opin Neurobiol. 2005;15(2):213-218. 7. Maeda F, Kleiner-Fisman G, Pascual-Leone A. Discussion Motor facilitation while observing hand actions: specificity of the effect and role of observer’s orientation. J Neuro physiol. 2002;87(3):1329-1335. The result suggests that understanding of task by mirror neuron system activation and learning through imitation take place not only in the observation of simple actions but also in the performance of task that requires action responding to stimulus such as SRTT. It was known that imitation and learning are involved even in the action that requires task understanding in humans. 8. Muthukumaraswamy SD, Johnson BW. Primary mo tor cortex activation during action observation revealed by wavelet analysis of the EEG. Clin Neurophysiol. 2004;115(8):1760-1766. 9. Hayes SJ, Ashford D, Bennett SJ. Goal-directed imi tation: the means to an end. Acta Psychol (Amst). 2008;127(2):407-415. Financial Disclosure 10. Perry A, Bentin S. Mirror activity in the human brain while observing hand movements: a comparison be tween EEG desynchronization in the mu-range and pre vious fMRI results. Brain Res. 2009;1282:126-132.i We certify that no party having a direct interest in the results of the research supporting this article has or will confer a benefit on us or on any organization with which we are as sociated. 11. Pineda JA. The functional significance of mu rhythms: translating “seeing” and “hearing” into “doing”. Brain Res Brain Res Rev. 2005;50(1):57-68. 12. Duntley SP, Kim AH, Silbergeld DL, Miller JW. Charac terization of the mu rhythm during rapid eye movement sleep. Clin Neurophysiol. 2001;112(3):528-531. References 1. Halsband U, Lange RK. Motor learning in man: a re view of functional and clinical studies. J Physiol Paris. 2006;99(4-6):414-424. 13. Oberman LM, McCleery JP, Ramachandran VS, Pineda JA. EEG evidence for mirror neuron activity during the observation of human and robot action: toward an analy sis of the human qualities of interactive robots. Neuro computing. 2007;70: 2194-2203. 2. Shumway-Cook A, & Woollacott MH. Motor Control: Translating Research into Clinical Practice, Baltimore : 2. Shumway-Cook A, & Woollacott MH. Motor Control: Translating Research into Clinical Practice, Baltimore : 118 Articles © The authors \| Journal compilation © J Clin Med Res and Elmer Press™ \| www.jocmr.org
W2985762302.txt	https://openreviewhub.org/sites/default/files/cte/1608/abstraktlviv.pdf	en		Depth of Application of a Microgranule for Precision Sowing on the Controlled Release of Nutrients, and the Growth of Maize Plants in the Initial Stage of Development	Chemical technology and engineering. Proceedings.2019.№1	2,019	cc-by	824	1 Depth of application of a microgranule for precision sowing on the controlled release of nutrients, and the growth of maize plants in the initial stage of development Natalia Matłok1, Józef Gorzelany2, Piotr Antos3, Maciej Balawejder 4 1. Department of Food and Agriculture Production Engineering, University of Rzeszow, Poland, 35-601 Rzeszów, Zelwerowicza 4, nmatlok@ur.edu.pl 2. Department of Food and Agriculture Production Engineering, University of Rzeszow, Poland, 35-601 Rzeszów, Zelwerowicza 4, gorzelan@ur.edu.pl 3. Department of Computer Engineering in Management, Rzeszow University of Technology, Poland, 35-959 Rzeszów, Aleja Powstańców Warszawy 12, p.antos@prz.edu.pl 4. Department of Chemistry and Food Technology, University of Rzeszow, Poland, 35-601 Rzeszów, Ćwiklińskiej 1a, maciejb@ur.edu.pl Abstract– The manuscript presents the results of vase experiments on the determination of the depth impact of soil microgranulate application for precision sowing on the growth of maize plants in the initial stage of development. Multi-component granule used in the experiment characterized by the controlled release of ingredients was made on the basis of an alternative source of a phosphate in accordance with the patent application No. P.429318. Kеуwords – alternative raw material, phosphorus, precision fertilization, soil-applied microgranula, maize Introduction The innovative simplified technology of maize cultivation consists of the utilization of a cultivating and seeding aggregate, thanks to which seed sowing is performed at the same time as a deep precise application of fertilizer to cultivated soil [Ochal i in., 2015]. Aim and scope The purpose of the pot experiments was to determine the effect of the depth of application for precision sowing of microgranules developed on the basis of an alternative source of phosphorus originating from thermally processed bone waste (patent application No. P. 429318) for the growth and development of corn at the initial stage of development. Methodology Table 1. Composition of microgranules for precision sowing Content of makro- and mikroelements P2O5 K2O CaO MgO 314,0 g∙kg-1 42,3 g∙kg-1 663,1 g∙kg-1 240,3 g∙kg-1 Na2O 5,4 g∙kg-1 Mn 6,5 mg∙kg-1 Fe 1 mg∙kg-1 Cu 3,2 mg∙kg-1 Zn 0,8 mg∙kg-1 The pot experiments were carried out under strict conditions in the vegetation greenhouse. Standardized soil, a mixture of sand and acidified peat with a pH of 5.5-6.5 in the ratio of 7: 1, was used in the experiment and certified maize seeds of the variety Farm Gigant (MTZ = 264g) were utilized. The developed microgranule (table 1) for precision sowing at a dose of 30 kg ∙ ha-1 was applied at the depth of: 2, 4, 6, 8 and 10 cm in relation to the kernel in the Fix system. The experiment was carried out at a constant soil moisture of 50% PPW. 2nd INTERNATIONAL SCIENTIFIC CONFERENCE “CHEMICAL TECHNOLOGY AND ENGINEERING – 2”, JUNE 24-28TH, 2019, LVIV, UKRAINE 2 Conclusions Based on the pot experiments on the depth of application of microgranules for precision sowing with controlled release of components, it was found that the highest growth and development of maize plants occurred in a variant in which the fertilizer was applied at a depth of 2 cm to the kernel. The location of microgranules at a depth of 2 cm from the kernel resulted in obtaining the highest parameters of biometric traits of maize plants in the 6th week of sowing (Fig. 1). 60 4 Height of plants Width of the leaf blade 3,5 50 Height [cm] 30 20 55,0 49,3 41,3 47,7 51,7 43,0 Width [cm] 3 40 10 2,5 2 3,5 1,5 1 2,4 2,3 4 6 8 10 0 kontrola 2 4 6 8 kontrola 10 Depth of application [cm] 2 Depth of application [cm] 4 Dry mass of aboveground parts 8 Dry mass of underground parts 3,5 7 3 Dry mass [g] 6 Dry mass [g] 2,4 0,5 0 5 4 7,41 3 2 2,6 2,0 5,47 6,00 4,77 3,95 3,46 2,5 2 3,14 1,5 1 3,37 2,76 3,11 2,26 1,86 0,5 1 0 0 kontrola 2 4 6 8 10 Depth of application [cm] kontrola 2 4 6 8 10 Depth of application [cm] Fig.1. Biometric features of maize plants depending on the depth of application of the fertilizer Summary The application of a multi-component fertilizer with a controlled release of ingredients at a depth of 2 cm in relation to the kernel, developed on the basis of an alternative source of phosphate, results in the best growth and development of maize plants in the first 6 weeks of sowing. Thanks The research was carried out as part of the project "Development of innovative fertilizers on the basis of an alternative source of raw material" No. BIOSTRATEG1 / 270963 // 6 / NCBR / 2015 co-financed from public funds at the disposal of the National Center for Research and Development under the "Environment, Agriculture and Forestry" Program - BIOSTRATEG. Literature [1] Ochal P., Jadczyszyn T., Jurga B., „Rozwój systemu korzeniowego kukurydzy w zależności od umieszczenia nawozu w glebie”, Polish Journal of Agronomy, 23, s.74– 81, 2015. 2nd INTERNATIONAL SCIENTIFIC CONFERENCE “CHEMICAL TECHNOLOGY AND ENGINEERING – 2”, JUNE 24-28TH, 2019, LVIV, UKRAINE
https://openalex.org/W4366463243	https://journals.plos.org/plosbiology/article/file?id=10.1371/journal.pbio.3001995&type=printable	English	null	The dynamic architecture of Map1- and NatB-ribosome complexes coordinates the sequential modifications of nascent polypeptide chains	PLoS biology	2,023	cc-by	18,685	Academic Editor: Jamie H. D. Cate, University of California, Berkeley, UNITED STATES Academic Editor: Jamie H. D. Cate, University of California, Berkeley, UNITED STATES Received: November 10, 2021 Accepted: January 10, 2023 Published: April 20, 2023 Copyright: © 2023 Knorr 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. Copyright: © 2023 Knorr et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All cryo-electron microscopy maps are available at the EM Data Bank with accession codes EMD-16182 for the Map1-C1-ribosome complex, EMD-16191 for the Map1-C2-ribosome complex, EMD-16090 for the NatB-RNC complex with two stably bound NatBs (class I) and EMD-16086 for the NatB-RNC map focused sorted on NatB-2 (class II). All models are available at the Protein Data Bank (PDB) with accession codes 8BQD for the Map1-C1-ribosome complex, 8BQX for the Map1-C2-ribosome The dynamic architecture of Map1- and NatB- ribosome complexes coordinates the sequential modifications of nascent polypeptide chains Alexandra G. Knorr☯, Timur Mackens-Kiani☯, Joanna Musial, Otto Berninghausen, Thomas Becker, Birgitta Beatrix, Roland BeckmannID Alexandra G. Knorr☯, Timur Mackens-Kiani☯, Joanna Musial, Otto Berninghausen, Thomas Becker, Birgitta Beatrix, Roland BeckmannID Department of Biochemistry, Gene Center, Ludwig-Maximilians University Munich, University of Munich, Munich, Germany Department of Biochemistry, Gene Center, Ludwig-Maximilians University Munich, University of Munich, Munich, Germany a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ☯These authors contributed equally to this work. * beatrix@genzentrum.lmu.de (BB); beckmann@genzentrum.lmu.de (RB) ☯These authors contributed equally to this work. * beatrix@genzentrum.lmu.de (BB); beckmann@genzentrum.lmu.de (RB) OPEN ACCESS Cotranslational modification of the nascent polypeptide chain is one of the first events during the birth of a new protein. In eukaryotes, methionine aminopeptidases (MetAPs) cleave off the starter methionine, whereas N-acetyl-transferases (NATs) catalyze N-terminal acetyla- tion. MetAPs and NATs compete with other cotranslationally acting chaperones, such as ribosome-associated complex (RAC), protein targeting and translocation factors (SRP and Sec61) for binding sites at the ribosomal tunnel exit. Yet, whereas well-resolved structures for ribosome-bound RAC, SRP and Sec61, are available, structural information on the mode of ribosome interaction of eukaryotic MetAPs or of the five cotranslationally active NATs is only available for NatA. Here, we present cryo-EM structures of yeast Map1 and NatB bound to ribosome-nascent chain complexes. Map1 is mainly associated with the dynamic rRNA expansion segment ES27a, thereby kept at an ideal position below the tun- nel exit to act on the emerging substrate nascent chain. For NatB, we observe two copies of the NatB complex. NatB-1 binds directly below the tunnel exit, again involving ES27a, and NatB-2 is located below the second universal adapter site (eL31 and uL22). The binding mode of the two NatB complexes on the ribosome differs but overlaps with that of NatA and Map1, implying that NatB binds exclusively to the tunnel exit. We further observe that ES27a adopts distinct conformations when bound to NatA, NatB, or Map1, together sug- gesting a contribution to the coordination of a sequential activity of these factors on the emerging nascent chain at the ribosomal exit tunnel. Citation: Knorr AG, Mackens-Kiani T, Musial J, Berninghausen O, Becker T, Beatrix B, et al. (2023) The dynamic architecture of Map1- and NatB- ribosome complexes coordinates the sequential modifications of nascent polypeptide chains. PLoS Biol 21(4): e3001995. https://doi.org/10.1371/ journal.pbio.3001995 Academic Editor: Jamie H. D. Cate, University of California, Berkeley, UNITED STATES Received: November 10, 2021 Accepted: January 10, 2023 Published: April 20, 2023 Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. Abbreviations: acetyl-CoA, acetyl-coenzyme A; AF2, AlphaFold 2; APD, amino peptidase domain; CTF, contrast transfer function; ES, expansion segment; MetAP, methionine aminopeptidase; NAC, nascent polypeptide-associated complex; NAT, N-acetyl-transferase; PDF, peptidyl- deformylase; RAC, ribosome-associated complex; RNC, ribosome nascent chain complex; rtRNC, RNaseI-treated RNC; SN, supernatant; SRP, signal recognition particle; TAP, tandem affinity purification; TEV, tobacco etch virus; TPR, tetratricopeptide repeat; TRAP, Translocon Associated Protein; wt, wild type. The fundamental importance of N-terminal methionine removal is reflected by the lethality caused by deletion of all MetAP-encoding genes in eubacteria [10,11] and yeast [4]. In baker’s yeast (Saccharomyces cerevisiae; S. cerevisiae), Map1 (a type I MetAP) represents the major iso- form, indicated by the higher copy number as well as by a much stronger slow growth pheno- type of map1 null mutants when compared to map2 null mutants [12–14]. Both Map1 and Map2 were previously shown to bind to ribosomes [8,15], and the ribosome interaction of Map1 was shown to be salt-sensitive and independent of the nascent polypeptide chain. More- over, the Map1-interacting region was mapped via cross-linking studies to the peptide exit tunnel periphery of the 60S subunit, likely contacting the region covered by uL23 and uL29 [16]. This position is overlapping with contact sites of various exit tunnel-binding factors such as the chaperones RAC (ribosome-associated complex) and NAC (nascent polypeptide-associ- ated complex), as well as the secretory pathway factors SRP (signal recognition particle) and the Sec61 protein-conducting channel. Furthermore, evidence was provided for an involve- ment of rRNA expansion segment ES27 in the interaction of MAPs with the ribosome since deletion of the longest helix of ES27 resulted in a decrease of Map1 and Map2 ribosome associ- ation in S. cerevisiae [17,18]. For unmodified nascent peptides but also for nascent peptides after methionine cleavage, Nα-acetylation is another highly frequent cotranslational modification in eukaryotes. It is cata- lyzed by Nα-acetyltransferases (NATs), which transfer an acetyl group from acetyl-coenzyme A (acetyl-CoA) to the α-amino group of the emerging nascent chain. With the exception of NatD, ribosome-associated NATs form dimeric or trimeric hetero-complexes and usually con- sist of a small catalytic and at least one additional large auxiliary subunit [19]. In humans, seven subtypes of NATs exist: NatA to NatH, with the first three of them, NatA, NatB, and NatC acetylating the majority of substrate proteins [19–21]. PLOS BIOLOGY PLOS BIOLOGY Cryo-EM structures of ribosome-bound Map1 and NatB which can play important roles in the targeting, folding, and stability of the newly made polypeptide. complex, 8BJQ for the NatB-RNC complex with two stably bound NatBs (class I) and 8BIP for the NatB-RNC map focused sorted on NatB-2 (class II). Translation of the vast majority of mRNAs starts on an AUG codon with a methionine- bound initiator tRNA (Met-tRNAi-Met), resulting in methionine as the first amino acid of most proteins. When this starter methionine is followed by a small and uncharged amino acid, such as Ala, Cys, Gly, Pro, Ser, Thr, or Val, it is usually removed cotranslationally by evolution- arily conserved methionine aminopeptidases (MetAPs) [1–3]. There are two types of MetAPs. While type I is found in eubacteria and type II in archaea, eukaryotes harbor both types of MetAPs. The two types differ in a characteristic insertion (approximately 60 aa) in the catalytic domain of type II enzymes [4]. The common catalytic domain belongs to the family of evolu- tionarily conserved metalloproteases and adopts the typical aminopeptidase fold also known as “pita-bread” protease fold [5]. Catalysis typically requires one or two divalent cations (for review, see [6,7]). In contrast to bacterial MetAPs, eukaryotic MetAP1s possess an additional N-terminal extension containing two zinc finger domains, a RING-finger-like Cys2-Cys2 zinc finger (aas 22 to 40 in yeast) and a Cys2-His2 zinc finger (aas 50 to 66 in yeast) related to RNA-binding zinc fingers. This extension has been suggested to be important for the correct functional alignment of Map1 on the ribosome in yeast [8]. Eukaryotic type II MetAPs also contain an N-terminal extension carrying a positively charged Lys-rich stretch [7,9]. Funding: A.G.K. received funding through Graduate School of Quantitative Biosciences Munich. R.B. is funded by grants from the Deutsche Forschungsgemeinschaft (GRK1721) and by an European Research Council (ERC) Advanced Grant (HumanRibogenesis, Reference Nr. 885711). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Introduction In all kingdoms of life, nascent polypeptide chains are subject to chemical modification as soon as they emerge from the ribosomal exit tunnel. The earliest and most common modifica- tions in eukaryotes are the cleavage of the N-terminal amino acid and Nα-acetylation, both of 1 / 28 PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001995 April 20, 2023 PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001995 April 20, 2023 Competing interests: The authors have declared that no competing interests exist. The most abundant member of the Nα-acetyltransferase family is NatA, which modifies nascent chains with an N-terminal Ser, Ala, Thr, Gly, Val, or Cys after the initiator methionine has been removed by MetAPs. In contrast, Nα-acetylation by NatB and NatC occurs without initiator methionine removal, since they directly acetylate this methionine when followed by Asp, Glu, Asn, or Gln (in case of NatB) or by large hydrophobic residues including Leu, Ile, Phe, and Tyr (in case of NatC). In yeast it was shown that whereas N α acetylation by NatA seems to play a role in sys In yeast, it was shown that whereas N-α-acetylation by NatA seems to play a role in sys- temic adaptation control, modification by NatB seems rather to be important for protein PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001995 April 20, 2023 2 / 28 PLOS BIOLOGY Cryo-EM structures of ribosome-bound Map1 and NatB folding [22]. Furthermore, depletion of NatB subunits to 30% of the wild-type level caused a 50% decrease in growth in Arabipopsis thaliana [23] and missense mutations in the catalytic domain NAA20 of human NatB were shown to cause autosomal recessive developmental delay, intellectual disability, and microcephaly, emphasizing the importance of NatB function for the cell [24]. Given the differences in substrate specificity and in requirement for initiator methionine removal, the question arises how access of MetAPs and the different NATs to the peptide exit site is spatially and temporally coordinated. Here, a recent cryo-EM structure of yeast NatA bound to native 80S ribosomes carrying a nascent chain showed that NatA is anchored directly at the exit tunnel by interactions with ribosomal rRNA expansion segments (ESs) [25]. NatA was found in a position sterically excluding concomitant binding of other cotranslationally acting chaperones (RAC/Ssb), SRP, as well as Sec61. Also recently, the structures of NatB from Candida albicans [26] and human NatB [27] as well as Chaetomium thermophilum Naa20 with a competitive inhibitor [28] were determined by X-ray crystallography and cryo-EM, respec- tively. However, for eukaryotic MAPs or the other NATs, structural information on their mode of ribosome interaction is largely lacking and the interplay of these factors at the exit tunnel is thus only poorly understood. Here, we present cryo-EM structures of nascent chain-carrying 80S ribosomes in complex with Map1 or the NatB complex from S. Competing interests: The authors have declared that no competing interests exist. cerevisiae at an overall resolution of 3.8 to 3.9 Å (for Map1-80S) and 3.1 to 3.8 Å (for NatB-80S), respectively. We observed Map1 mainly flexibly bound to the dynamic rRNA expansion segment ES27a that positions it directly juxtaposed the peptide exit tunnel. This position would allow for a very early encounter with substrate nascent chains and explain their modification as soon as they emerge from the ribosome tun- nel. Moreover, the observed binding mode of Map1 likely excludes concomitant NatA binding for subsequent Nα-acetylation of the new N-terminus. Despite also being recruited to the peptide tunnel exit periphery, NatB shows a substantially different binding mode at the exit site compared to NatA. Interestingly, we observe two copies of NatB, one bound directly below the tunnel exit site and to ES27a (NatB-1) and one bound to the second universal adapter site (UAS-II) for exit factors (NatB-2) [29]. In contrast to NatA, NatB-2 engages also via contacts to rigid parts of rRNA and the ribosomal protein eL31. For both NatBs, the catalytic subunits are positioned below the tunnel exit such that nascent chains could engage them at a length of approximately 55 amino acids. Yet, we speculate that NatB-2 is more likely to engage the substrate, while NatB-1 might serve to position NatB-2 via ES27a. PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001995 April 20, 2023 Cryo-EM structure of the Map1-ribosome complex Cryo-EM samples for endogenous MetAP-ribosome complexes were obtained from native pullouts of ribosome-bound TAP-tagged Map1 essentially as described before [25,30] (Figs 1A and S1). After elution by cleavage of the Map1-tag using tobacco etch virus (TEV) protease, the Map1-ribosome complexes were stabilized by treatment with the chemical crosslinker glu- taraldehyde prior to cryo-grid preparation. As observed before in native pullouts with cyclo- heximide-treated samples [25,30], 3D classification revealed classes with programmed ribosomes predominantly in the pre-translocational state with tRNAs present in the canonical A and P sites, but also classes representing termination/pre-recycling complexes (with eRF1 and ABCE1) and idle (tRNA-free) ribosomes (S2 Fig). Notably, in the majority of classes, ES27a was found in the position below the peptide exit tunnel (ES27-exit), and attached to ES27a, we observed an additional density reaching to the peptide exit tunnel. We joined those PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001995 April 20, 2023 3 / 28 PLOS BIOLOGY Cryo-EM structures of ribosome-bound Map1 and NatB Fig 1. Cryo-EM structures of Map1 bound to ribosomes via expansion segment ES27a. (A) Concentrated eluate obtained from Map1-TAP affinity purification shown on a 12% Nu-PAGE. Mass spectrometry analysis confirmed the presence of Map1 and ribosomal proteins. A contamination from a viral protein is marked with an asterisk. See S1 Raw Images for the raw gel image. (B) Cryo-EM structures of Map1 in complex with a translating 80S ribosome in two different conformations (left: Map1-C1-80S; center: Map1-C2-80S; right: bottom view on the peptide exit tunnel). The maps were filtered according to local resolution. Isolated densities were extracted after the final CTF refinement. (C) Cartoon representation of bottom views with overlay (left) and separate views (right) as shown in (B). (D) An AlphaFold 2 model for Map1 was fitted into the density, shown as front view. The maps were filtered to 20 Å using a Gaussian low-pass filter. (E-H) Two views on the tunnel exit highlighting the position of Map1-C1 (E, G) and Map1-C2 (F, H) with respect to tunnel exit surrounding ribosomal proteins coloured as indicated in the legend below. (I) In vitro binding assay addressing the contribution of ES27a to ribosome-Map1 binding. Samples from the pelleting assay using recombinant Map1 and purified RNaseI-treated (rtRNC) or nontreated RNCs were applied to a 15% SDS-PAGE. For Map1 alone the supernatant (SN) fraction and for all other samples the pellet (P) fraction is shown. Co-pelleting of Map1 with the ribosome was quantified by densitometry. Cryo-EM structure of the Map1-ribosome complex When ESs were digested by RNAseI, Map1 binding was significantly decreased to about 40% compared to Map1 binding to untreated 80S ribosomes. TE: tunnel exit. ZF: zinc finger domain. APD: aminopeptidase domain. Map1-C1: light green, Map1-C2: dark green, eL22: purple, H59: orange, 40S SU: light yellow, 60S SU: grey, ES27a: cyan, tRNAs: dark blue, nascent chain (NC): pink. See S1 Raw Images for raw gel images and S1 Data for numerical data underlying the quantification. Fig 1. Cryo-EM structures of Map1 bound to ribosomes via expansion segment ES27a. (A) Concentrated eluate obtained from Map1-TAP affinity purification shown on a 12% Nu-PAGE. Mass spectrometry analysis confirmed the presence of Map1 and ribosomal proteins. A contamination from a viral protein is marked with an asterisk. See S1 Raw Images for the raw gel image. (B) Cryo-EM structures of Map1 in complex with a translating 80S ribosome in two different conformations (left: Map1-C1-80S; center: Map1-C2-80S; right: bottom view on the peptide exit tunnel). The maps were filtered according to local resolution. Isolated densities were extracted after the final CTF refinement. (C) Cartoon representation of bottom views with overlay (left) and separate views (right) as shown in (B). (D) An AlphaFold 2 model for Map1 was fitted into the density, shown as front view. The maps were filtered to 20 Å using a Gaussian low-pass filter. (E-H) Two views on the tunnel exit highlighting the position of Map1-C1 (E, G) and Map1-C2 (F, H) with respect to tunnel exit surrounding ribosomal proteins coloured as indicated in the legend below. (I) In vitro binding assay addressing the contribution of ES27a to ribosome-Map1 binding. Samples from the pelleting assay using recombinant Map1 and purified RNaseI-treated (rtRNC) or nontreated RNCs were applied to a 15% SDS-PAGE. For Map1 alone the supernatant (SN) fraction and for all other samples the pellet (P) fraction is shown. Co-pelleting of Map1 with the ribosome was quantified by densitometry. When ESs were digested by RNAseI, Map1 binding was significantly decreased to about 40% compared to Map1 binding to untreated 80S ribosomes. TE: tunnel exit. ZF: zinc finger domain. APD: aminopeptidase domain. Map1-C1: light green, Map1-C2: dark green, eL22: purple, H59: orange, 40S SU: light yellow, 60S SU: grey, ES27a: cyan, tRNAs: dark blue, nascent chain (NC): pink. See S1 Raw Images for raw gel images and S1 Data for numerical data underlying the quantification. https://doi.org/10.1371/journal.pbio.3001995.g001 PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001995 April 20, 2023 Cryo-EM structure of the Map1-ribosome complex In both classes, the position of Map1 at the exit tunnel is similar to that of the homologs of Map1 involved in 60S subunit biogenesis, yeast Arx1 (associated with ribosomal export com- plex protein 1) and human EBP1 (ErbB3-binding protein 1) [32–35]. Moreover, Map1-C1 superimposes well with the bacterial Map visualized in a PDF-Map-70S ribosome complex from Escherichia coli [36] (S4 Fig). Thus, despite the rather low resolution, the reconstructions allowed us to fit a model of Map1 generated by AlphaFold 2 (AF2) [37] into the respective densities, thereby providing an idea of the overall positioning of Map1 with respect to the ribosome (Fig 1D). The fits were guided by high-resolution cryo-EM structures of ribosome-bound EBP1 [34,35,38] (S4 Fig). After fitting the human 80S-EBP1 models into our densities for an overall orientation determi- nation of Map1, we superimposed the AF2 model for Map1 and rigid body fitted it separately into isolated densities (Figs 1D and S5A–S5D). This resulted in positioning of the globular amino peptidase domain (APD) of Map1 below the peptide exit tunnel contacting ES27a and H59. Density for the APD spans from UAS II (comprising eL31 and eL22) to UAS I (compris- ing uL23 and uL29) (Fig 1E–1H) [29] and is thus in agreement with published cross-linking data [16]. In addition, density for the nascent chain was visible, representing a broad variety in composition and lengths of nascent chains obtained through the native pullout. Therefore, a possible influence of differences in the nature and length of the nascent chain on the two observed Map1 states cannot be addressed. In addition to the large APD, AF2 also predicts the structure of the two zinc finger (ZF) domains of Map1 at a position that coincides with extra density observed in our maps, locating the zinc fingers adjacent to ribosomal protein eL22 (Fig 1D–1F). As stated above, ES27a is the main contact site for Map1 to the 60S ribosomal subunit. ES27 consists of three A helices, which can change their position flexibly around the three-way junc- tion connecting rRNA helices H63, ES27a, and ES27b (nomenclature according to Petrov and colleagues [39]). The longest helix, ES27a, thereby undergoes the most severe conformational changes. In yeast, so far, two main positions are known, one with the tip of ES27a facing towards the L1 stalk of the 60S (L1-position) and one facing towards the peptide exit tunnel (exit-position) [31]. Cryo-EM structure of the Map1-ribosome complex https://doi.org/10.1371/journal.pbio.3001995.g001 classes and subjected them to focused 3D classification using a soft mask for the exit tunnel/ ES27a region. Two classes were enriched, showing a prominent globular, nonribosomal extra density attached to ES27a in different conformations below the peptide exit. These two classes differed mainly in the position of ES27a and its attached nonribosomal density, but not in the overall ribosomal state. Although the additional density could not be better resolved due to its classes and subjected them to focused 3D classification using a soft mask for the exit tunnel/ ES27a region. Two classes were enriched, showing a prominent globular, nonribosomal extra density attached to ES27a in different conformations below the peptide exit. These two classes differed mainly in the position of ES27a and its attached nonribosomal density, but not in the overall ribosomal state. Although the additional density could not be better resolved due to its PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001995 April 20, 2023 4 / 28 PLOS BIOLOGY Cryo-EM structures of ribosome-bound Map1 and NatB flexibility, based on its position, overall shape, and dimension, we assigned it to Map1 (Figs 1B, 1C, and S2). ES27a in the exit position serving as the principal binding site for Map1 is in agreement with deletion experiments where the tip of ES27a was shortened, leading to reduced levels of ribosome associated Map1 [17,18]. Furthermore, our assignment agrees with bio- chemical findings based on chemical cross-linking, showing Map1 close to uL29, a protein located adjacent to the tunnel exit [16]. While both classes could be refined to an overall resolution of 3.8 and 3.9 Å (referred to as Map1-C1-80S and Map1-C2-80S), respectively, local resolution of Map1 bound to ES27a was limited to 7 Å and below (S3 Fig). This indicates a high degree of flexibility of the Map1-ribo- some interaction, most likely owing in part to the flexibility of its binding partner ES27a, which can cover a continuous conformational space between tunnel exit site and L1 protuber- ance [31]. However, since local refinement attempts using the multibody approach in RELION also failed, we concluded that the flexibility of ES27a does not solely prevent higher resolution but that the binding of Map1 itself to ES27a is flexible. Accordingly, we were not able to gain higher local resolution required to build a molecular model for the regions comprising ES27a and Map1. PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001995 April 20, 2023 Cryo-EM structure of the NatB-ribosome complex We purified RNCs carrying a well-established NatB substrate as nascent chain, in which the free N-termi- nus ends with the amino acid sequence MDEL (RNCMDEL). The same sequence was used in form of a CoA-Ac-MDEL inhibitor for co-crystallization with Chaetomium thermophilum Naa20 [28]. High salt-washed RNCMDEL were reconstituted with an 18× molar excess of recombinantly purified NatB (Naa25/Naa20) and subjected to cryo-EM and single particle analysis (S7 Fig). 3D variability analysis in CryoSPARC and focused sorting on the exit tunnel region revealed classes with extra density accounting for the NatB complex associated with the 80S ribosome, yet displaying a high degree of compositional and conformational heterogeneity (S8 Fig). Classes containing additional density corresponding to NatB could be divided into two sets: one set with extra density for only one copy of NatB (NatB-1; consisting of Naa20-1 and Naa25-1) flexibly attached to ES27a and one set with additional density for a second NatB complex bound to UAS-II [29] (Fig 2A). The second NatB complex (henceforth referred to as NatB-2) generally exhibited low conformational variance in classes where it was present, and h h b ll l d T dd h f h S b d its interaction with the ribosome was well resolved. To address the fact that ES27a-bound NatB-1 exhibited greater conformational heterogeneity, we performed focused sorting on this density, revealing one class (9.645 particles) in which both NatB complexes showed secondary structure resolution. Here, the ES27a-bound NatB-1 complex was positioned in direct vicinity to NatB-2 and exhibited much lower conformational flexibility than in other classes. This class (class I) was refined to a final overall resolution of 3.8 Å (local resolution ranging from approx- imately 4 to 9 Å for the NatB complexes; S9 Fig, left panel), which unambiguously revealed the architecture of both NatB complexes. In addition, we subjected all particles containing NatB-2 (and flexible NatB-1) to 3D variability analysis focusing on the NatB-2 area, yielding a class (class II) containing particularly well-resolved NatB-2 (45.530 particles) and refined this class to an overall resolution of 3.1 Å (local resolution ranging from below 3 to 6 Å for NatB-2; S9 Fig, right panel). This revealed α-helical secondary structure in regions proximal to the ribosome and allowed us to unambiguously identify the disc-shaped α-helical tetratricopeptide repeat (TPR) containing Naa25 subunit for both ribosome-bound NatBs in class I (S5E–S5J Fig). Cryo-EM structure of the Map1-ribosome complex Interestingly, we observed ES27a-exit in two novel stabilized conforma- tions, when bound to Map1. Compared to ES27a-bound NatA complexes, in Map1 complexes, ES27a is rotated with the three-way junction as a pivot by 31 degrees for conformation 1 and by 19 degrees for conformation 2 (S6 Fig). 5 / 28 PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001995 April 20, 2023 PLOS BIOLOGY Cryo-EM structures of ribosome-bound Map1 and NatB To confirm the major contribution of ES27a to Map1 ribosome binding, we performed in vitro binding assays with purified Map1 and ribosome nascent chain complexes (RNCs) or RNaseI-treated RNCs (rtRNCs), as done before for NatA [25]. In rtRNCs, rRNA ESs are clipped off by the RNAseI treatment, as previously shown [25]. Map1 binding to rtRNCs was significantly reduced by about 60% when comparing to untreated RNCs, again confirming that ES27a is a major binding site for Map1 recruitment to the ribosomal exit site (Fig 1I). Taken together, our analysis shows that Map1 is bound to the 80S ribosome in the immedi- ate vicinity of the ribosomal tunnel exit mainly via a flexible association with the dynamic rRNA ES27a. This brings Map1 in an ideal position to act on nascent polypeptide chains as soon as they emerge from the ribosomal tunnel into the cytoplasm. Cryo-EM structure of the NatB-ribosome complex y p To gain further insight into the coordination between methionine cleavage and N-acetylation, we followed an in vitro reconstitution approach using purified components. We purified RNCs carrying a well-established NatB substrate as nascent chain, in which the free N-termi- nus ends with the amino acid sequence MDEL (RNCMDEL). The same sequence was used in form of a CoA-Ac-MDEL inhibitor for co-crystallization with Chaetomium thermophilum Naa20 [28]. High salt-washed RNCMDEL were reconstituted with an 18× molar excess of recombinantly purified NatB (Naa25/Naa20) and subjected to cryo-EM and single particle analysis (S7 Fig). 3D variability analysis in CryoSPARC and focused sorting on the exit tunnel region revealed classes with extra density accounting for the NatB complex associated with the 80S ribosome, yet displaying a high degree of compositional and conformational heterogeneity (S8 Fig). Classes containing additional density corresponding to NatB could be divided into two sets: one set with extra density for only one copy of NatB (NatB-1; consisting of Naa20-1 and Naa25-1) flexibly attached to ES27a and one set with additional density for a second NatB complex bound to UAS-II [29] (Fig 2A). The second NatB complex (henceforth referred to as NatB-2) generally exhibited low conformational variance in classes where it was present, and its interaction with the ribosome was well resolved. To address the fact that ES27a-bound NatB-1 exhibited greater conformational heterogeneity, we performed focused sorting on this density, revealing one class (9.645 particles) in which both NatB complexes showed secondary structure resolution. Here, the ES27a-bound NatB-1 complex was positioned in direct vicinity to NatB-2 and exhibited much lower conformational flexibility than in other classes. This class (class I) was refined to a final overall resolution of 3.8 Å (local resolution ranging from approx- imately 4 to 9 Å for the NatB complexes; S9 Fig, left panel), which unambiguously revealed the architecture of both NatB complexes. In addition, we subjected all particles containing NatB-2 (and flexible NatB-1) to 3D variability analysis focusing on the NatB-2 area, yielding a class (class II) containing particularly well-resolved NatB-2 (45.530 particles) and refined this class to an overall resolution of 3.1 Å (local resolution ranging from below 3 to 6 Å for NatB-2; S9 Fig, right panel). To gain further insight into the coordination between methionine cleavage and N-acetylation, we followed an in vitro reconstitution approach using purified components. PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001995 April 20, 2023 Cryo-EM structure of the NatB-ribosome complex We further noticed that the globular catalytic Naa20 subunits are less well resolved (when compared to Naa25), indicative of flexibility, especially in the ES27a-bound NatB-1. We then performed rigid-body fitting of an AF2 model which is highly similar to the crystal structure of C. albicans NatB (PDB 5K18) [26]. In brief, the 12 tetratrico (TPR-) repeats (α0-α29) of Naa25 together with its C-terminal helical domain (α30-α36) form a ring-like structure with N-and C-termini in close vicinity. Naa20 is located in and protrudes from a circular pocket formed by the PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001995 April 20, 2023 6 / 28 PLOS BIOLOGY Cryo-EM structures of ribosome-bound Map1 and NatB Fig 2. Cryo-EM structure and model of the NatB-ribosome complex. (A) Composite map showing cryo-EM structures of NatB in complex with translating 80S ribosomes (RNCMDEl) filtered according to local resolution. Isolated densities of NatB-1 (from class I) and NatB-2 (from class II) were extracted after the final refinement. Views are shown on the peptide tunnel exit (left; bottom view), rotated 70˚ horizontally (middle; front view), and rotated 60˚ vertically (right; side view). (B) Zoom on the peptide exit site showing the NatB-ribosome molecular model (NatB-1 and NatB-2) in front (left) and side views (right) as indicated in (A). Overview (upper left panel (C)) and zoomed views (D-F) focusing on the Naa25-2 ribosome contact sites. Interactions of (D) helix α35 at the C-terminus of Naa25-2 with the H100/101-and H94/98 junctions, (E) the Naa25-2 α34-α35 loop (Lys720) with Asp6 in the N-terminus of eL31, and (F) Lys791 and Arg794 at the very C-terminus (α36) of Naa25-2 with U3153 and U3293 within H94/98-junction are shown. NatB-1 was omitted for clarity. (G) Same view as (C) showing the model for the NatB-2 ribosome complex docked in density and highlighting Naa25-2 C-terminus (orange). (H) Model of the Naa25 C- terminus highlighting the position of the four positive patches (PPs). All patches contain two charged amino acids as indicated. Charge inversion double mutants were generated (PP1, PP3, PP4, and PPall). (I) Western blot analysis of sedimentation assays (triplicates) using recombinant wild-type or mutant NatB Fig 2. Cryo-EM structure and model of the NatB-ribosome complex. (A) Composite map showing cryo-EM structures of NatB in complex with translating 80S ribosomes (RNCMDEl) filtered according to local resolution. Isolated densities of NatB-1 (from class I) and NatB-2 (from class II) were extracted after the final refinement. Naa25 TPR repeats. This structure could be fitted with only minor adjustments into both NatB densities (S5E–S5J Fig and S1 Table). Here, the bases U3153 and U3293 within the H94/98-junction were contacted by Lys 791 and Arg794 at the very C-terminus of Naa25-2, likely via a cation-π stack. To test the contribution of the before-mentioned residues to ribosome binding, we selected positive patches (each patch containing two closely spaced basic amino acids) at the C-termi- nus of Naa25 (Fig 2G) and an unrelated positive patch in the same area (Lys 747 and Lys 751). We created double charge inversion mutants (Lys or Arg to Glu) for each patch (PP1 to PP4; Fig 2H) or for all patches (PPall) similar to as described in ref [41]. Purified wild type (wt) and mutant NatB complexes carrying an N-terminal His-tag on Naa25 (for western blot detection) were used for in vitro binding assays. To prevent any bias by a specific nascent chain, we chose purified idle 80S ribosomes over RNCs in these assays (S7 Fig). The western blot analysis showed that binding of NatB to 80S ribosomes was significantly reduced by mutation of K723E, K725E (positive patch PP1; 47% of wt binding) and K791E, R794E (PP4; 31%), The third site comprises bases at the junction of H94 and H98 that bind the very C-terminus (α36) of Naa25-2 (Fig 2F). Here, the bases U3153 and U3293 within the H94/98-junction were contacted by Lys 791 and Arg794 at the very C-terminus of Naa25-2, likely via a cation-π stack. To test the contribution of the before-mentioned residues to ribosome binding, we selected positive patches (each patch containing two closely spaced basic amino acids) at the C-termi- nus of Naa25 (Fig 2G) and an unrelated positive patch in the same area (Lys 747 and Lys 751). We created double charge inversion mutants (Lys or Arg to Glu) for each patch (PP1 to PP4; Fig 2H) or for all patches (PPall) similar to as described in ref [41]. Purified wild type (wt) and mutant NatB complexes carrying an N-terminal His-tag on Naa25 (for western blot detection) were used for in vitro binding assays. To prevent any bias by a specific nascent chain, we chose purified idle 80S ribosomes over RNCs in these assays (S7 Fig). Cryo-EM structure of the NatB-ribosome complex Views are shown on the peptide tunnel exit (left; bottom view), rotated 70˚ horizontally (middle; front view), and rotated 60˚ vertically (right; side view). (B) Zoom on the peptide exit site showing the NatB-ribosome molecular model (NatB-1 and NatB-2) in front (left) and side views (right) as indicated in (A). Overview (upper left panel (C)) and zoomed views (D-F) focusing on the Naa25-2 ribosome contact sites. Interactions of (D) helix α35 at the C-terminus of Naa25-2 with the H100/101-and H94/98 junctions, (E) the Naa25-2 α34-α35 loop (Lys720) with Asp6 in the N-terminus of eL31, and (F) Lys791 and Arg794 at the very C-terminus (α36) of Naa25-2 with U3153 and U3293 within H94/98-junction are shown. NatB-1 was omitted for clarity. (G) Same view as (C) showing the model for the NatB-2 ribosome complex docked in density and highlighting Naa25-2 C-terminus (orange). (H) Model of the Naa25 C- terminus highlighting the position of the four positive patches (PPs). All patches contain two charged amino acids as indicated. Charge inversion double mutants were generated (PP1, PP3, PP4, and PPall). (I) Western blot analysis of sedimentation assays (triplicates) using recombinant wild-type or mutant NatB PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001995 April 20, 2023 7 / 28 PLOS BIOLOGY Cryo-EM structures of ribosome-bound Map1 and NatB complex and idle (80S) or RNaseI-treated 80S ribosomes (rt80S). Top: fraction of NatB bound to ribosomes quantified by densitometric analysis of western blot images. Bottom: representative western blot displaying supernatant (SN) and pellet (P) fractions of two such experiments. See S1 Raw Images for all raw western blot images and S1 Data for numerical data underlying the quantification. complex and idle (80S) or RNaseI-treated 80S ribosomes (rt80S). Top: fraction of NatB bound to ribosomes quantified by densitometric analysis of western blot images. Bottom: representative western blot displaying supernatant (SN) and pellet (P) fractions of two such experiments. See S1 Raw Images for all raw western blot images and S1 Data for numerical data underlying the quantification. https://doi.org/10.1371/journal.pbio.3001995.g002 https://doi.org/10.1371/journal.pbio.3001995.g002 Naa25 TPR repeats. This structure could be fitted with only minor adjustments into both NatB densities (S5E–S5J Fig and S1 Table). Naa25 TPR repeats. This structure could be fitted with only minor adjustments into both NatB densities (S5E–S5J Fig and S1 Table). Overall, the two NatB densities cover the area below the 60S exit site spanning from ES27a to the second UAS for exit factors (eL31 and uL22) [29]. NatB-1 is anchored between H59 of 25S rRNA and the long arm of the ES27a A-helix (Fig 2B). Here, contacts are established by the loops of Naa25-1 N-terminal TPRs (TPR1 and 2) that are sandwiched between the two rRNA elements. Another contact to the ES27a tip is established by the TPR-helices of the Naa25-1 C-terminus (α34-α36). In this conformation, the catalytic subunit Naa20-1 faces towards the exit tunnel, but density is only visible at the well-conserved contact interface with Naa25-1 (including highly conserved Thr2 and Glu48 of Naa20 and Arg296 of Naa25) [40], indicating that it is largely delocalized. NatB-2 is anchored to the ribosomal surface somewhat offset from the tunnel exit of the 60S and is arranged such that the two catalytic Naa20 subunits face each other. In contrast to NatB-1, ribosomal contacts are in this case established mainly via rRNA but also to the ribo- somal protein eL31, yet involving only the C-terminal TPRs of Naa25-2 (Fig 2C). In detail, we identified three distinct interaction sites of Naa25-2 with the ribosomal exit site in the map after focused refinement on NatB-2 (class II): The first site was established around the junction of H100 and H101 of 25S rRNA and the N-terminal part of α35 of Naa25-2 (Fig 2D) that con- tains a series of positively charged amino acids (Lys725, Lys729, Lys732, and Lys736). The sec- ond contact site is established between the Naa25-2 Lys720, located in the loop between α34 and α35, and Asp6 of the N-terminus of ribosomal protein eL31 (Fig 2E). The third site comprises bases at the junction of H94 and H98 that bind the very C-terminus (α36) of Naa25-2 (Fig 2F). Here, the bases U3153 and U3293 within the H94/98-junction were contacted by Lys 791 and Arg794 at the very C-terminus of Naa25-2, likely via a cation-π stack. The third site comprises bases at the junction of H94 and H98 that bind the very C-terminus (α36) of Naa25-2 (Fig 2F). PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001995 April 20, 2023 Naa25 TPR repeats. This structure could be fitted with only minor adjustments into both NatB densities (S5E–S5J Fig and S1 Table). The absence of ESs indeed reduced the binding of NatB to 8% com- pared to NatB binding to untreated 80S ribosomes, confirming an important role of the ES for recruitment of both NatB copies to the ribosome (Fig 2I). We next compared ribosome-bound NatB-1 and NatB-2 with the NatA complex. Here, sev- eral observations were made. (i) The overall space occupied below the exit site is overlapping, indicating that in the observed conformations NatA and NatB can only bind exclusively (Fig 3A). (ii) The architecture of ribosome-bound NatB clearly differs from the NatA-ribo- some complex and displays a distinct 60S binding mode. NatA mainly employs 25S rRNA ES for 60S binding. Here, ES27a and ES39 anchor the auxiliary Naa15 (Nat1) subunit to the exit site, and Naa50 (Nat5)—which has no equivalent in NatB—makes a third contact to ES7. While NatB-1 also binds to ES27a, NatB-2 engagement of the 60S differs compared to NatA binding and does not involve ES27a or other ESs. (iii) Like Naa10 (Ard1), the catalytic subunit of the NatA complex, both Naa20-1 and Naa20-2 of NatB have no direct contact to the ribo- some. We further note that Naa20-2 is better resolved, while Naa20-1, apart from the contact site with Naa25-1, is largely delocalized. Notably, a rigid body fit of the NatB-2 model into NatB-1 would lead to a clash between Naa20-1 and Naa25-2, indicating that Naa20-1 needs to adjust its orientation with respect to Naa25 compared to NatB-2 (and the X-ray structure [26]). Nevertheless, in order to assess their principal potential to contribute catalytic activity, we compared this rigid-body fit of Naa20-1 with our models for Nat20-2 and Naa10 of NatA, since it represents a sufficiently accurate approximation of the overall position of Naa20-1 (Figs 3C and S10). Interestingly, it would require roughly the same minimum length of the nascent chain of about 55 amino acids to reach either one of the catalytic centers, assuming a direct path from the 30-CCA end of the tRNA to the tunnel exit and from there into the Naa20 catalytic center (30 aa inside and 25 aa outside the exit tunnel) (Fig 3C). Naa20-2 is oriented similarly to Naa10 of NatA [25] with respect to the position of acetyl-CoA and accessibility for the nascent chain N-terminus (Fig 3B and 3C). Naa25 TPR repeats. This structure could be fitted with only minor adjustments into both NatB densities (S5E–S5J Fig and S1 Table). The western blot analysis showed that binding of NatB to 80S ribosomes was significantly reduced by mutation of K723E, K725E (positive patch PP1; 47% of wt binding) and K791E, R794E (PP4; 31%), whereas K747E, K751E that in our structure are not directly involved in ribosome binding showed only a very weak effect (PP3; 83%). When all positive patches (PPall) were mutated, binding was almost entirely abolished (6% of wt binding), confirming the contribution of these positive patches to the interaction of NatB with the ribosome (Fig 2I). This indicated that the positive charges on the surface of the Naa25 C-terminus have an additive effect on ribo- some binding by establishing a composite binding patch for rRNA interaction. The results of these binding assays confirm the significance of the described interaction patches between Naa25 and the ribosome. Whereas in Naa25-1, these positively charged amino acids interact with ES27a, in Naa25-2, they enable the binding to H94/H98 and H100/H101 junctions (Fig 2D–2F). p p g whereas K747E, K751E that in our structure are not directly involved in ribosome binding showed only a very weak effect (PP3; 83%). When all positive patches (PPall) were mutated, binding was almost entirely abolished (6% of wt binding), confirming the contribution of these positive patches to the interaction of NatB with the ribosome (Fig 2I). This indicated that the positive charges on the surface of the Naa25 C-terminus have an additive effect on ribo- some binding by establishing a composite binding patch for rRNA interaction. The results of these binding assays confirm the significance of the described interaction patches between Naa25 and the ribosome. Whereas in Naa25-1, these positively charged amino acids interact with ES27a, in Naa25-2, they enable the binding to H94/H98 and H100/H101 junctions (Fig 2D–2F). 8 / 28 PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001995 April 20, 2023 PLOS BIOLOGY Cryo-EM structures of ribosome-bound Map1 and NatB Interestingly, as observed for NatA and also Map1, in the class showing the stable assembly with two NatB complexes (class I), ES27a adopts a specific conformation. Compared to the Map1-C1 position of ES27a that is closest to the tunnel exit, the NatB-bound position is rotated 37˚ away from the tunnel exit (S6 Fig). We thus assessed the contribution of ESs to NatB binding by performing quantitative bind- ing assays using empty 80S or RNAse-I-treated 80S (depleted of ES as described in [25] and for Map1 in Fig 1I) (S7 Fig). PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001995 April 20, 2023 Naa25 TPR repeats. This structure could be fitted with only minor adjustments into both NatB densities (S5E–S5J Fig and S1 Table). While the substrate could enter Naa20-2 in a straight path from the tunnel exit, it would need to form a turn to reach into the center of Naa20-1, the entrance to which is located on the lateral side (S10 Fig). Thus, both copies of Naa20 (in NatB- 1 and in NatB-2) could in principle be catalytically active. Yet, taking into account the delocali- zation and high degree of flexibility of Naa20-1 with respect to its auxiliary subunit in contrast to the more stably positioned Naa20-2, and given the more direct path that the nascent chain can take to enter Naa20-2, we speculate that Naa20-2 rather than Naa20-1 would act to N-acet- ylate most NatB substrates. While binding of NatA and NatB appears mutually exclusive we wondered to what extent concomitant binding of Map1 would be sterically allowed. Unlike stated before [25], compari- son of the binding modes reveals that Map1 in both C1 and C2 positions would possibly clash with NatA, although clashes between Map1-C1 and NatA would be rather minor. Yet, both NatB complexes, especially the ES27a-bound NatB-1, would severely overlap with both observed Map1 positions (S11 Fig). Thus, this comparison is rather suggestive for competitive binding of Map1 and NATs. This notion is further supported by the observation that ES27a orientations are apparently different for every ligand. 9 / 28 PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001995 April 20, 2023 PLOS BIOLOGY Cryo-EM structures of ribosome-bound Map1 and NatB Fig 3. Comparison of ribosome-bound NatB and NatA complexes. (A) Bottom view (upper panel) and front view (lower panel) showing an overlay of the NatB-1 (pink) and NatB-2 (orange) ribosome structure with isolated densitie for ribosome-bound NatA (bright green) (EMD-0201; [25]). (B) Comparison of positions for the NatA and NatB-2 catalytic subunits (Naa10 and Naa20) with respect to the 60S subunit shown as front and top view. The position of acetyl-CoA (Ac-CoA) and a putative model for the nascent chains is shown. For clarity, only Naa20 of NatB-2 is shown. (C) Left panel: cut front view of the NatB ribosome cryo-EM map highlighting the nascent polypeptide chain and the position NatB-1 and NatB-2 (left panel). Right panels: Zoom-in views highlighting the catalytic Naa20-2 subunit and illustration of the minimal distance that a nascent chain has to span to reach the active site of Naa20-2. https://doi.org/10.1371/journal.pbio.3001995.g003 Fig 3. Comparison of ribosome-bound NatB and NatA complexes. PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001995 April 20, 2023 https://doi.org/10.1371/journal.pbio.3001995.g003 Discussion During translation of a nascent polypeptide, various factors are dynamically interacting with the ribosomal peptide exit site to probe the biochemical and biophysical properties of the emerging nascent chain. For example, the activity of the modifying enzymes Map1 and the NATs is dependent on the properties of the amino acids following the N-terminal methionine. The ribosome-associated complex RAC containing the Hsp70 homolog Ssz1 binds various (partially unfolded) nascent chains, whereas the SRP recognizes hydrophobic, partially helical N-terminal signal sequences. Commonly, all these factors are able to interact with the ribosome employing fast on- and off-rates even in the absence of the nascent chain in order to scan the ribosome for the emerg- ing nascent chain substrate. Yet, a number of structural studies showed that binding sites for most exit site factors on the ribosome are overlapping. Based on those structures, neither NatA nor RAC or SRP can bind together to the exit site, at least not in the observed conformations. This implies a dynamic and sequential or collaborative ribosome interaction and activity of these factors on the nascent chain dependent on the presence of the cognate nascent chain N- terminus substrate, which will change the apparent off-rates of the respective factors (see scheme in Fig 4). This is best documented for SRP, which, after an initial encounter, remains bound to the RNC for the entire targeting cycle, but only after engaging a sufficiently hydro- phobic signal sequence [42–44]. This study investigating ribosome binding for Map1 and NatB expands our knowledge on how exit site factors on the ribosome may be orchestrated by the dynamic rRNA expansion segment ES27a. We noted that, as observed for the NatA complex, Map1 and NatB directly bind to ES27a and intriguingly position the rRNA A-helix in a very specific conformation with respect to the tunnel exit (see S6 Fig). Map1, which cleaves the N-terminal methionine, is likely one of the earliest nascent chain binders, since cleavage at least for bacterial MAP has been shown to occur as soon as the nascent chain has reached a length of 44 amino acids [49]. We find it mainly bound to ES27a and were able to enrich two distinct conformations positioning the Map1-bound tip of ES27a closer to the tunnel exit than observed with NatA. In contrast, when bound to the two NatB complexes, ES27a moves even further away from the tunnel exit than in case of NatA binding. Naa25 TPR repeats. This structure could be fitted with only minor adjustments into both NatB densities (S5E–S5J Fig and S1 Table). (A) Bottom view (upper panel) and front view (lower panel) showing an overlay of the NatB-1 (pink) and NatB-2 (orange) ribosome structure with isolated densities for ribosome-bound NatA (bright green) (EMD-0201; [25]). (B) Comparison of positions for the NatA and NatB-2 catalytic subunits (Naa10 and Naa20) with respect to the 60S subunit shown as front and top view. The position of acetyl-CoA (Ac-CoA) and a putative model for the nascent chains is shown. For clarity, only Naa20 of NatB-2 is shown. (C) Left panel: cut front view of the NatB ribosome cryo-EM map highlighting the nascent polypeptide chain and the position NatB-1 and NatB-2 (left panel). Right panels: Zoom-in views highlighting the catalytic Naa20-2 subunit and illustration of the minimal distance that a nascent chain has to span to reach the active site of Naa20-2. https://doi.org/10.1371/journal.pbio.3001995.g003 10 / 28 PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001995 April 20, 2023 PLOS BIOLOGY Cryo-EM structures of ribosome-bound Map1 and NatB PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001995 April 20, 2023 Discussion We thus hypothesize that ES27a, by adopting exit factor-specific conformations, plays a role in providing specificity and probably exclusive binding for the dif- ferent factors. Apart from the ES27a interaction, most exit site factors employ electrostatic interactions to bind to 25S/28S rRNA. For example, positively charged patches are present in NAC [29,48,50], NatA, bacterial Map [49], and the RAC/Ssb1 complex [46,51,52]. Interestingly, the precise electrostatic interaction sites on the ribosome differ from factor to factor. Both NatA and NatB employ charged patches on their TPR-repeat containing subunits. Naa15 of NatA contacts a binding pocket formed by rRNA H24 and H47 close to eL31 with its charged N-terminus, and the region close to ES39 (H98-H100 region) with a patch of lysines on its C-terminus. As described above, both Naa25 subunits in our structure employ positively charged patches to engage with rRNA, whereby Naa25-2 binds to a similar but not the same region of the exit site (see Fig 2C–2F) as Naa15 (NatA), thus showing a different specific binding pattern. We propose a model according to which the exit site factors Map1, NatA, and NatB all require an interaction with the flexible arm of ES27a. In addition, however, binding of these factors to the ribosomal exit site specifically positions both the factors themselves and also ES27a in distinct states, thereby enabling probing of the nature of the nascent chain as potential substrates. We thus speculate that, in analogy to the mobile P-stalk for translation factors [53–55], ES27a could be a primary binding hub for modifying enzymes, maintaining them in close PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001995 April 20, 2023 11 / 28 PLOS BIOLOGY Cryo-EM structures of ribosome-bound Map1 and NatB Fig 4. Scheme depicting the possible interplay of ribosome exit site factors. The translating ribosome exposing a nascent polypeptide chain is engaged by primary exit site factors (NAC, Map1, NatB, SRP) depending on the properties of the emerging chain’s N-terminus. After these primary factors have performed their activity, secondary factors (e.g., NatA, RAC, Sec61) gain access to the nascent chain. Whereas some of them can coexist on the ribosome (e.g., NatA and Map1-C1 or NAC and SRP), others cannot or have to bind sequentially (e.g., NatA and NatB). ES: expansion segment, RNC: ribosome-nascent chain complex. The tunnel exit is indicated by a yellow dotted circle. Color codes for factors and entry codes for the electron microscopy database (EMD) are given below. Discussion Reference for EMD 2844 is [45], for EMD 6105 is [46], for EMB 1651 is [47], and EMD 4938 is [48]. https://doi.org/10.1371/journal.pbio.3001995.g004 Fig 4. Scheme depicting the possible interplay of ribosome exit site factors. The translating ribosome exposing a nascent polypeptide chain is engaged by primary exit site factors (NAC, Map1, NatB, SRP) depending on the properties of the emerging chain’s N-terminus. After these primary factors have performed their activity, secondary factors (e.g., NatA, RAC, Sec61) gain access to the nascent chain. Whereas some of them can coexist on the ribosome (e.g., NatA and Map1-C1 or NAC and SRP), others cannot or have to bind sequentially (e.g., NatA and NatB). ES: expansion segment, RNC: ribosome-nascent chain complex. The tunnel exit is indicated by a yellow dotted circle. Color codes for factors and entry codes for the electron microscopy database (EMD) are given below. Reference for EMD 2844 is [45], for EMD 6105 is [46], for EMB 1651 is [47], and EMD 4938 is [48]. Fig 4. Scheme depicting the possible interplay of ribosome exit site factors. The translating ribosome exposing a nascent polypeptide chain is engaged by primary exit site factors (NAC, Map1, NatB, SRP) depending on the properties of the emerging chain’s N-terminus. After these primary factors have performed their activity, secondary factors (e.g., NatA, RAC, Sec61) gain access to the nascent chain. Whereas some of them can coexist on the ribosome (e.g., NatA and Map1-C1 or NAC and SRP), others cannot or have to bind sequentially (e.g., NatA and NatB). ES: expansion segment, RNC: ribosome-nascent chain complex. The tunnel exit is indicated by a yellow dotted circle. Color codes for factors and entry codes for the electron microscopy database (EMD) are given below. Reference for EMD 2844 is [45], for EMD 6105 is [46], for EMB 1651 is [47], and EMD 4938 is [48]. properties of the emerging chain s N terminus. After these primary factors have performed their activity, secondary factors (e.g., NatA, RAC, Sec61) gain access to the nascent chain. Whereas some of them can coexist on the ribosome (e.g., NatA and Map1-C1 or NAC and SRP), others cannot or have to bind sequentially (e.g., NatA and NatB). ES: expansion segment, RNC: ribosome-nascent chain complex. The tunnel exit is indicated by a yellow dotted circle. Color codes for factors and entry codes for the electron microscopy database (EMD) are given below. Discussion Reference for EMD 2844 is [45], for EMD 6105 is [46], for EMB 1651 is [47], and EMD 4938 is [48]. https://doi.org/10.1371/journal.pbio.3001995.g004 vicinity to the nascent chain substrates for probing their properties. More rigid ribosome bind- ing can then occur via factor-specific interactions mainly involving electrostatic interactions with the exit site rRNA, leading to both specific fixation of ES27a and temporary exclusion of other competing exit site factors. Substrates of NatA-mediated N-acetylation require prior removal of the first methionine by Map1, whereas for NatB activity, the N-terminal methionine still needs to be present. Interest- ingly, in our structures, we observe two copies of NatB, one rigidly bound next to the peptide exit tunnel (at UAS-II) with no contact to ES27a, and one more flexibly bound mainly to PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001995 April 20, 2023 12 / 28 PLOS BIOLOGY Cryo-EM structures of ribosome-bound Map1 and NatB ES27a. We further show that presence of ES27a is necessary for both NatBs to bind to ribosomes (Fig 2I), since removal of ES27a by RNaseI almost completely abolishes ribosome binding. This suggests that spatial constraining and correct positioning of NatB-2 (which does not contact ES27a) is likely to depend on the presence of NatB-1 and ES27a. This is further supported by the observation that, during classification, all classes that showed NatB-2 also contained extra density for NatB-1 at ES27a, whereas not all classes with NatB-1 showed NatB-2. This opens the possibility that NatB-1 binds first to the ribosome via ES27a and is needed to stably position NatB-2 next to the tunnel exit. This would be analogous to a recent study showing that exit fac- tors such as NAC and SRP can in principle cooperate [56]. For cotranslational ER-targeting, NAC acts as a gatekeeper to shield nascent chains, which are not substrates for SRP, while facili- tating recruitment of SRP to the ribosome. Another study shows that the Translocon Associated Protein (TRAP) may help recruiting ribosomes to the ER and subsequently aids in stabilizing the RNC-Sec61 complex and contributes to membrane protein biogenesis [57]. In contrast to NatB, concomitant binding of NatA and Map1-C1, but not Map1-C2, is still possible since they would barely sterically clash (S11 Fig). Interestingly, bacterial Map also occupies two different positions on the ribosomal surface close to the exit tunnel, only one of which allows binding of peptidyl-deformylase (PDF) at the same time [36]. Discussion Given that yeast Map1 is bound to the ribosome mainly via ES27a and its position is dependent on ES27a movement, we favor a model for a Map1-NatA interplay, in which ES27a orchestrates a sequential action of Map1 and NatA on their substrate. This is in line with a failure of all our attempts to visualize Map1 and NatA together on the ribosome. A possible reason for the somewhat puzzling observation of two NatB copies on the ribo- some might reflect a function in efficient discrimination between Map1, NatA, and NatB sub- strates. Among the cotranslationally acting nascent chain modifying factors discussed in this study, Map1 is the most abundant protein (average copy number per cell according to the Sac- charomyces Genome Database (www.yeastgenome.org; 14,218 +/−5,474) followed by NatA (Naa15; 8,398 +/−4,076) and NatB (Naa25; 5,693 +/−1,503). Thus, the likelihood for an RNC to be probed by Map1 or NatA is higher. If, however, the first NatB binds to RNCs with a vacant ES27a, it would automatically exclude Map1 or NatA (re) binding and thereby prime this RNC as a possible substrate for the second, likely the catalytically active, NatB-2. At this point, however, we cannot clearly decide whether both or only one of the NatB copies are active in modifying nascent chains. Taking into account the observed conformational distor- tion of Naa20 of NatB-1 and the rather obscured path of the nascent chain to its catalytic sub- unit, we speculate that NatB-2 may be the more active complex that provides the majority of the modifying activity. Taken together, we propose that the primary and secondary interactors of the nascent chain (see Fig 4) may follow either a collaborative (as in case of NAC and SRP) or a sequential mode (as in case of Map1 and NatA) for the productive interplay of the various modifying enzymes. At least for the nascent chain modifying enzymes, ES27a plays a central role in their recruitment to and orchestration at the ribosomal peptide exit site. However, for a complete understanding, a quantitative assessment of the kinetic properties of the different modifying, chaperoning, and targeting factors with respect to nascent chain-dependent RNC binding and dissociation will be necessary. Purification of recombinant Map1 Map1 containing an N-terminal His8-tag followed by a linker and a HRV 3C cleavage site was expressed from a pET28a vector. E. coli cells were harvested by centrifugation in a SCL6000 13 / 28 PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001995 April 20, 2023 PLOS BIOLOGY Cryo-EM structures of ribosome-bound Map1 and NatB rotor (Sorvall) for 10 min at 4,500 rpm and 4˚C. After washing with 1× PBS, the pellet was resuspended in cold lysis buffer (50 mM Tris (pH 8.0), 500 mM NaCl, 1 mM PMSF, 1 EDTA- free protease inhibitor cocktail pill/50 ml (Roche)) and subjected to mechanical lysis using a Microfluidizer (Micro Fluidics). Clarified lysate was obtained after spinning for 15 min at 15,000 rpm in a SS34 rotor (Sorvall) at 4˚C. After filtering the lysate, it was applied on a HisTrap HP column (GE Healthcare) equilibrated with HT-20 buffer (50 mM Tris (pH 7.5), 500 mM NaCl, 20 mM imidazol, 10 mM β-mercaptoethanol). After elution with HRV 3C pro- tease under high salt conditions, the main fractions were loaded onto a SP FF column (GE Healthcare) equilibrated with 20 mM HEPES at pH 7.0. Elution was performed by a linear salt gradient up to 1 M NaCl, the main fractions showing purified Map1 protein were pooled and the buffer was adjusted to 20 mM HEPES (pH 7.5), 100 mM KOAc, 2.5 mM Mg(OAc)2, 1 mM DTT, 0.5 mM PMSF, 10 μg/ml cycloheximide including a protease inhibitor cocktail tablet (Roche). Purification of recombinant NatB complexes Naa25 containing an N-terminal His8-tag followed by a linker and a HRV 3C cleavage site was expressed from the MCS1 of pRSFDuet-1 vector (Novagen). A gene encoding a codon-opti- mized version of catalytically inactive E25A and H74A double mutant of Naa20 [26] for E. coli expression was synthesized by Eurofins. An additional M36L mutation was introduced to pre- vent internal translation initiation. This modified NAA20 gene was cloned into the MCS2 of the same pRSFDuet-1 vector. To overcome a disproportion of auxiliary to catalytic subunit, Naa20 was also cloned into pET21a separately and was used for coexpression with the dimeric NatB construct. After transformation of both plasmids into E. coli BL21(DE3) cells, cultures were grown in LB medium and induction was performed with 1 mM IPTG at 16˚C overnight. Harvesting and lysate preparation was done as described for Map1. NatB and its mutants (PP1 to PPall) were purified via Ni-NTA following the manufacturer’s protocol with the exception that after washing proteins were eluted in 50 mM MES (pH 6.0), 500 mM NaCl, and 500 mM imidazole. Eluted NatB was subsequently subjected to size exclu- sion chromatography on a Superdex 200 (GE Healthcare) in GF buffer (10 mM MES (pH 6.0), 400 mM KOAc, 5 mM Mg(OAc)2, and 1 mM DTT). NatB containing fractions were pooled and concentrated using Ultra-4 centrifugal filter devices (Amicon, MWCO 50 kDa) and stored in GF buffer at −80˚C. For the cryo-EM sample, the His-tag was removed by 3C protease. About 200 μg of His8- NatB in GF buffer were incubated with 25 μg His6-3C protease for 45 min at 20˚C on a turning wheel. Cleaved His8-tag and His6-3C protease were removed using 10 μl of magnetic beads (His-tag Isolation & Pulldown; Thermo Fisher), and the supernatant was used for reconstitu- tion of the NatB-RNCMDEL complex for single particle analysis. PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001995 April 20, 2023 Generation and purification of charge inversion mutant NatB complexes Several positive patches on Naa25 were identified as potential candidates for ribosome bind- ing: patch1 containing K723 and K725, patch2 containing K729 and K736, patch3 containing K747 and K751, and patch4 containing K791 and R794. We generated charge inversion mutants of all four positive patches (PP1 to PP4) mutated to E (similar to Magin and col- leagues [41]), as well as one mutant with all PP amino acids mutated to E. Mutations were per- formed using a site-directed mutagenesis kit (New England BioLabs). Mutation in the DNA sequence were introduced by PCR. To that end, the pRSFDuet-1 vector harboring the NAA25 insert (see above) was amplified using primers introducing charge inversions. Site-directed PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001995 April 20, 2023 14 / 28 PLOS BIOLOGY Cryo-EM structures of ribosome-bound Map1 and NatB mutagenesis was performed according to the manufacturer’s manual. Expression and purifica- tion were performed in the same way as described above. Mass spectrometry of native Map1-ribosome complexes Proteins were in-gel reduced for 30 min at 55˚C using 45 mM dithioerythritol (DTE) in 50 mM NH4HCO3. Cysteines were carbamidomethylated for 30 min at room temperature in 100 mM iodoacetamide/50 mM NH4HCO3. In-gel digestion was performed at 37˚C overnight with 70 ng porcine trypsin (Promega, Fitchburg, WI, USA). Peptides were extracted using 70% ACN. Prior to liquid chromatography, the samples were dried using a SpeedVac vacuum con- centrator. Peptide chromatography was performed on an Ultimate 3000 nano-LC system (Thermo Fisher Scientific) using an EasySpray separation column (PepMap RSLC C18, 50 cm length, 75 μm ID, Thermo Fisher Scientific) at a flow rate of 250 nl/min. As solvent A 0.1% for- mic acid was used. The chromatography method consisted on gradients from 3% to 25% sol- vent B (0.1% formic acid in acetonitrile) in 30 min and from 25% to 40% B in 5 min. Mass spectrometry was performed on a Q Exactive HF-X mass spectrometer (Thermo Fisher Scien- tific) using a top 12 data-dependent acquisition method. Spectra were searched using MAS- COT V2.4 (Matrix Science, London, UK) and the S. cerevisiae subset of the UniProt database. The results were filtered for an FDR <1%. Purification of native Map1-ribosome complexes For native pullouts of Map1-ribosome complexes, a S. cerevisiae strain expressing C-terminally tandem affinity purification (TAP)-tagged Map1 from Euroscarf (genotype SC0000; MATa; ura3-52; leu2-3,112; YLR244c::TAP-KlURA3; accession number SC1694) was used. Cells were grown in YPD medium to an OD600 of 4.0 and 5 g of wet cells were resuspended in lysis buffer (LB-2.5; 20 mM HEPES (pH 7.5), 100 mM KOAc, 2.5 mM Mg(OAc)2, 1 mM DTT, 0.5 mM PMSF, 10 μg/ml cycloheximide, protease inhibitor cocktail tablet (Roche)). Cell disruption was performed using a Freezer Mill (6970 EFM). The powder was resuspended in 15 ml LB- 2.5 and the lysate spun for 15 min at 4˚C in an SS-34 rotor (Sorvall) at 15,000 rpm to clarify the lysate. The SN was loaded onto several 600 μl sucrose cushions (750 mM sucrose in LB- 2.5) and centrifuged for 1 h at 100,000 rpm in a TLA 100.3 (Sorvall) at 4˚C. The pellets were resuspended in LB-2.5 and pooled for subsequent TAP purification. Approximately 150 μl of magnetic IgG-coupled Dynabeads M-270 Epoxy (Life Technologies) were equilibrated with 300 μl LB-2.5 containing 0.5% TritonX-100 (LB-2.5+T) twice and added to the pooled sample. The sample was incubated with the beads for 1 h at 4˚C on a rotating wheel, harvested on a magnet, and resuspended in 500 μl LB-2.5+T. After three washing steps with 500 μl LB-2.5 and three washing steps with 500 μl LB-2.5 (sample: W4-6), ribosome-Map1 complexes were eluted in 120 μl LB-2.5 containing 70 units of Ac-TEV protease (Thermo Fisher) for 1 h at 20˚C. A concentration of 14 A260/ml was measured on the NanoDrop (Implen). Samples were subse- quently analyzed on a 12% Nu-PAGE gel followed by western blot analysis. In vitro translation and purification of ribosome nascent chain complex Ribosome nascent chain complexes were purified after in vitro translation of an mRNA reporter in a cell-free yeast translation extract. For reconstitution with Map1, the previously described truncated uL4 construct [25] containing an N-terminal His8-HA tag for purification and immunoblotting followed by a TEV cleavage site, the first 64 amino acids of uL4 and a “CMV” stalling sequence was used (His-HA-TEV-CMV-uL4 mRNA). For reconstitution with NatB, the abovementioned construct was modified to code for a NatB substrate. The first five PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001995 April 20, 2023 15 / 28 PLOS BIOLOGY Cryo-EM structures of ribosome-bound Map1 and NatB residues of uL4 were replaced by a MDEL sequence, which is preceded by a His8-V5 tag fol- lowed by a Factor Xa cleavage site. His-V5-Xa-CMV-uL4 mRNA and His-HA-TEV-CMV-uL4 mRNA was produced using the T7 Message Machine Kit (Thermo Fisher). For preparation of uL4-CMV-RNCs or uL4-CMV-RNCMDEL, ribosomes were programmed using a yeast cell-free translation extract, either from ski2Δ cells (for uL4-RNC) or BY4741 cells (for uL4-RNCMDEL). The in vitro trans- lations were performed at 17˚C for 75 min as described before and stopped by adding 200 μg/ ml cycloheximide (only for uL4-CMV-RNCs). The uL4-CMV-RNCs were affinity purified using magnetic Ni-NTA beads (Dynabeads). For this, the translation reaction was mixed with preequilibrated Dynabeads in 250 buffer (50 mM Tris/HCl (pH 7.0), 250 mM KOAc, 25 mM Mg(OAc)2, 5 mM β-mercaptoethanol, 250 mM sucrose, 10 μg/ml cycloheximide, 0.1% Nikkol, 0.1% EDTA-free protease inhibitor cocktail pill (Roche), 0.1% SUPERase-In, 20 U/l (Thermo Fisher)) containing 10 μg ml−1 yeast tRNA mix (Sigma-Aldrich) for 15 min at 4˚C, using a 800 μl slurry of beads for a 1,250-μl sample. The bead resin was washed 3 to 4 times with 250 buffer. Elution was performed using 250 buffer with 350 mM imidazole over the course of 5 min. The sample was loaded onto 400 μl of high salt sucrose cushion (1 M sucrose 50 mM Tris/HCl (pH 7.0), 500 mM KOAc, 25 mM Mg(OAc)2, 5 mM β-mercaptoethanol, 10 μg/ml cycloheximid, 0.1% Nikkol, 0.1% EDTA-free protease inhibitor cocktail pill (Roche)), and ribosomes were pelleted by centrifugation using a TLA 120.2 rotor (Beckman) for 45 min at 100,000 rpm and 4˚C and resuspended in 30 μl 250 buffer on ice for 30 min while shaking. Subsequently, the N-terminal His-HA-tags were cleaved using TEV protease in 250 buffer for 45 min at room temperature. In vitro translation and purification of ribosome nascent chain complex The mixture was again spun through a 600-μl sucrose cushion in a TLA 100 rotor (Beckman) for 45 min at 100,000 rpm and 4˚C. Afterwards, the pellet was resuspended in 30 μl grid buffer (20 mM Tris/HCl (pH 7.0), 50 mM KOAc, 2.5 mM Mg(OAc)2, 1 mM DTT, 125 mM Sucrose, 100 μg/mL cycloheximide, 0.05% Nikkol) on ice while shaking for 30 to 45 min. uL4-CMV-RNCMDEL were purified as described above with following modifications: In all buffers, 50 mM HEPES/KOAc (pH 7.5) was used instead of Tris/HCl (pH 7.0), and cyclohexi- mide was omitted from all buffers. Before elution, an additional high-salt wash was added (with 50 mM HEPES/KOAc (pH 7.5), 500 mM KOAc, 25 mM Mg(OAc)2, 5 mM β-mercap- toethanol, 250 mM sucrose, 0.1% Nikkol, 0.1% EDTA-free protease inhibitor cocktail pill (Roche)). After centrifugation, the ribosomal pellet was resuspended in Factor Xa cleavage buffer (20 mM HEPES/KOH, 150 mM KOAc, 5 mM Mg(OAc)2, 5 mM Ca(Cl)2 125 mM Sucrose, 5 mM β-mercaptoethanol, 0.1% Nikkol). To cleave the His8-V5 tag and to obtain the free MDEL N-terminus, Factor Xa protease was added to resuspended RNCs to a final concen- tration of 0.25 mg/ml, and the sample was incubated for 3 h at room temperature on a rotating wheel. Subsequently, the reactions were spun again through the high salt sucrose cushion (see above) uL4-CMV-RNCMDEL (in short RNCMDEL), and pellets we resuspended in grid buffer (20 mM HEPES/KOH, 100 mM KOAc, 5 mM Mg(OAc)2, 125 mM sucrose, 5 mM β-mercap- toethanol, 0.05% Nikkol). RNase I treatment of 80S ribosomes and ribosome nascent chain complexes For selective removal of eukaryotic specific expansion segments, RNCs or nonprogrammed ribosomes (80S) were incubated with 40 U RNase I (Thermo Fisher) per 1 A260 unit of ribo- somes for 45 min at 25˚C. The reaction was stopped with 0.5 U SUPERase-In (Thermo Fisher) RNase inhibitor per 1 U of RNase I, and the sample was placed on ice. The RNase I-treated 80S and RNCs are referred to as rt80S and rtRNC, respectively. In vitro reconstitution of Map1-ribosome complexes To characterize binding of Map1 to ribosomes, Map1 was in vitro reconstituted with uL4-RNC or RNaseI-treated uL4-RNC (rtRNC). Binding reactions were performed using 2 pmol of ribosomes (80 nM) and a 30-fold molar excess (2,4 μM) of purified Map1. The binding buffer was adjusted to a final concentration of 50 mM Tris (pH 7.0), 150 mM KOAc, and 2.5 mM Mg(OAc)2. After preincubation of all components but ribosomes for 5 min on ice, the 2 pmol ribosomes were added and the samples were incubated for 15 min at room temperature. The reaction was loaded onto 600 μl sucrose cushion (750 mM sucrose, 20 mM HEPES (pH 7.5), 150 mM KOAc, 2.5 mM Mg(OAc)2, 1 mM DTT) and spun for 2.5 h in a SW55Ti rotor (Beckman) at 40,000 rpm and 4˚C. The tubes were immediately frozen in liquid nitrogen and cut at 1/3 from the bottom. The upper 2/3 contained the supernatant (SN), whereas the lower 1/3 contained the pellet fraction (P). After TCA precipitation, Map1 binding to ribosomes was analyzed by SDS-PAGE after staining with SimplyBlue SafeStain (Thermo Fisher), and densi- tometry was performed using ImageJ version 1.53Q. See S1 Raw Images for all raw gel images and S1 Data for numerical data underlying the densitometric quantification. Purification of nonprogrammed 80S ribosomes Idle, nonprogrammed 80S ribosomes were purified from S. cerevisiae W303-1a cells. Cells were harvested at logarithmic growth, resuspended in lysis buffer (20 mM HEPES-KOH (pH 7.5), 100 mM KOAc, 10 mM Mg(OAc)2, 1 mM DTT, protease inhibitor (Roche)), and opened up using a French press. Cell debris was separated by centrifugation (SS34 rotor, 15,000 rpm, 20 min at 4˚C). The supernatant was cleared by another centrifugation (Ti70 rotor, 37,000 rpm, 30 min at 4˚C) resulting in an “S100 extract.” About 3 ml of this S100 supernatant were PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001995 April 20, 2023 16 / 28 PLOS BIOLOGY Cryo-EM structures of ribosome-bound Map1 and NatB loaded on 1 ml sucrose cushion (20 mM HEPES-KOH (pH 7.5), 500 mM KOAc, 10 mM Mg(OAc)2, 1 mM DTT, protease inhibitor (Roche), and 1.5 M sucrose), and ribosomes were pelleted (TLA110 rotor, 100,000 rpm, 1 h, 4˚C). The pellet was resuspended in 300 μl Buffer A (20 mM HEPES-KOH (pH 7.5), 500 mM KOAc, 12.5 mM Mg(OAc)2, 1 mM DTT), mixed with an equal volume of 2× puromycin buffer (20 mM HEPES-KOH (pH 7.5), 500 mM KOAc, 12.5 mM Mg(OAc)2, 1 mM DTT, 2 mM puromycin, and 0.01% RNasin), and incubated for 30 min at 25˚C. The reaction was then loaded on a 10% to 40% sucrose gradient in buffer A and spun for 20 h (SW40 rotor, 13,400 rpm, 4˚C). The gradient was harvested using a gradient station (Biocomp), the 80S peak was collected, the ribosomes were concentrated by pelleting (TLA110, 100,000 rpm, 1 h, 4˚C), and the 80S ribosomes were resuspended in lysis buffer. PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001995 April 20, 2023 Cryo-electron microscopy of the Map1-ribosome complex Sample preparation. Approximately 4 pmol of the freshly prepared samples from the native Map1-TAP pullout were adjusted to 40 μl total sample volume with LB-2.5 containing Nikkol to a final concentration of 0.05% (w/v). For cross-linking, glutaraldehyde was added to a final concentration of 0.02% (w/v), and the sample was incubated for 15 min on ice. After a short spin of 5 min at 4˚C and 14,000 rpm in a table top centrifuge (Eppendorf), 3.5 μl were applied to 2 nm precoated Quantifoil R3/3 holey carbon support grids. Before sample applica- tion, the grids were glow discharged for 30 s at 0.3 mbar. Vitrification and data processing. Vitrification was performed by plunge freezing the grid into liquid ethane using Vitrobot Mark IV (FEI Company/Thermo Fisher) with an incu- bation time of 45 s and blotting for 2 to 3 s at 4˚C and a humidity of 95%. Data were collected on a Titan Krios G3 (Thermo Fisher) equipped with a K2 direct detector (Gatan) at 300 keV using the semiautomated data acquisition software EPU (Thermo Fisher). A total of 48 frames with a dose of 1.17 e−/Å2 per frame were collected in a defocus range of −0.5 to −3.2 μm. Mag- nification settings resulted in a pixel size of 1.059 Å/pixel. Frame alignment was executed with MotionCor2 [58], and the estimation of the contrast transfer function (CTF) was performed with Gctf [59]. Micrographs were screened manually for ice quality, and the resulting 8,358 micrographs were used for automated particle picking in Gautomatch (https://www2.mrc-lmb.cam.ac.uk/ download/gautomatch-056/). After a two-dimensional (2D) classification in RELION 3.0 [60] to discard nonribosomal particles, in total, 115,082 particles were subjected to an initial refine- ment. Subsequent 3D classification into eight classes led to three classes only containing poorly resolved 80S ribosomes (7% with 8,234 particles, 1% with 1,226 particles, 0.01% with 149 parti- cles) and one class showing the 60S subunit only (6%, 6,728 particles). The remaining four classes showed well-resolved 80S ribosomes, all of which were programmed with tRNAs (mainly in A/A and P/P states) and showed the mobile expansion segment ES27a solely in the exit position and a weak extra density between ES27a and the tunnel exit. One of these classes (12%, 13,804 particles) contained eRF1 and ABCE1/Rli1. These four classes were merged for further processing (86%, 98,745 particles). In vitro reconstitution of NatB-ribosome complexes Binding assays were performed using 80S and purified NatB complex (either with wt Naa25 or PP mutant Naa25 variants). All assays were performed using 2 pmol (80 nM) of ribosomes and 20-fold molar excess of NatB (1.6 μM). The binding buffer for NatB-ribosome complex formation was adjusted to 50 mM Tris (pH 7.5), 150 mM KOAc, 5 mM Mg(OAc)2, and 1 mM acetyl-CoA. Sample reactions were pelleted through a sucrose cushion (750 mM sucrose, 20 mM HEPES (pH 7.5), 150 mM KOAc, 5 mM Mg(OAc)2, 1 mM DTT, and 10 μg/ml cyclohexi- mide). Supernatant and pellet fractions were separated on Nu-PAGE gels and transferred onto PVDF membranes. After transfer, the lower half of the membrane was decorated with a poly- clonal antiserum against uL29 and the upper half was analyzed using an anti-His antibody (Roche). Secondary antibodies were conjugated with horseradish peroxidase. Western blots were analyzed via chemiluminescence on an Amersham LAS Imager 600, and densitometry was performed using ImageJ version 1.53Q. See S1 Raw Images for all raw western blot images and S1 Data for numerical data underlying the densitometric quantification. PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001995 April 20, 2023 17 / 28 PLOS BIOLOGY Cryo-EM structures of ribosome-bound Map1 and NatB PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001995 April 20, 2023 Cryo-electron microscopy of the NatB-ribosome complex Sample preparation. Around 2.5 pmol uL4-RNCMDEL were in vitro reconstituted with an 18-times molar excess (44 pmol) of recombinantly purified inactive NatB complex. Final buffer conditions were 50 mM HEPES (pH 7.5), 116 mM KOAc, 5 mM Mg(OAc)2, 1 mM DTT, 0.05% Nikkol (w/v), 1 mM acetyl-CoA. The total reaction volume for cryo-grid making was 25 μl. Vitrification and data processing. The freshly prepared sample was applied to 2 nm pre- coated Quantifoil R3/3 holey carbon support grids and plunge frozen under the same condi- tion as described for the Map1-ribosome complex. Data were collected on a Titan Krios G3 (Thermo Fisher) equipped with a K2 direct detector (Gatan) at 300 keV using the semiauto- mated data acquisition software EPU (Thermo Fisher). A total of 40 frames with a dose of 1.409 e−/Å2 per frame were collected in a defocus range of 0.5 to 3.5 μm. Magnification settings resulted in a nominal pixel size of 1.049 Å/pixel. Frame alignment was performed with MotionCor2 [58]. All further processing steps were carried out in CryoSPARC, version 4.0.0 [61] unless oth- erwise specified. For a total of 10,380 selected micrographs, CTF estimation was performed using the patch-based CTF estimator in CryoSPARC. 80S ribosomal particles were picked by first generating templates from a subset of micrographs using CryoSPARC’s Blob Picker and performing 2D classification, then picking from all micrographs using the Template Picker with the thusly generated 2D templates. After 2D classification, 447,470 particles were selected for ab initio reconstruction and homogenous refinement of a consensus map. The aligned particles were then subjected to 3D variability analysis using a soft mask around the peptide tunnel exit region on the large ribo- somal subunit. Roughly half of all particles (45.5%, 203,513 particles) were sorted into classes exhibiting either no additional density in the masked region, no ES27a in the exit position, or only noisy signal likely corresponding to NatB-1 around ES27a and were thus discarded. Two of the three remaining classes (77,918 particles and 50,791 particles) showed density for a rigidly bound copy of NatB (NatB-2) at the second universal adapter site on the 60S tunnel exit and only fuzzy density for NatB-1. A third class of 115,238 particles contained density only for NatB-1, but no signal for NatB-2. All three of these classes were subjected to additional rounds of focused sorting and refinement using a mask around the expected position of NatB-1. Cryo-electron microscopy of the Map1-ribosome complex A subsequent 3D classification using a binary soft mask enclosing ES27a and the region below the TE revealed one class (25%, 25,245 particles) that showed a well-shaped ES27a as well as a defined extra density where Map1 was expected. All other classes showed either poorly resolved ES27a or fragmented densities at the expected Map1 position (24% with 23,499 particles, 28% with 27,590 particles, 23% with 22,411 parti- cles). The well-resolved class was further refined and subjected to another round of local 3D classification, this time with a mask covering only the tip of ES27a and the putative Map1 den- sity. The two resulting classes exhibited Map1 in two different conformations, either tightly connected to ES27a (47%, 11,938 particles) or with a looser connection to ES27a (53%, 13,307 particles). Both classes were CTF refined to an overall resolution of 3.9 Å and 3.8 Å according to the gold standard resolution criterion (FSC = 0.143) and comprising 10% or 12% of the total particle number after 2D classification, respectively. Subsequently local resolution was calcu- lated using RELION. Model of the Map1-ribosome complex. To interpret the density for Map1, a homology model was generated by AF2 [37]. To position this model inside the low-resolution density, it was superimposed on the high- resolution cryo-EM model of human Ebp1 bound to the ribosome (PDB 6SXO; [35]). This was followed by a rigid-body fit of the Map1 model into the two isolated densities. 18 / 28 PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001995 April 20, 2023 PLOS BIOLOGY Cryo-EM structures of ribosome-bound Map1 and NatB PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001995 April 20, 2023 Supporting information S1 Fig. Affinity purification of native Map1-ribosome complexes. (A) Amido black stained PVDF membrane of Map1-TAP purification samples separated on a 12% Nu-PAGE gel. (B) ECL-developed PVDF membrane after western blotting using antibodies against the CaMBD (α-CAB) moiety of the TAP tag and against ribosomal protein uL29. A shift of the α-CAB sig- nal upon TEV cleavage indicates the successful cleavage of the Protein A domain from the TAP tag leaving Map1-CaMBD and copurified ribosomes in the elution fraction. (C) 12% Nu- PAGE of the elution fraction from the Map1-ribosome purification. Ly, lysate; SN, superna- tant; P, pellet; FT, flow through; R, resuspension; W, wash; E, elution; B, boiled beads. 0.1 A260 of E was loaded; for wash fractions, 1/17 of the volume was loaded on the gel; for Ly, SN, P, and FT, 3 μl of the sample were loaded corresponding to 1/6,000 for L, 1/8,000 for SN, and 1/ 1,000 for P and FT. TAP = tandem affinity purification; CaMBD = Calmodulin-binding domain. *, contamination from a viral protein. See S1 Raw Images for all raw gel and western blot images shown in (A, B, and C). S2 Fig. Classification scheme for native Map1-ribosome complexes. Particles were picked with Gautomatch followed by 2D classification in RELION to discard nonribosomal particles. Subsequent refinement and 3D classification into eight classes resulted in four high-resolution classes all showing in the exit position. These classes, comprising 86% of all particles, were joined and subjected to a masked classification on ES27 and the region around the TE. One- quarter of the particles formed a stable class with a defined ES27 and an additional density for Map1. This class was further subclassified applying a mask covering the tip of ES27 and the Map1 density. The resulting two stable classes showed Map1 in two conformations (red, blue), harboring 10% and 13% of all particles. Both classes were CTF refined to an overall resolution of 3.9 Å and 3.8 Å, respectively. All maps are shown at the same contour level; percentages refer to the previous processing step. S3 Fig. Local resolution and FSC curves for the Map1-C1-80S and Map1-C2-80S cryo-EM reconstructions. (A) Cryo-EM maps of Map1-C1-80S and Map1-C2-80S before postproces- sing and colored according to local resolution as determined by RELION. Local resolution for Map1 in both maps ranged from 6.5 Å to below 12.5 Å, indicating a high degree in flexibility. Cryo-electron microscopy of the NatB-ribosome complex In this process, a majority of the particles (224,091 or 91.9% of the particles selected after initial sorting) showed a high continuous conformational heterogeneity of NatB-1 and was not processed further. One subclass of 9,645 particles, however, showed a defined density for both NatB-2 and NatB-1 in which NatB1 was positioned in direct vicinity to NatB-2 and directly below the tunnel exit. This class was termed “Class I” and refined to a resolution of 3.8 Å according to gold standard (FSC = 0.143). All initial classes containing NatB-2 were also subjected to focused sorting on NatB-2 in a similar fashion and with the resulting class of particles with rigidly bound NatB-2 (45,530 par- ticles), termed “Class II”, a reconstruction of the NatB2-ribosome complex at a resolution of 3.1 Å was obtained. Model of the NatB-ribosome complex. The model of the ribosome was generated by adapting a model of the yeast 80S ribosome stalled on the CGA-CCG inhibitory codon combi- nation [62] for the 60S ribosomal subunit and of ES27a in the exit position from NatA-ribo- some complex [25]. A homology model of NatB (comprising Naa25 and Naa20) was obtained by using AF2 in multimer mode [37]. All models were first rigid-body fitted in ChimeraX [63] after minor adjustments, such as rearranging both ES27a and the C-terminal helices of Naa25 PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001995 April 20, 2023 19 / 28 PLOS BIOLOGY Cryo-EM structures of ribosome-bound Map1 and NatB based on the higher-resolution reconstruction of Class II. The model for Class II, containing only NatB-2 but not NatB-1, was then refined using real space refinement in Phenix [64] and manual adjustment in WinCoot [65] using ProSmart and RCrane modules [66,67]. A model for Class I was then generated by rigid-body fitting both the model for Class II and the Alpha- fold model for NatB-1 into the corresponding density and performing one round of real space refinement in Phenix, followed by manual adjustments in WinCoot. All cryo-EM structures and models were displayed with ChimeraX [63]. PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001995 April 20, 2023 Supporting information (B) FSC curves for both refined classes; the average resolution was estimated according to the gold standard. (T ) S4 Fig. Comparison of Map1 bound to the ribosome tunnel exit with Map1 homologs. (A) Zoomed view focusing on the tunnel exit region of the Map1-C1-80S map compared to S.c. Arx1-containing pre-60S (EMD-6615), the H.s. EBP1-bound 80S (EMD-10344 and 10609) and the PDF-Map-70S ribosome complex from E. coli (EMD-9753). (B) Overlays of the Map1-C1-80S map with isolated densities for ES27a, Arx1, EBP1, and bacterial Map (bMap) PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001995 April 20, 2023 20 / 28 20 / 28 PLOS BIOLOGY Cryo-EM structures of ribosome-bound Map1 and NatB S5 Fig. Fitting of the Map1 and NatB models into density. (A-D) Two views showing the fit of the Map1 AlphaFold-2 model into the isolated density from the Map1-C1 class (A, B) and Map1-C2 class (C, D). (E) Fit of the model for ES27a-bound NatB-1 into isolated density from Class I (Naa25-1 in rainbow, Naa20 purple, ES27a blue). (F) Side view of (E). (G) View show- ing the fit of both NatB models into the density of Class I. (H) Fit of the model for NatB-2 into isolated density from Class II (after focused sorting on NatB-2). Naa25-1 is shown in rainbow, Naa20 in grey. (I) Side view of (H). (J) View highlighting the interaction of the C-terminal α- helices of Naa25-2 (from NatB-2) with ribosomal RNA. (H, I). All maps were filtered accord- ing to local resolution. (TIF) S5 Fig. Fitting of the Map1 and NatB models into density. (A-D) Two views showing the fit of the Map1 AlphaFold-2 model into the isolated density from the Map1-C1 class (A, B) and Map1-C2 class (C, D). (E) Fit of the model for ES27a-bound NatB-1 into isolated density from Class I (Naa25-1 in rainbow, Naa20 purple, ES27a blue). (F) Side view of (E). (G) View show- ing the fit of both NatB models into the density of Class I. (H) Fit of the model for NatB-2 into isolated density from Class II (after focused sorting on NatB-2). Naa25-1 is shown in rainbow, Naa20 in grey. (I) Side view of (H). (J) View highlighting the interaction of the C-terminal α- helices of Naa25-2 (from NatB-2) with ribosomal RNA. (H, I). All maps were filtered accord- ing to local resolution. (TIF) S6 Fig. Conformation of ES27a in Map1-, NatA-, and NatB-bound ribosomal complexes. Supporting information View focusing on the exit tunnel with the position of ES27a as observed in the NatA-ribosome structure [25], in the Map1-ribosome structures (classes C1 and C2), and in the NatB-ribo- some structure (class I with two stable NatBs bound) outlined. Relative rotation angles around the H63, ES27a, and ES27b three-way junction as well as the distances between the respective ES27a tip positions are shown. S7 Fig. NuPAGE analysis of NatB, 80S ribosomes, RNCs, and NatB mutants. 12% Nu- PAGE gels showing purified components used for the NatB project. (A) Lane 1, RNCMDEL; lane 2, Marker (PAGE Ruler Unstained, Thermo Fisher, #26614); lane 3, rt80S ribosomes; lane 4, 80S ribosomes, lane 5, empty; lane 6, NatBwt. (B) Lane 1, marker; lane 2, NatBPP1, lane 3, NatBPP3; lane 4, NatBPP4; lane 5 NatBPPall. See S1 Raw Images for all raw gel images. (TIF) S8 Fig. Classification scheme for in vitro reconstituted NatB-RNC complex. After particle picking and 2D classification in CryoSPARC, 447,470 particles were selected for ab initio reconstruction and homogenous refinement of a consensus map. Based on 3D variability anal- ysis of the region below the peptide exit tunnel, four principal classes were isolated. Two classes showed density for a rigidly bound copy of NatB (NatB-2) at the second universal adapter site on the 60S tunnel exit and fuzzy density for NatB-1. A third class of 115,238 particles contained density only for NatB-1, but no signal for NatB-2. All three of these classes were subjected to additional rounds of focused sorting and refinement using a mask around the expected posi- tion of NatB-1. This revealed one subclass showing a defined density for both NatB-2 and NatB-1. This subclass (class I; 9,645 particles) was refined to a resolution of 3.8 Å according to gold standard (FSC = 0.143). Other subclasses showed a high degree of conformational hetero- geneity for ES27a and the bound NatBs, as exemplified by a few selected classes displayed here. In an additional, independent classification branch all initial classes containing NatB-2 were subjected to focused sorting on NatB-2 resulting in class II (45,530 particles), which was fur- ther refined to a resolution of 3.1 Å. S9 Fig. Local resolution and FSC curve of NatB-ribosome complexes. (A) Cryo-EM maps of the two main classes of the NatB-ribosome complex were low-pass filtered and colored accord- ing to local resolution in CryoSPARC. PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001995 April 20, 2023 Supporting information Local resolution ranged from approximately 4 Å to 9 Å for the two NatBs in class I (left) and from approximately 3 Å to 6 Å for the focused refined NatB-2 in class II (right). (B) FSC curves for both refined NatB-ribosome classes; the average 21 / 28 PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001995 April 20, 2023 PLOS BIOLOGY Cryo-EM structures of ribosome-bound Map1 and NatB resolution was estimated according to the gold standard to 3.8 Å and 3.1 Å, respectively. (TIF) S10 Fig. Comparison of ribosome-bound NatB-1 and NatB-2. (A) Position of the NatB-1 and NatB-2 models (Naa25 grey, Naa20-1 blue, Naa20-2 red) with respect to the 60S subunit (shown as grey density). In addition, the position of acetyl-CoA (Ac-CoA) bound to each Naas20 subunit as well as putative models for the nascent chain are shown, once reaching into the catalytic center in Naa20-1 (green), once into Naa20-2 (yellow). (B) Same view as (A) but zoomed and showing only the two catalytic Naa20 subunits. (C, D) Top views of (A) and (B) (from the tunnel exit down) on the entire NatB complexes (C) or focusing only on the two Naa20 subunits (D). (TIF) S11 Fig. Comparison of ribosome-bound NAT and Map1 complexes. (A, B) Bottom view showing an overlay of the NatA-ribosome structure (A) NatB-ribosome structure (B) with iso- lated densities for ribosome-bound Map1 in C1 and C2 position. (C) and (D) show side views. (TIF) S1 Table. Cryo-EM data collection, refinement, and validation statistics for NatB-ribo- some complexes. Overview over cryo-EM data collection, data processing, and model fitting parameters for the NatB-RNCMDEL structures. Class I represents the data subset with two sta- bly bound NatBs, class I the data subset focused sorted on NatB-1. (PDF) S1 Table. Cryo-EM data collection, refinement, and validation statistics for NatB-ribo- some complexes. Overview over cryo-EM data collection, data processing, and model fitting parameters for the NatB-RNCMDEL structures. Class I represents the data subset with two sta- bly bound NatBs, class I the data subset focused sorted on NatB-1. (PDF) S1 Raw Images. Raw gel and western blot images. (A) Raw image of 12% Nu-PAGE gel from the Map1-TAP affinity purification. The lane shown in Figs 1A and S1C is labeled with “S.” Lanes labeled with “X” were not shown or discussed in the manuscript. (B) Raw image of the Coomassie-stained 15% SDS-PAGE gel used in Fig 1I. PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001995 April 20, 2023 Supporting information Shown are samples from the Map1 co- sedimentation assay with RNCs, RNaseI-treated RNCs (rtRNCs), and nonprogrammed ribo- somes (np80S). Lanes labeled with “X” were not shown or discussed in the manuscript. (C) Raw images of the western blot from the first replicate of NatB binding assay using wild type (WT) NatB and PP1, PP4, and PP_all NatB mutants. Top: Chemiluminescence image; levels were adjusted for optimum contrast. This image was also used in the quantification of NatB binding shown in Fig 2I (see also S1 Data). Bottom: Overlay of adjusted chemiluminescence image and visible light image for marker. Lanes labeled with “X” were not used for quantifica- tion. (D) Raw images of the western blot from the second replicate of NatB binding assay using wild type (WT) NatB and PP1, PP4, and PP_all NatB mutants. Top: Chemiluminescence image; levels were adjusted for optimum contrast. This image was also used in the quantifica- tion of NatB binding shown in Fig 2I (see also S1 Data) and is displayed as representative west- ern blot in Fig 2I. Bottom: Overlay of adjusted chemiluminescence image and visible light image for marker. Lanes labeled with “S” were shown as representative signals in Fig 2I. Lanes with “X” were not used for quantification or shown in the manuscript. (E) Raw images of the western blot from the third replicate of NatB binding assay using wild type (WT) NatB and PP1, PP4, and PP_all NatB mutants. Top: Chemiluminescence image; levels were adjusted for optimum contrast. This image was also used in the quantification of NatB binding shown in Fig 2I (see also S1 Data). Bottom: Overlay of adjusted chemiluminescence image and visible light image for marker. Lanes labeled with “X” were not used for quantification. (F) Raw images of the western blot of NatB binding assay using wild type (WT) NatB and the PP3 NatB mutant. Top: Chemiluminescence image; levels were adjusted for optimum contrast. This image was also used in the quantification of NatB binding shown in Fig 2I (see also S1 Data). Bottom: Overlay of adjusted chemiluminescence image and visible light image for marker. 22 / 28 PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001995 April 20, 2023 PLOS BIOLOGY Cryo-EM structures of ribosome-bound Map1 and NatB Lanes labeled with “X” were not used for quantification. (G) Raw images of the western blot of NatB binding assay using idle ribosomes (80S) or RNaseI-treated idle ribosomes (rt80S). Acknowledgments We thank Charlotte Ungewickell and Susanne Rieder for technical support and Dr. Thomas Fro¨hlich for mass spectrometry analysis. Supporting information Top: Chemiluminescence image; levels were adjusted for optimum contrast. This image was also used in the quantification of NatB binding shown in Fig 2I (see also S1 Data). Bottom: Overlay of adjusted chemiluminescence image and visible light image for marker. Lanes labeled with “S” were shown as representative signals in Fig 2I. Lanes labeled with “X” were not used for quantification or shown in the manuscript. (H) Raw image of PVDF membrane stained with Amido black from western blot of affinity purification of native Map1-ribosome complexes as used in S1A Fig. (I) Raw chemiluminescence images from western blot of affinity purification of native Map1-ribosome complexes as shown in S1B Fig. Top: raw image after incubation with anti-CAB antibody. Bottom: raw image after incubation with anti-uL29 antibody. Levels were uniformly adjusted for optimal contrast. (J) Raw image of Coomassie-stained 12% NuPAGE gel showing input samples used for NatB binding assays (Fig 2I). Wild type NatB (NatBWT), RNCs, RNaseI-treated ribosomes (rt80S), and untreated ribosomes (80S) are labeled and were used in S7A Fig. Lanes labeled with “X” were not shown or discussed in the manuscript. (K) Raw image of the Coomassie-stained 12% NuPAGE gel showing purified NatB positive-patch mutants. Lanes shown in S7B Fig are labeled. Lanes labeled with “X” were not shown or discussed in the manuscript. (PDF) S1 Data. Numerical data for densitometric quantification. Numerical data obtained from densitometric quantification of gel and western blot images using ImageJ version 1.53Q and calculations of averages and errors shown in Figs 1I and 2I. For Fig 1I, band intensities relative to background of the Map1 band in the Coomassie-stained gel were determined and normal- ized to the control with untreated RNCs. For Fig 2I, band intensities over background for bands corresponding to Naa25 and uL29 were determined for supernatant and pellet fraction, and the ratio of the Naa25 band intensities was calculated for each pair of fractions. Ratios were normalized by the band intensity measured for the ribosomal protein in each corre- sponding pellet fraction. Averages of these ratios were determined from the replicates and nor- malized to the wild type control experiment, where the binding efficiency in the control was set to 100%. Errors were calculated as standard deviations of the averages determined from replicates. Author Contributions Conceptualization: Thomas Becker, Birgitta Beatrix, Roland Beckmann. Data curation: Timur Mackens-Kiani, Thomas Becker. Data curation: Timur Mackens-Kiani, Thomas Becker. Formal analysis: Alexandra G. Knorr, Timur Mackens-Kiani, Thomas Becker, Roland Beckmann. Funding acquisition: Roland Beckmann. Funding acquisition: Roland Beckmann. Funding acquisition: Roland Beckmann. Investigation: Alexandra G. Knorr, Timur Mackens-Kiani, Joanna Musial. 23 / 28 PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001995 April 20, 2023 PLOS BIOLOGY Cryo-EM structures of ribosome-bound Map1 and NatB Methodology: Alexandra G. Knorr, Timur Mackens-Kiani, Joanna Musial, Otto Berninghausen, Birgitta Beatrix. Project administration: Thomas Becker, Birgitta Beatrix, Roland Beckmann. Resources: Roland Beckmann. Software: Alexandra G. Knorr, Timur Mackens-Kiani. Software: Alexandra G. Knorr, Timur Mackens-Kiani. Supervision: Thomas Becker, Birgitta Beatrix, Roland Beckmann. Validation: Alexandra G. Knorr, Timur Mackens-Kiani. Visualization: Alexandra G. Knorr, Timur Mackens-Kiani. Writing – original draft: Alexandra G. Knorr, Thomas Becker, Birgitta Beatrix, Roland Beckmann. Writing – review & editing: Timur Mackens-Kiani, Thomas Becker, Birgitta Beatrix, Roland Beckmann. 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https://openalex.org/W1965000937	https://europepmc.org/articles/pmc3180451?pdf=render	English	null	Diffusion of Myosin V on Microtubules: A Fine-Tuned Interaction for Which E-Hooks Are Dispensable	PloS one	2,011	cc-by	12,149	Introduction diffusion of myo V on microtubules is mediated by an electrostatic interaction of the positively charged loop 2 and the negatively charged E-hooks. To understand the molecular basis of this interaction three major questions have been addressed here: Does indeed the charge of loop 2 contribute to microtubule binding? Or does rather the amino acid composition of loop 2 make the difference? And most importantly, after the initial binding to the microtubule, what biophysical feature enables myo V to start diffusing along the filament? Efficient long-range intracellular transport of organelles is powered by processive motors of the kinesin, dynein and myosin superfamilies [1,2]. Studies over the past two decades have also shown a coordinated interplay between both microtubule- and actin-based transport systems [3,4,5,6]. For example, in extracts of the squid giant axon, a vesicle moving along a microtubule can suddenly leave its track and continue to move on an invisible track believed to be an actin filament [7]. In fish and amphibian melanophores, pigment granules (melanosomes) are transported on microtubules towards the cell periphery by kinesin-2 where their movement on actin filaments is driven by myosin V (myo V) [8,9,10,11]. To date, the regulation of this crosstalk remains elusive. We used single molecule Total Internal Reflection Fluorescence Microscopy (TIRFM) to characterize the association and diffusion of myo V mutants containing negatively charged loop 2 motifs on microtubules. The previously proposed electrostatic model pre- dicts that myo V mutants containing a net negative charge on their loops would cease to interact with the negatively charged microtubules. Surprisingly, myo V diffusion on microtubules is neither determined nor limited by the charge of loop 2 as both, the positively charged Wildtype myo V and the negatively charged loop 2 mutants, bind to and diffuse on microtubules. Most strikingly, neither for the initial association nor for the subsequent diffusion of myo V along microtubules E-hooks are required. Additional analysis of the microtubule binding and diffusion behavior of our oppositely charged constructs suggests that in addition to charge- charge interactions between myo V and the microtubule also non- ionic (e.g. van-der-Waals) attraction co-determines the interaction between myo V and microtubules, while hydrophilic effects by loop 2 merely play a subordinate role in facilitating diffusion on intact microtubules. Studies by Ali et al. Abstract Organelle transport in eukaryotes employs both microtubule and actin tracks to deliver cargo effectively to their destinations, but the question of how the two systems cooperate is still largely unanswered. Recently, in vitro studies revealed that the actin-based processive motor myosin V also binds to, and diffuses along microtubules. This biophysical trick enables cells to exploit both tracks for the same transport process without switching motors. The detailed mechanisms underlying this behavior remain to be solved. By means of single molecule Total Internal Reflection Microscopy (TIRFM), we show here that electrostatic tethering between the positively charged loop 2 and the negatively charged C-terminal E- hooks of microtubules is dispensable. Furthermore, our data indicate that in addition to charge-charge interactions, other interaction forces such as non-ionic attraction might account for myosin V diffusion. These findings provide evidence for a novel way of myosin tethering to microtubules that does not interfere with other E-hook-dependent processes. Citation: Zimmermann D, Abdel Motaal B, Voith von Voithenberg L, Schliwa M, O¨ kten Z (2011) Diffusion of Myosin V on Microtubules: A Fine-Tuned Interaction for Which E-Hooks Are Dispensable. PLoS ONE 6(9): e25473. doi:10.1371/journal.pone.0025473 Editor: Magdalena Bezanilla, University of Massachusetts Amherst, United States of America Editor: Magdalena Bezanilla, University of Massachusetts Amherst, United States of America Received May 9, 2011; Accepted September 5, 2011; Published September 26, 2011 Received May 9, 2011; Accepted September 5, 2011; Published September 26, 2011 Received May 9, 2011; Accepted September 5, 2011; Published September 26, 2011 mermann et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits tion, and reproduction in any medium, provided the original author and source are credited. Copyright: 2011 Zimmermann et al. This is an open-access article distributed under the terms of the Creative Commons Attribut unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: 2011 Zimmermann et al. This is an open-access article distributed under the t unrestricted use, distribution, and reproduction in any medium, provided the original author Funding: This work was supported by a Long Term European Molecular Biology Organization (EMBO) fellowship to ZO¨ (#1134-2006) and grants from the Deutsche Forschungsgemeinschaft (DFG, #OE 501/1-1) and the Friedrich-Baur-Stiftung (#01/09) to ZO¨ and MS. Z.O¨ . acknowledges support from SFB 863. URL EMBO: http://www.embo.org; URL DFG: http://www.dfg.de; URL Friedrich-Baur-Stiftung: http://www.baur-stiftung.de. Abstract The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: zoekten@ph.tum.de * E-mail: zoekten@ph.tum.de Dennis Zimmermann, Basma Abdel Motaal, Lena Voith von Voithenberg, Manfred Schliwa, Zeynep O¨ kten* Institute for Anatomy and Cell Biology, Ludwig-Maximilians-University of Munich, Munich, Germany September 2011 \| Volume 6 \| Issue 9 \| e25473 A negative net charge on loop 2 impairs the myosin V interaction with actin filaments To assess the functionality of our constructs, we performed ATPase activity and in vitro single molecule motility measurements on actin. The in vitro single-molecule velocity and runlength Figure 1. Schematic representations of the myosin V loop 2 constructs. (A) Schematic diagram depicting the domain structure of myo V with its surface-exposed loop 2 shown in yellow. The C-terminal GCN4 motif ensures dimerization. (B) Schematic domain model of truncated myo V in its homodimeric form. The regions are color-coded for each structural motif. The same set of colors is used in A and B. (C) Comparison of the loop 2 sequences from myo V Wildtype, Minus4 and Minus13. Mutants Minus4 (blue) and Minus13 (green) were designed by altering positively charged amino acids (lysines and arginines) to alanine and glutamic/aspartic acid, respectively. (+) and (2) indicate positively and negatively charged amino acids, respectively. The net charge of each construct is indicated in brackets next to the respective construct. doi:10 1371/journal pone 0025473 g001 Taken together, our results now make testable predictions for myo V’s capability to diffuse on the microtubule. If, as previously proposed [12], the interaction between myo V and microtubules were exclusively dependent on electrostatic forces, only Wildtype myo V (positive loop 2 net charge) would be expected to show diffusion on microtubules. However, if this interaction depends either on additional attraction forces or/and domain regions other than the proposed loop 2, the two negatively charged constructs, Minus4 and Minus13, should as well diffuse along the negatively charged microtubule lattice. Figure 1. Schematic representations of the myosin V loop 2 constructs. (A) Schematic diagram depicting the domain structure of myo V with its surface-exposed loop 2 shown in yellow. The C-terminal GCN4 motif ensures dimerization. (B) Schematic domain model of truncated myo V in its homodimeric form. The regions are color-coded for each structural motif. The same set of colors is used in A and B. (C) Comparison of the loop 2 sequences from myo V Wildtype, Minus4 and Minus13. Mutants Minus4 (blue) and Minus13 (green) were designed by altering positively charged amino acids (lysines and arginines) to alanine and glutamic/aspartic acid, respectively. (+) and (2) indicate positively and negatively charged amino acids, respectively. The net charge of each construct is indicated in brackets next to the respective construct. Indeed, irrespective of their net charge, all three myo V constructs showed diffusion along microtubules (Figure 2, black bars). Design of the myosin V loop 2 mutants Loop 2 of myo V is a well-defined region of ,44 amino acids near the so-called 50/20 kDa junction of the head domain. It has been implicated in binding the motor domain to actin [14,15] and recently has been suggested to also mediate the interaction of myo V with microtubules [12]. It is surface-exposed and exhibits a net positive charge of +5. To dissect the contribution of the myo V loop 2 on the interaction with microtubules, we have generated two heavy mero myosin (HMM)-like myo V mutants with surplus net negative charge by substituting the positively charged residues of loop 2 with either alanine (Minus4, four negative net charges) or glutamic and aspartic acid (Minus13, thirteen negative net charges) (Figure 1). Loop 2 is not the prime determinant of the interaction between myosin V and microtubules y To uncover the role of electrostatic interactions via myo V loop 2, we assessed the effects of increasing KCl-concentrations on the Wildtype and loop 2 mutants by quantifying the number of microtubule-interacting myo V molecules. Here, we discriminated between microtubule-colocalization events (defined as association events) and events where association was followed by subsequent diffusion (defined as diffusion events). To this end, the number of events per microtubule unit length and time period was determined at KCl concentrations of 25, 50, 100 and 200 mM. An increase in ionic strength significantly decreased microtubule association of all three constructs (gray bars in Figure 2, A–C and Table S2). The Minus13 mutant, in particular, showed the most dramatic reduction of microtubule association, even at moderate ionic strength (Figure 2C). We attribute this behavior to a pronounced repulsion between the highly negatively charged motor loop and the negatively charged surface of the microtubule [29]. Although this finding indicates that the surface-exposed loop 2 may indeed participate in myo V’s affinity for microtubules, for two obvious reasons, the observed attraction cannot be primarily attributed to simple charge-charge interactions between the negatively charged microtubule and the normally positively charged loop 2: first, the positively and the negatively charged loop 2 constructs display similar salt-dependent decrease in microtubule association (gray bars in Figure 2, A–C); second, despite carrying opposite net charges on their loop 2, Wildtype and Minus4 show comparable levels of association at 25 mM salt (Table S2). Introduction [12] have added a further facet to the interaction between actin and microtubule motor systems, where myo V was found to switch from an actin filament onto an intersecting microtubule followed by one-dimensional diffusive motion over large distances in vitro. In previous studies it was shown, that a positively charged structural element (loop 2) of the myo V catalytic head stabilizes the weakly bound state of myo V to negative patches on the actin filament via electrostatic interactions [13,14,15,16]. Likewise, several kine- sins interact via a positively charged motor domain structure (K- loop) [17,18,19,20,21,22] with the negatively charged C- Terminus (E-hook) of microtubules extending from the surface [23,24]. Based on these findings, and to explain the observed myo V-microtubule interaction, the authors proposed that the PLoS ONE \| www.plosone.org September 2011 \| Volume 6 \| Issue 9 \| e25473 September 2011 \| Volume 6 \| Issue 9 \| e25473 1 E-Hook-Independent Diffusion of Myosin V Results measurements (Figure S1, A-B and Table S1), as well as the ATPase activity values of HMM-like Wildtype myo V (Figure S1C and Table S1) were in good agreement with those reported previously [13,15,25,26,27]. Several previous studies showed that decreasing the positive net charge on loop 2 leads to a decreased affinity for actin [14,15,28]. In line with that, the ATPase activity on F-actin was significantly decreased (Minus4) or abolished (Minus13) for the myo V constructs carrying a negative net charge on their loop 2. (Figure S1C and Table S1). Accordingly, in vitro motility studies did not yield any single molecule runs for those two constructs. Thus only a positively charged loop 2 promotes a productive interaction with actin. Design of the myosin V loop 2 mutants Myosin V diffusion takes place without the help of E-hooks E-hooks give rise to a pronounced negatively charged mantle around the microtubule [34]. They have long been known to facilitate one-dimensional diffusion of a number of microtubule- associated proteins by tethering them electrostatically to microtu- bules [17,18,20,36,37]. Besides the fact that the observed electrostatic attraction towards microtubules is not mediated by positive charges on loop 2 of myosin, additional non-ionic attraction forces might also be involved in myo V’s interaction with microtubules. To test if the negatively charged E-hooks play a substantial role in microtubule association and diffusion of the myo V constructs, we made use of limited proteolysis by subtilisin to generate S-microtubules that lack the E-hook [23,31]. E-hook removal was confirmed by SDS-PAGE and Western blot analysis (Figure 4A) [17,23,38]. Figure 2. Interaction of myosin V with microtubules under increasing ionic strength conditions. TIRFM movie sequences of single-molecule experiments with 100 nM Cy3-labeled myo V Wildtype (A), Minus4 (B) and Minus13 (C) on Atto488-labeled microtubules under increasing salt concentrations (i.e. 25, 50, 100 and 200 mM KCl), were analyzed for microtubule-association and subsequent diffusion over a 70-seconds time period. For this, the total number of microtubule- associated myo V particles (shaded) and the number of diffusing particles (solid) per unit length of the microtubules and time of measurement are plotted as a function of KCl concentration. () indicates that in those cases only one event in total was counted. Error bars represent mean 6 confidence interval (a = 0.95). doi:10.1371/journal.pone.0025473.g002 In favor of the non-ionic attraction model, the absence of E- hooks does not interfere with the ability of Wildtype to associate with microtubules (Figure 4B and Table S3). Moreover, for all three loop 2 constructs the number of associated motors per microtubule unit length leveled off (gray bars in Figure 4B and Table S3). For the most negative Minus13 construct, E-hook removal resulted in a significant increase in microtubule association by 75% (p,0.005, Table S2 vs. Table S3). Most importantly, the removal of E-hooks did not interfere with the ability of Wildtype myo V to diffuse along S-microtubules. The diffusion behavior of Wildtype on S-microtubules meets all the criteria of a one-dimensional diffusion process (Figure 5A and Figure S4) [12,31,32,33,34]. Compared to untreated microtubules, with 1.2160.09 mm/s myo V’s diffusion on S-microtubules E-Hook-Independent Diffusion of Myosin V We next assessed the proposed role of the negatively charged E- hooks [12] in tethering myo V to the microtubule surface. E-Hook-Independent Diffusion of Myosin V E-Hook-Independent Diffusion of Myosin V diffusion constant (D = 0.113 mm2/s) which is in good agreement with previous work [30] (Table 1 and Video S2). Strikingly, our analysis demonstrates that the D-values for the loop 2 mutants were similar compared with D of Wildtype (Table 1). Additional support came from the linear increase in mean square displace- ment (MSD) over time (Figure S2, B–D) [31,32,33]. As expected, by calculating D from the obtained slopes in the MSD-plots, we could confirm the validity of the Gaussian-based D-value calculations. Figure 2. Interaction of myosin V with microtu increasing ionic strength conditions. TIRFM movie single-molecule experiments with 100 nM Cy3-labeled m (A), Minus4 (B) and Minus13 (C) on Atto488-labeled micro increasing salt concentrations (i.e. 25, 50, 100 and 200 analyzed for microtubule-association and subsequent d 70-seconds time period. For this, the total number o associated myo V particles (shaded) and the numbe particles (solid) per unit length of the microtubules measurement are plotted as a function of KCl c () indicates that in those cases only one event in tota Error bars represent mean 6 confidence interval (a = 0.9 doi:10.1371/journal.pone.0025473.g002 The diffusion-derived single displacements of all three con- structs distributed as zero-centered Gaussians (Figure 3B), as expected for one-dimensional Brownian motion [34]. Zero net displacement was also confirmed by boxplots (Figure S2A). During diffusion, the time of association with the microtubule (tA) for all four constructs distributed exponentially (Figure S3, A–C). Individual mean values of tA (Table 1) ranged from 14.1 s to 23.8 s, with tA of Wildtype being the shortest. Finally, the average distance scanned by the respective constructs during diffusional events did not vary significantly amongst Wildtype and Minus13 and was only slightly larger for Minus4 (Table 1). As expected, diffusion did not require ATP [12,31,35]. Control experiments confirmed that the observed diffusion was not caused by the antibody that coupled the motor to the fluorophore, nor did the antibody display any specific association with microtubules. In line with the above myo V-microtubule association analysis, the diffusion analysis performed here strongly supports the notion that the interaction between the motor and the microtubule is not restricted to probable loop 2-conveyed electrostatic forces only. Most notably, Minus13, a construct carrying 13 negative net charges on its loop 2, shows a highly similar diffusion behavior as the positively charged Wildtype (Figure 3B). A negative net charge on loop 2 impairs the myosin V interaction with actin filaments Due to the clear salt-dependent interactions shown here, charge-charge interactions are likely to dominate the binding of myo V to microtubules. Notably, not only at low-salt conditions but also, at 100 mM KCl (Wildtype and Minus13) and 200 mM KCl (Wildtype) did we observe diffusion (Figure 2, black bars, Table S2 and Video S1). Hence it can be concluded that electrostatic attraction mediated by loop 2 cannot be considered the sole molecular determinant for microtubule association. Therefore, it cannot be ruled out that other attraction forces (e.g. non-ionic) co- determine myo V’s affinity toward microtubules. doi:10.1371/journal.pone.0025473.g001 September 2011 \| Volume 6 \| Issue 9 \| e25473 September 2011 \| Volume 6 \| Issue 9 \| e25473 PLoS ONE \| www.plosone.org 2 Discussion Compared to motor-driven directed movement, unbiased one- dimensional diffusion is faster over short distances and does not consume energy, thus representing an efficient and supportive mechanism for intracellular transport processes [33,35,37,40,41]. Recent studies by Ali et al. [12,30] suggested that one-dimensional diffusion of myo V on microtubules is based on electrostatic interaction between the positively charged loop 2 of myo V and the negatively charged E-hooks of microtubules. Here we probed the proposed model of electrostatic myo V-tethering to the microtubule via complementary charged stretches. For this myo V mutants carrying either moderate or high surplus negative charges on their loop 2 were generated. To gain detailed mechanistic insights into the unbiased diffusion of myo V, we distinguished between association of myo V with microtubules (i.e. binding without additional quantifi- cation of subsequent diffusion events) and diffusion per se. From our analyses now a more complex and multilayered picture of the interaction modes between myo V and microtubules emerges. Figure 3. One-dimensional diffusion of myosin V loop 2 constructs on microtubules. (A) Kymographs of sequential frames depicting diffusive movement of single Cy3-labeled myo V Wildtype, Minus4 and Minus13 molecules (top to bottom, pseudo-colored green) on Atto488-labeled microtubules in 25 mM KCl. Microtubules are not shown for this purpose. Control represents a stationary, non-diffusing motor molecule on the microtubule. (B) TIRFM movie sequences of single myo V molecules on microtubules in 25 mM KCl were analyzed and then plotted as a displacement histogram. A single Gaussian (solid color-coded lines) was fitted to the data using equation 1 (Methods). From the obtained fit, the variance s was used to calculate the diffusion coefficient D according to the equation, D = s/2t, where t is the time interval between images, resulting in DWt = 0.113 mm2/s (n = 464), DMinus4 = 0.089 mm2/s (n = 801) and DMinus13 = 0.081 mm2/s (n = 425). Black, blue and green color-coded fit-lines depict the Gaussian fit for the individual displacement distribution of myo V Wildtype, Minus4 and Minus13, respectively. Figure 3. One-dimensional diffusion of myosin V loop 2 constructs on microtubules. (A) Kymographs of sequential frames depicting diffusive movement of single Cy3-labeled myo V Wildtype, Minus4 and Minus13 molecules (top to bottom, pseudo-colored green) on Atto488-labeled microtubules in 25 mM KCl. Microtubules are not shown for this purpose. Control represents a stationary, non-diffusing motor molecule on the microtubule. All myosin V loop 2 charge mutants show unperturbed diffusion on microtubules Next, we characterized the behavior of the diffusion events for myo V Wildtype and the loop 2 mutants on microtubules (Figure 3A, Videos S2 and S3). Diffusion of Wildtype yielded a September 2011 \| Volume 6 \| Issue 9 \| e25473 PLoS ONE \| www.plosone.org 3 E-Hook-Independent Diffusion of Myosin V Figure 3. One-dimensional diffusion of myosin V loop 2 constructs on microtubules. (A) Kymographs of sequential frames depicting diffusive movement of single Cy3-labeled myo V Wildtype, Minus4 and Minus13 molecules (top to bottom, pseudo-colored green) on Atto488-labeled microtubules in 25 mM KCl. Microtubules are not shown for this purpose. Control represents a stationary, non-diffusing motor molecule on the microtubule. (B) TIRFM movie sequences of single myo V molecules on microtubules in 25 mM KCl were analyzed and then plotted as a displacement histogram. A single Gaussian (solid color-coded lines) was fitted to the data using equation 1 (Methods). From the obtained fit, the variance s was used to calculate the diffusion coefficient D according to the equation, D = s/2t, where t is the time interval between images, resulting in DWt = 0.113 mm2/s (n = 464), DMinus4 = 0.089 mm2/s (n = 801) and DMinus13 = 0.081 mm2/s (n = 425). Black, blue and green color-coded fit-lines depict the Gaussian fit for association time of 12.4 s with microtubules remained essentially unchanged (Table 1 and Figure S4A). Notably, the diffusion coefficient D = 0.226 mm2/s was twice as high as that of Wildtype on untreated microtubules (Table 1 and Figure 5). Non-electrostatic attraction forces contribute to the interaction of myosin V with microtubules (A) Kymographs of sequential frames depicting diffusive movement of single Cy3-labeled myo V Wildtype, Minus4 and Minus13 molecules (top to bottom, pseudo-colored green) on Atto488-labeled microtubules in 25 mM KCl. Microtubules are not shown for this purpose. Control represents a stationary, non-diffusing motor molecule on the microtubule. (B) TIRFM movie sequences of single myo V molecules on microtubules in 25 mM KCl were analyzed and then plotted as a displacement histogram. A single Gaussian (solid Figure 3. One-dimensional diffusion of myosin V loop 2 constructs on microtubules. (A) Kymographs of sequential frames depicting diffusive movement of single Cy3-labeled myo V Wildtype, Minus4 and Minus13 molecules (top to bottom, pseudo-colored green) on Atto488-labeled microtubules in 25 mM KCl. Microtubules are not shown for this purpose. Control represents a stationary, non-diffusing motor molecule on the microtubule. (B) TIRFM movie sequences of September 2011 \| Volume 6 \| Issue 9 \| e25473 Non-electrostatic attraction forces contribute to the interaction of myosin V with microtubules The striking observation that irrespective of the net charge of loop 2 all three constructs bind to S-microtubules, while only Wildtype (Figures 4B and 5, Table S3 and Video S4) diffuses, raises the question whether besides electrostatics also additional attraction forces contribute to the interaction between myo V and microtubules. We propose that due to the E-hook removal, the formerly homogeneously negatively charged microtubule is now marked by negative and also positive surface charges [29,39]. Hence, formerly prevalent electrostatic repulsion forces (negative loop 2 vs. negative E- hooks) are now eliminated and electrostatic attraction forces (negative loop 2 vs. positive patches on the microtubule surface) are now free to exert their effects. The absence of the ,4 nm E-hook spacers would enhance additional, non-ionic attraction forces that have strong effects over short distances, thereby preventing the individual molecule from advancing from an associative to a diffusive state (as is the case for Minus4 and Minus13) (Figure 4B). Under such circumstances, only constructs bearing a residual ionic repulsive capacity (positive loop 2 vs. positive patches on the microtubule surface), such as the Wildtype, display diffusion (Figure 4B). Taken together, we suggest that additional, non-electrostatic attraction forces contribute to myo V’s interaction with microtubules. Figure 3. One-dimensional diffusion of myosin V loop 2 constructs on microtubules. (A) Kymographs of sequential frames depicting diffusive movement of single Cy3-labeled myo V Wildtype, Minus4 and Minus13 molecules (top to bottom, pseudo-colored green) on Atto488-labeled microtubules in 25 mM KCl. Microtubules are not shown for this purpose. Control represents a stationary, non-diffusing motor molecule on the microtubule. (B) TIRFM movie sequences of single myo V molecules on microtubules in 25 mM KCl were analyzed and then plotted as a displacement histogram. A single Gaussian (solid color-coded lines) was fitted to the data using equation 1 (Methods). From the obtained fit, the variance s was used to calculate the diffusion coefficient D according to the equation, D = s/2t, where t is the time interval between images, resulting in DWt = 0.113 mm2/s (n = 464), DMinus4 = 0.089 mm2/s (n = 801) and DMinus13 = 0.081 mm2/s (n = 425). Black, blue and green color-coded fit-lines depict the Gaussian fit for the individual displacement distribution of myo V Wildtype, Minus4 and Mi 13 ti l Figure 3. One-dimensional diffusion of myosin V loop 2 constructs on microtubules. Discussion (B) TIRFM movie sequences of single myo V molecules on microtubules in 25 mM KCl were analyzed and then plotted as a displacement histogram. A single Gaussian (solid color-coded lines) was fitted to the data using equation 1 (Methods). From the obtained fit, the variance s was used to calculate the diffusion coefficient D according to the equation, D = s/2t, where t is the time interval between images, resulting in DWt = 0.113 mm2/s (n = 464), DMinus4 = 0.089 mm2/s (n = 801) and DMinus13 = 0.081 mm2/s (n = 425). Black, blue and green color-coded fit-lines depict the Gaussian fit for the individual displacement distribution of myo V Wildtype, Minus4 and Minus13, respectively. d l The observation that not only positively but also negatively charged loop 2 constructs displayed a salt-dependent decrease in microtubule association (Figure 2) allows the following two conclusions. First, electrostatic interaction is indeed the prevailing force mediating the association of myo V with microtubules. Second, loop 2 is not the site responsible for such attraction because the oppositely charged loop 2 constructs Wildtype and Minus4 (+5 vs. 24) display equivalent salt-sensitive binding behavior. Along with the observation that the Wildtype and Minus4 associate at equivalent levels with microtubules further supports the notion that loop 2 neither mediates nor maintains the interaction between myo V and microtubules (Figure 2A and 2B). Thus another charged patch (or even multiple patches) on the myo V surface, mediating the observed salt-dependent interaction with the microtubule, need to be considered. Interestingly, as soon as enough negative charges were introduced into myo V loop 2 (e.g. doi:10.1371/journal.pone.0025473.g003 exhibits on average larger maximum displacements per frame interval (i.e., maximum speed) and shows a trend toward prolonged scan distances (3.0260.34 mm), while the average PLoS ONE \| www.plosone.org September 2011 \| Volume 6 \| Issue 9 \| e25473 September 2011 \| Volume 6 \| Issue 9 \| e25473 4 E-Hook-Independent Diffusion of Myosin V Table 1. Key parameters of diffusion of myosin V (Wildtype) and two myosin V loop 2 mutants. Discussion Most strikingly, and contrary to the predictions inferred from the electrostatic model, the charge of loop 2 neither determines nor limits the diffusion behavior of myo V. The narrow range of the diffusion constants (0.113 mm2/s of Wildtype to 0.089 mm2/s of Minus13) (Figure 3B, and Table 1) argues against a loop 2-biased charge-dependence of diffusion. In analogy to recent findings by Minoura et al. [34] and based on our results, we propose the following two-phase model for diffusion of myo V on microtubules. Phase One is the initial association with the filament. This step represents a prerequisite for diffusion and is accomplished as long as electrostatic surface effects exerted from both, the interactor and the substrate, cause attraction rather than repulsion. Phase Two is the advancement to the diffusive state. This phase heavily depends on a balanced interplay between attraction and repulsion of myo V to and from the microtubule. Here, it is crucial that the strength of attraction is restricted to such an extent that the motor is free to move laterally. Simply put, strong attraction forces may bind a high number of motor molecules to the surface but they also prevent those motors from moving (Minus4 and Minus13 on S-microtubules); conversely, weak attraction favors the diffusion along filaments, but at the same time gives the molecule a hard time to initially bind (Minus13 on untreated microtubules). Since the dosage makes the difference, in this specific case the actual dosage of electrostatic vs. non-ionic attraction on the surface determines the strength of binding and the likelihood of moving (Figure 6). The fact that on the side of the interactor (i.e., myo V) loop 2 as the potential electrostatic tether structure is neither required for the binding to nor for the diffusion along microtubules, prompted us to dissect the potential contributions of the substrate (i.e., microtubule) to myo V association and diffusion. If indeed attraction forces other than electrostatic tethering contribute to the interaction between myo V and microtubules, then microtubule E-hooks representing the proposed electrostatic tethering structures should be dispens- able. Indeed, on S-microtubules lacking E-hooks, association levels for Wildtype and Minus4 remained unchanged compared to untreated microtubules (Figure 4B vs. 2A and 2B), demonstrating that E-hook-mediated tethering is not involved in microtubule association of myo V. Removing the E-hooks only affected the Minus13 construct containing an unusually high negative charge on its surface (as discussed above). Discussion Diffusion was quantified by single displacement measurements. Parameters of myo V diffusion on subtilisin- treated microtubules (S-microtubules) for Wildtype are shown in the bottom part. All parameters were obtained from experiments in 25 mM KCl. n represents the total number of analyzed diffusion and displacement events. Values represent mean 6 S.E.M. Statistical significance at P.0.05, P,0.05 and P,0.005 vs. Wildtype was determined using Student’s t-Test. doi:10.1371/journal.pone.0025473.t001 Minus13), microtubule ‘‘affinity’’ for myo V was significantly reduced. This finding points to strong repulsion forces arising from the evenly distributed negative charges on its loop 2 motif (Figure 2C). This effect is readily revoked after the repulsive microtubule element (i.e., E-hooks) is removed (compare Figure 2C with Figure 4B). Taken together, we suggest that the dominating force that tethers myo V to the microtubule is due to charge- charge interactions. However, those interactions are by no means mediated by loop 2. In addition to electrostatic forces, non- electrostatic forces may exert significant influence at the myo V- microtubule interface. Minus13), microtubule ‘‘affinity’’ for myo V was significantly reduced. This finding points to strong repulsion forces arising from the evenly distributed negative charges on its loop 2 motif (Figure 2C). This effect is readily revoked after the repulsive microtubule element (i.e., E-hooks) is removed (compare Figure 2C with Figure 4B). Taken together, we suggest that the dominating force that tethers myo V to the microtubule is due to charge- charge interactions. However, those interactions are by no means mediated by loop 2. In addition to electrostatic forces, non- electrostatic forces may exert significant influence at the myo V- microtubule interface. myo V constructs, while the overall affinity remains comparable to that of Wildtype myo V on untreated microtubules. In addition, when E-hooks are present, they may act as negatively charged 4 nm spacers [29,34] that via repulsion force facilitate transitions from the stationary to the diffusion phase. Hence on microtubules that lack E-hooks, constructs containing a pronounced negatively charged loop 2 region (Minus4 and Minus13) are now free to productively interact with the un-shielded positive patches on the ‘‘naked’’ microtubule surface [29,39] (Figure 4B). In contrast, the Wildtype construct with its net positive loop 2 charge, retains some residual ionic repulsive capacity (positive loop 2 vs. positive patches on the microtubule surface) and thereby on S-microtubules still manages to advance into the diffusive state (Figure 4B and 5, Video S4). PLoS ONE \| www.plosone.org September 2011 \| Volume 6 \| Issue 9 \| e25473 Discussion Diffusion coefficient (D) (mm2 s21) Maximum speed (Vmax) (mm s21) Scan distance (xscan) (mm) Association time (tA) (s) n Diffusion events n Displacement events MyoV (Wildtype) 0.11360.004* 1.0460.06** 2.2160.27* 14.160.5* 31 464 MyoV (Minus4) 0.08960.003* 0.9960.06* 2.9560.28* 23.863.2 * 35 801 MyoV (Minus13) 0.08160.003* 0.8460.06** 2.0760.23* 16.260.8 32 425 on S-microtubules MyoV (Wildtype) 0.22660.006* 1.2160.09** 3.0260.34* 12.462.7* 20 327 Diffusion coefficients (D) were measured from the variance of the Gaussian fit function of the displacement histogram (Figure 3B). Values for maximum speed (Vmax) reflect the average maximum displacement along a microtubule during one frame interval (i.e., 5 frames per s) and were calculated from the respective number of analyzed diffusion events. Scan distances (xscan) were calculated as the distance between the two extreme positions the motor has scanned on the microtubule during one diffusion event. Association times (tA) represent the total time a myo V molecule spent on the microtubule during one diffusion event. Mean values for tA were measured from the exponential fit of the plotted histogram of single tA-values (Figure S3). Diffusion events are defined as events during which the myo V molecule has bound to the filament and subsequently started diffusing. Diffusion was quantified by single displacement measurements. Parameters of myo V diffusion on subtilisin- treated microtubules (S-microtubules) for Wildtype are shown in the bottom part. All parameters were obtained from experiments in 25 mM KCl. n represents the total number of analyzed diffusion and displacement events. Values represent mean 6 S.E.M. Statistical significance at P.0.05, P,0.05 and *P,0.005 vs. Wildtype was determined using Student’s t-Test. doi:10.1371/journal.pone.0025473.t001 Diffusion coefficients (D) were measured from the variance of the Gaussian fit function of the displacement histogram (Figure 3B). Values for maximum speed (Vmax) reflect the average maximum displacement along a microtubule during one frame interval (i.e., 5 frames per s) and were calculated from the respective number of analyzed diffusion events. Scan distances (xscan) were calculated as the distance between the two extreme positions the motor has scanned on the microtubule during one diffusion event. Association times (tA) represent the total time a myo V molecule spent on the microtubule during one diffusion event. Mean values for tA were measured from the exponential fit of the plotted histogram of single tA-values (Figure S3). Diffusion events are defined as events during which the myo V molecule has bound to the filament and subsequently started diffusing. Discussion (A) Removal of the Carboxy-terminal E-hook from microtubules. (Left panel) SDS/12% PAGE gel of untreated microtubules (lane 1), and after subtilisin-treatment (lane 2). (Middle and right panel) Western blots of these two lanes with anti-a or anti-b tubulin antibodies. Subtilisin- treatment resulted in the complete loss of epitope reactivity, hence complete E-hook removal can be assumed. (B) TIRFM movie sequences of single-molecule experiments with 100 nM Cy3-labeled myo V Wildtype, Minus4 and Minus13 on Atto488-labeled S-microtubules, in 25 mM KCl were analyzed for microtubule-association and subsequent diffusion over a 70-seconds time period. The total number of S- microtubule-associated and diffusing myo V particles per unit length of the microtubules and time of measurement is plotted as category plot for the respective myo V constructs. Error bars represent mean 6 confidence interval (a = 0.95). doi:10.1371/journal.pone.0025473.g004 Figure 5. One-dimensional diffusive motion of myosin V on microtubules lacking the E-hook. (A) The displacement between successive image frames of diffusive movements for myo V Wildtype on S- microtubules was determined. TIRFM movie sequences of 100 nM Cy3- labeled myo V Wildtype on Atto488-labeled microtubules were analyzed and plotted as a displacement histogram. The diffusion coefficient D was calculated according to the equation, D = s/2t, where t represents the time interval between images and s the variance. s was obtained from the Gaussian fit (solid line), resulting in D = 0.226 mm2/s (n = 327). (B) Kymograph of sequential frames depicting the diffusive motion of a single Cy3-labeled myo V Wildtype molecule (pseudo-colored green) on Atto488-labeled S-mmicrotubules (for this purpose not visualized). Control represents a stationary, non-diffusing motor molecule on the microtubule. doi:10.1371/journal.pone.0025473.g005 Figure 4. Interaction of myosin V with S-microtubules. (A) Removal of the Carboxy-terminal E-hook from microtubules. (Left panel) SDS/12% PAGE gel of untreated microtubules (lane 1), and after subtilisin-treatment (lane 2). (Middle and right panel) Western blots of these two lanes with anti-a or anti-b tubulin antibodies. Subtilisin- treatment resulted in the complete loss of epitope reactivity, hence complete E-hook removal can be assumed. (B) TIRFM movie sequences of single-molecule experiments with 100 nM Cy3-labeled myo V Wildtype, Minus4 and Minus13 on Atto488-labeled S-microtubules, in 25 mM KCl were analyzed for microtubule-association and subsequent diffusion over a 70-seconds time period. Discussion While the capacity to interact with S-microtubules did not differ significantly, diffusion was observed exclusively with the Wildtype construct (Figure 4B and Table S3). In other words, on microtubules lacking E-hooks, the fraction of associated motors remains high, though fewer of the attached myo V molecules advance to the diffusive state and thus remain stationary. As soon as the nearly homogeneous mantle of negatively charged E-hooks is removed, non-ionic forces (e.g. van-der-Waals interactions) that show strong effects over short distances [reviewed in 42,43] become a substantial attraction force. We therefore suggest that non-ionic attraction forces account for the observed decrease in diffusion for all three The fact that only Wildtype is still capable of diffusing on S- microtubules (Figure 5) suggests that only the native form is capable of balancing the interplay between ionic and non-ionic effects, making Wildtype almost ‘‘immune’’ to harsh changes on the microtubule (Figure 6). A closer look at the diffusion behavior of Wildtype reveals that compared to untreated microtubules, its diffusion along S-microtubules in fact is smoother (larger single displacements), yielding an increased diffusion constant (Figure 5A and Table 1). The E-hook-independent mode of interaction displayed by myo V offers an attractive explanation for the recent observation of myo V-aided movement of kinesin. Myo V was PLoS ONE \| www.plosone.org September 2011 \| Volume 6 \| Issue 9 \| e25473 5 E-Hook-Independent Diffusion of Myosin V Figure 4. Interaction of myosin V with S-microtubules. (A) Removal of the Carboxy-terminal E-hook from microtubules. (Left panel) SDS/12% PAGE gel of untreated microtubules (lane 1), and after subtilisin-treatment (lane 2). (Middle and right panel) Western blots of these two lanes with anti-a or anti-b tubulin antibodies. Subtilisin- treatment resulted in the complete loss of epitope reactivity, hence complete E-hook removal can be assumed. (B) TIRFM movie sequences of single-molecule experiments with 100 nM Cy3-labeled myo V Wildtype, Minus4 and Minus13 on Atto488-labeled S-microtubules, in 25 mM KCl were analyzed for microtubule-association and subsequent diffusion over a 70-seconds time period. The total number of S- microtubule-associated and diffusing myo V particles per unit length of the microtubules and time of measurement is plotted as category plot for the respective myo V constructs. Error bars represent mean 6 confidence interval (a = 0.95). doi:10.1371/journal.pone.0025473.g004 shown to increase kinesin’s run length on microtubules in vitro [30] Figure 4. Interaction of myosin V with S-microtubules. Discussion The total number of S- microtubule-associated and diffusing myo V particles per unit length of the microtubules and time of measurement is plotted as category plot for the respective myo V constructs. Error bars represent mean 6 confidence interval (a = 0.95). Figure 5. One-dimensional diffusive motion of myosin V on microtubules lacking the E-hook. (A) The displacement between successive image frames of diffusive movements for myo V Wildtype on S- microtubules was determined. TIRFM movie sequences of 100 nM Cy3- labeled myo V Wildtype on Atto488-labeled microtubules were analyzed and plotted as a displacement histogram. The diffusion coefficient D was calculated according to the equation, D = s/2t, where t represents the time interval between images and s the variance. s was obtained from the Gaussian fit (solid line), resulting in D = 0.226 mm2/s (n = 327). (B) Kymograph of sequential frames depicting the diffusive motion of a single Cy3-labeled myo V Wildtype molecule (pseudo-colored green) on Atto488-labeled S-mmicrotubules (for this purpose not visualized). Control represents a stationary, non-diffusing motor molecule on the microtubule. doi:10.1371/journal.pone.0025473.g005 doi:10.1371/journal.pone.0025473.g004 shown to increase kinesin’s run length on microtubules in vitro [30]. Our results point to a synergism between E-hook-independent tethering by myo V to microtubules that enhances the E-hook- dependent processive movement of kinesin. Buffers Buffers used in this study are defined as: Buffer A (80 mM Pipes, pH 6.9, 1 mM MgCl2, and 1 mM EGTA); Buffer B (25 mM imidazole, pH 7.4, 25 mM KCl, 4 mM MgCl2, 1 mM EGTA, and 10 mM DTT); Buffer C (Buffer B plus oxygen scavenging system composed of 1700 U/ml catalase, 26 U/ml glucose oxidase, and 0.4 mg/ml glucose); Buffer D (0.3 M KCl, 25 mM imidazole, pH 7.4, 4 mM MgCl2, 1 mM EGTA, and 10 mM DTT); Buffer E (Buffer B plus 5 mg/ml BSA); Buffer F (50 mM KCl, 10 mM imidazole, pH 7.0, 1 mM EGTA, 1 mM MgCl2, and 1 mM DTT); Buffer G (25 mM potassium acetate, 12.5 mM Aces?KOH, 2 mM magnesium acetate, 1 mM EGTA). In this respect unbiased diffusion of myo V contrasts with biased diffusion of certain kinesins such as MCAK along microtubules, which is E-hook-dependent [20,44]. A different kinesin motor, kip3 from yeast, which belongs to the kinesin-8 class, also diffuses on microtubules, but does so without the help of E-hooks [45]. Thus different types of motors can diffuse on microtubules, but the underlying mode of interaction differs. PLoS ONE \| www.plosone.org Reagents All reagents were the highest purity commercially available and were obtained from Sigma-Aldrich (Munich, Germany), unless mentioned otherwise. The constructs described below were cloned from the p2Bac/ pFastBac-dhM5-CaM plasmid [46] and contain the first 1098 amino acids (D1098) of chicken myo Va HMM (6 IQ), with a PLoS ONE \| www.plosone.org September 2011 \| Volume 6 \| Issue 9 \| e25473 6 E-Hook-Independent Diffusion of Myosin V Figure 6. The balance between attraction forces determines the diffusive state of myosin V on microtubules. (Left part) Strong attraction forces prevent microtubule-bound myo V molecules from advancing to the diffusive state. This Trapped State is achieved, if in addition to electrostatic also non-ionic attraction forces become increasingly dominant (Minus4 and Minus13 on S-microtubules). (Middle part) Diffusion takes place if for myo V the attraction toward the microtubule is of moderate strength. This Diffusive State in general is achieved when attraction and repulsion outweigh each other. Two different possibilities might account for that behavior. First, electrostatic and non-electrostatic interaction forces at the myo V binding-interface are well-balanced (Wildtype on untreated and S-microtubules); second, strong loop 2-derived ionic attraction is dominated by ionic repulsion elements (E-hooks) on the microtubule binding-interface (Minus4 on untreated microtubules). (Right part) Weak attraction toward microtubules prevents myo V from binding effectively to the filament, and hence diffusion becomes unlikely. This Dissociative State is given, if electrostatic repulsion via hydrophilic surface structures (E-hooks) becomes predominant (Minus13 on untreated microtubules). Red and blue colors indicate strong and weak attraction forces toward the microtubule surface, respectively. doi:10.1371/journal.pone.0025473.g006 Figure 6. The balance between attraction forces determines the diffusive state of myosin V on microtubules. (Left part) Strong attraction forces prevent microtubule-bound myo V molecules from advancing to the diffusive state. This Trapped State is achieved, if in addition to electrostatic also non-ionic attraction forces become increasingly dominant (Minus4 and Minus13 on S-microtubules). (Middle part) Diffusion takes place if for myo V the attraction toward the microtubule is of moderate strength. This Diffusive State in general is achieved when attraction and repulsion outweigh each other. Two different possibilities might account for that behavior. First, electrostatic and non-electrostatic interaction forces at the myo V binding-interface are well-balanced (Wildtype on untreated and S-microtubules); second, strong loop 2-derived ionic attraction is dominated by ionic repulsion elements (E-hooks) on the microtubule binding-interface (Minus4 on untreated microtubules). Reagents (Right part) Weak attraction toward microtubules prevents myo V from binding effectively to the filament, and hence diffusion becomes unlikely. This Dissociative State is given, if electrostatic repulsion via hydrophilic surface structures (E-hooks) becomes predominant (Minus13 on untreated microtubules). Red and blue colors indicate strong and weak attraction forces toward the microtubule surface, respectively. doi:10.1371/journal.pone.0025473.g006 leucine zipper fused in frame to the native coiled coil to ensure dimerization. To facilitate purification, a FLAG-tag epitope (DYKDDDDK) was introduced at the N-terminus. Myo Va was co-expressed with the human essential light chain using the Baculovirus Expression System (Invitrogen, Darmstadt, Germany) and purified as described [46]. The loop 2 mutations were introduced by custom DNA synthesis (Sloning BioTechnology, Puchheim, Germany). For myo V Minus4, all lysines and arginines within loop 2 were changed to alanines (K607A, R619A, R624A, K628A, K631A, R633A, K639A, K642A, K643A). For myo V Minus13, all lysines and arginines were substituted with glutamic or aspartic acid (K607E, R619D, R624E, K628E, K631D, R633E, K639E, K642E, K643E). Both loop 2 mutants were cloned into the p2Bac/pFastBac-dhM5-CaM plasmid. Expression and purifi- cation of the constructs were performed as described [46,47]. The purified proteins were dialyzed for 1 h at 4uC in the absence of ATP against Buffer B (plus 50% glycerol) and stored at 220uC. leucine zipper fused in frame to the native coiled coil to ensure dimerization. To facilitate purification, a FLAG-tag epitope (DYKDDDDK) was introduced at the N-terminus. Myo Va was co-expressed with the human essential light chain using the Baculovirus Expression System (Invitrogen, Darmstadt, Germany) and purified as described [46]. The loop 2 mutations were introduced by custom DNA synthesis (Sloning BioTechnology, Puchheim, Germany). For myo V Minus4, all lysines and arginines within loop 2 were changed to alanines (K607A, R619A, R624A, K628A, K631A, R633A, K639A, K642A, K643A). For myo V Minus13, all lysines and arginines were substituted with glutamic or aspartic acid (K607E, R619D, R624E, K628E, K631D, R633E, K639E, K642E, K643E). Both loop 2 mutants were cloned into the p2Bac/pFastBac-dhM5-CaM plasmid. Expression and purifi- cation of the constructs were performed as described [46,47]. The purified proteins were dialyzed for 1 h at 4uC in the absence of ATP against Buffer B (plus 50% glycerol) and stored at 220uC. Western blot analysis After gel electrophoresis, proteins were transferred onto a nitrocellulose membrane (Protran-Whatman, Dassel, Germany) using a semi-dry transfer apparatus (Peq Lab, Erlangen, Germany). Incubation with primary (anti a- and b-tubulin) and secondary (anti-rat IgG and anti-mouse IgG) antibodies was Fluorescent labeling of proteins FLAG-purified myo V protein was conjugated to monoclonal anti-FLAG Cy3 antibody (#A9594 clone M2) by incubating 500 nM myo V protein with 11 mg/ml antibody at room temperature for 5 min in Buffer B and subsequent storage on ice. The mixture was further diluted in Buffer C to the desired final myo V concentration before use. Subtilisin-treatment of microtubules Microtubules were prepared from porcine brain tubulin [48]. Labeling of tubulin with Atto488 (AttoTec, Siegen, Germany) and the polymerization of microtubules was performed as described in [49]. For the preparation of fluorescent microtubules, Atto488- tubulin was mixed with unlabeled tubulin at 1:50 ratio. Tubulin (3.5 mg/ml) and Atto488 tubulin at a 50:1 ratio was polymerized in Buffer A (plus 1 mM GTP) at 36uC for 90 min. To stabilize the microtubules, 20 mM taxol (Invitrogen, Darmstadt, Germany) was added, followed by an incubation at 36uC for 40 min. The C-terminal ends of a- and b-tubulin (E-hook) were removed by incubation (36uC for 45 min) of 0.6 mg/ml with subtilisin A (#P5380) at 1:0.8 ratio in Buffer A (plus 20 mM taxol). The reaction was stopped by the addition of 2 mM PMSF (dissolved in isopropanol) and incubation at room temperature for 10 min. A previous study [12] used a nine- to twenty-three-fold higher concentration, which in our hands not only removes E-hooks but also affects microtubule integrity. Subtilisin-treated microtubules were pelleted at 27,0006g for 25 min. Pellets were washed with and resuspended in Buffer A (plus 20 mM taxol and 1 mM GTP). Samples were resolved on a 12% SDS-PAGE with subsequent Coomassie-stain or Western Blot analysis. Actin from chicken pectoralis was extracted from an acetone powder according to [50]. Purified G-actin (5 mM) was polymerized in Buffer B (plus 5 mM phalloidin) for 1 h at room temperature. For the preparation of fluorescent actin filaments, polymerization was performed in the presence of 5 mM TRITC-phalloidin. Reagents assays with microtubules as the cytoskeletal track, fluorescent microtubules (1 mM) (Buffer A plus 5 mM taxol) were infused into the flow cell and incubated for 3 min, followed by a wash with Buffer A (plus 5 mM taxol, and 0.7 mg/ml casein). 100 nM fluorescent-labeled myo V in Buffer C was added to the flow cell and incubated for 1 min. For experiments in which different ionic strength-conditions were applied to the flow cell, the required volume of KCl (1 M) was added to Buffer C yielding a final assay concentration of 50, 100 or 200 mM KCl. For assays in which actin filaments served as cytoskeletal tracks, flow cells were pre-incubated with 0.1 mg/ml N-ethyl maleimide (NEM)-modified heavy mero myosin (HMM) [51] in Buffer D for 3 min, rinsed with Buffer B, incubated with 0.1 mM TRITC- labeled actin filaments in Buffer B for 3 min, rinsed with Buffer B and then incubated with Buffer E for 5 min. 100 nM fluorescent- labeled myo V in Buffer C was applied and incubated for 1 min, followed by a wash step with Buffer C containing 1 mM ATP. Data acquisition Distances and single displacements were measured by brightest centroid tracking, using IMAGEJ. Single-displacement boxplots as well as all other data plotting and fitting, but also the statistical analysis was performed with IgorPro software (WaveMetrics, Inc., Portland, OR). Single-molecule motility was observed at room temperature using a total internal reflection fluorescent microscope (IX71, Olympus Biosystems, Planegg, Germany) equipped with a Plan objective lens (1006; numerical aperture, 1.65) and linked to a front-illuminated CCD camera (C-9100, Hamamatsu Photonics, Herrsching, Germany). Fluorophores were excited with a solid- state laser at wavelengths of 532 or 488 nm. The optical resolution was 160 nm per 262 -binned pixel, the integration time 200 ms. Typically, 350 images were recorded for a total of 70 s. Supporting Information Figure S1 Movement and activity of myosin V Wildtype and loop 2 mutants on actin filaments. Velocity (A) and runlength (B) distributions of myo V Wildtype were plotted as histograms. Data were obtained from single-molecule TIRFM experiments, where 100 nM Cy3-labeled myo V was incubated with Atto488-labeled F-actin in 25 mM KCl and 1 mM ATP. In (A) the data was fitted to a single Gaussian (according to equation 1, Methods section), yielding a mean velocity of 0.23 mm/s (n=62) for Wildtype. For the runlength distribution in (B) an exponential curve was fitted to the histograms (solid line), resulting in a mean value of 1.41 mm (n=62) for Wildtype. (C) Actin-activated ATPase for myo V Wildtype (black, open circles), Minus4 (blue, open diamonds) and Minus13 (green, open triangles) were measured with the NADH-coupled assay and plotted as a function of actin concentration (myo V concentration, 100 nM). The data were fitted to the Michaelis-Menten equation to determine the maximum ATPase rate (kcat) and the actin concentra- tion at which myo V is activated half-maximally (Km). Data shown is representative and was reproducible. (D) Kymograph of representa- tive motions of single Cy3-labeled myo V Wildtype on Atto488-labeled F-actin in buffer containing 25 mM KCl and 1 mM ATP. On actin, no movement for Minus4 and Minus13 was observed and hence no histograms (A and B) or kymographs (D) are depicted. All data obtained from A–C are summarized in Table S1. (TIF) Flow cell preparation For single-molecule motility assays 15-ml flow cells (area 1865 mm), covered by a nitrocellulose coverslip, were used. For PLoS ONE \| www.plosone.org September 2011 \| Volume 6 \| Issue 9 \| e25473 7 E-Hook-Independent Diffusion of Myosin V E-Hook-Independent Diffusion of Myosin V bules were generated with the MultipleKymograph macro for IMAGEJ. performed overnight at 4uC and at room temperature for 1 h, respectively. Monoclonal anti-a-Tubulin (clone YL 1/2) was a kind gift from Prof. Schleicher, M. (Ludwig-Maximilians Univer- sity, Munich, Germany) and monoclonal anti-b-Tubulin (clone SAP.4G5) was purchased from Santa Cruz Biotech., Inc. (Heidelberg, Germany). Quantification of microtubule association and diffusion was carried out with the data obtained from the performed single- molecule experiments on microtubules. For each microtubule, the numbers of diffusing and stationary particles during a 70-seconds period were counted. The numbers of diffusing and stationary particles on microtubule lengths of 1000–3000 mm were summed and divided by the corresponding microtubule lengths and time of measurement. By this procedure, the number of associated and diffusing myo V particles per microtubule unit length and time at the respective salt concentration (25, 50, 100 and 200 mM KCl) was obtained. Quantification on S-microtubules is based on data obtained from experiments in 25 mM KCl. Steady-State ATPase assays Microtubule- and actin-activated ATPase activity of myo V was determined in a coupled enzymatic assay [14,52] with a final MgATP concentration of 1 mM. ATPase activity assays for myo V on microtubules were performed with 100 nM myo V in Buffer F and increasing amounts of microtubules (0–35 mM in Buffer G). ATP hydrolysis by myo V (100 nM) in the presence or absence of F-actin (0–20 mM) was examined in Buffer F. Measurements were carried out in 96-well plates (Greiner, Frickenhausen, Germany) using a spectrophotometer (Biotek, Friedrichshall, Germany) at an excitation wavelength of 340 nm and 23uC. The data was analyzed with Kaleidagraph 3.6 (Synergy Software, Reading, PA) software and fitted to the Michaelis-Menten function [53]. On actin, only events of individual myo V molecules (n$25) walking along the filaments with an interaction time $2 s were classified as processive. The length of a processive run was determined manually with CellR software (Olympus Biosystems, Planegg, Germany). Velocities and runlength distributions were obtained from the single Gaussian fit according to equation 1 and the single exponential fit (as for tA of myo V on microtubules), respectively. PLoS ONE \| www.plosone.org Image and data analysis For details of the conditions for counting see Methods. n.a., not applicable. (DOC) represents the median. As expected for one-dimensional diffusion motions, no net displacement for the respective constructs was observed and hence all respective median values center at zero. (B– D) The mean-squared displacement (MSD) data of myo V Wildtype, Minus4 and Minus13 is plotted versus time, with the individual slopes providing an estimate of the respective D-values. The following D- values were calulated: DWt = 0.11 mm2/s (60.004 mm2/s S.D.), DMinus4 = 0.06 mm2/s (60.002 mm2/s S.D.) and DMi- nus13 = 0.07 mm2/s (60.002 mm2/s S.D.). Data were obtained from single-molecule TIRFM experiments with 100 nM Cy3-labeled myo V on Atto488-labeled microtubules in 25 mM KCl. Error bars represent the S.E.M. of the squared displacement values. Diffusive motion of myo V constructs on microtubules. Color-code: myo V Wildtype (black), Minus4 (blue) and Minus13 (green). (TIF) represents the median. As expected for one-dimensional diffusion motions, no net displacement for the respective constructs was observed and hence all respective median values center at zero. (B– D) The mean-squared displacement (MSD) data of myo V Wildtype, Minus4 and Minus13 is plotted versus time, with the individual slopes providing an estimate of the respective D-values. The following D- values were calulated: DWt = 0.11 mm2/s (60.004 mm2/s S.D.), DMinus4 = 0.06 mm2/s (60.002 mm2/s S.D.) and DMi- nus13 = 0.07 mm2/s (60.002 mm2/s S.D.). Data were obtained from single-molecule TIRFM experiments with 100 nM Cy3-labeled myo V on Atto488-labeled microtubules in 25 mM KCl. Error bars represent the S.E.M. of the squared displacement values. Diffusive motion of myo V constructs on microtubules. Color-code: myo V Wildtype (black), Minus4 (blue) and Minus13 (green). (TIF) Table S3 Summary of association and diffusion of various constructs on S-microtubules. Values for microtu- bule association and diffusion were calculated as described in Table S2. Data were obtained from single-molecule studies on subtilisin-treated microtubules (S-microtubules) in 25 mM KCl. Significance levels in association and diffusion on S-microtubules vs. untreated microtubules (P.0.05, P,0.05 and *P,0.005, Table S2) were determined using Student’s t-Test. For details of the counting conditions applied see Methods. (DOC) Video S1 One-dimensional diffusion of Wildtype myo- sin V on microtubules under high ionic strength. Cy3- labeled myosin V Wildtype (bright particles) was infused into a flow cell containing surface-attached Atto 488-labeled microtubules (dim filaments). Assay was performed in 100 mM KCl. Image and data analysis Diffusion events were defined as follows: Those events in which the myo V moves on microtubules in both directions (for .300 nm) were classified as diffusive events. For the characterization of myo V diffusion on microtubules, the following parameters were determined: (i) maximum speed of diffusion for a given encounter was defined as the maximum displacement along the microtubule during one frame interval (i.e., 5 frames per s); (ii) scan distance for a given encounter was calculated as the distance between the two extreme positions of the microtubule, on which the respective myo V molecule has diffused along; (iii) association time (tA) was defined as the total time an individual myo V molecule spent on the microtubule during recording. The individual values for tA of the respective myo V constructs were obtained from an exponential fit as described [20]. The mean square displacement (MSD) for all diffusion events was calculated as described in [54], plotted as a function of time and fitted to linear function. Single displacements of individual diffusion events for a given myo V construct were plotted as a displacement histogram. A single Gaussian was fitted to the data according to equation 1. y~a: exp : { x{x0 ð Þ=b ð Þ2 h i ð1Þ Figure S2 Diffusive motion of myosin V constructs on microtubules. (A) Box-Whisker plot of the diffusion-derived displacement distribution for myo V on microtubules. Upon the analysis of TIRFM movie sequences of single myo V molecules on microtubules in 25 mM KCl, single displacements between successive image frames were determined (Figure 2B). The displacement distribution of the respective myo V constructs (as indicated) is plotted as box-whisker plot, where the top and bottom of the boxes indicate the 75 and 25 percentile, the whiskers indicate the 90 and 10 percentile, while the solid line within the boxes ð1Þ Based on the obtained Gaussian fit curve for the respective displacement histogram, the variance s = b2 was calculated. From these data, the diffusion coefficient (D) was determined according to the 1st law of diffusion-derived equation, D = s/2t, where t is the time interval between successive images. Kymographs of representative movie -sequences from the performed single-molecule TIRFM assays on actin and microtu- PLoS ONE \| www.plosone.org September 2011 \| Volume 6 \| Issue 9 \| e25473 8 E-Hook-Independent Diffusion of Myosin V number of microtubule-associated particles (right column). Image and data analysis (A) The distribution of the values for tA is plotted as histogram with an exponential curve fit (solid line), yielding a mean tA-value of 12.4 s (n=20). (B) In this graph, the displacement distribution of myo V Wildtype on S-microtubules is plotted as Box-Whisker Plot, where the top and bottom of the boxes indicate the 75 and 25 percentile, the whiskers indicate the 90 and 10 percentile, while the solid line within the box represents the median. As it was observed for myo V Wildtype on untreated microtubules (Figure S2A), also on S-microtubules myo V Wildtype exhibits no net displacement during diffusion and hence the median centers at zero. (TIF) Video S3 One-dimensional diffusion of Minus13 myosin V on microtubules. Cy3-labeled myosin V Minus13 (bright particles) was infused into a flow cell containing surface-attached Atto 488-labeled microtubules (dim filaments). Assay was per- formed in 25 mM KCl. Excitation wavelength was 532 nm and representative image sequences were false-colored. This movie (272 frames) was recorded at 5 frames s21 and is displayed at three-fold speed. Scale bar represents 2 mm. (AVI) Table S1 Summary of behavior of various constructs on F-actin in 25 mM KCl. Velocities and runlengths of the single- molecule measurements on F-actin were obtained at 1 mM ATP. Values for velocity and runlength are mean 6 S.E.M. from Gaussian and exponential fits to the data (Figure S1, A and B), respectively. n is the number of processive runs. Km represents the actin concentration at which the ATPase rate is half the maximal rate, determined from the Michaelis-Menten curve fit (Figure S1C). kcat shows the maximum rate of ATP turnover as determined from fitting the data to the Michaelis-Menten equation (Figure S1C). n.m., not measurable. (DOC) Video S4 One-dimensional diffusion of Wildtype myosin V on microtubules lacking E-hooks. Cy3-labeled myosin V Wildtype (bright particles) was infused into a flow cell containing surface-attached Atto 488-labeled subtilisin-treated microtubules (dim filaments). Assay was performed in 25 mM KCl. Excitation wavelength was 532 nm and representative image sequences were false-colored. This movie (328 frames) was recorded at 5 frames s21 and is displayed at three-fold speed. Scale bar represents 2 mm. (AVI) Image and data analysis Excitation wavelength was 532 nm and representative image sequences were false-colored. This movie (89 frames) was recorded at 5 frames s21 and is displayed at three-fold speed. Scale bar represents 2 mm. (AVI) Figure S3 Interaction lifetime of diffusing myosin V on microtubules. (A–D) The distribution of the association times (tA) for myo V Wildtype, Minus4 and Minus13 were plotted as histograms. Data were obtained from single-molecule TIRFM experiments with 100 nM Cy3-labeled myo V on Atto488-labeled microtubules in 25 mM KCl. Exponential curves fitted to the respective histograms (solid lines) yield mean tA-values of 14.1 s (n = 31), 23.8 s (n = 35) and 16.2 s (n = 32) for myo V Wildtype (A), Minus4 (B) and Minus13 (C), respectively. (TIF) Video S2 One-dimensional diffusion of Wildtype myo- sin V on microtubules. Cy3-labeled myosin V Wildtype (bright particles) was infused into a flow cell containing surface-attached Atto 488-labeled microtubules (dim filaments). Assay was per- formed in 25 mM KCl. Excitation wavelength was 532 nm and representative image sequences were false-colored. This movie (236 frames) was recorded at 5 frames s21 and is displayed at three-fold speed. Scale bar represents 2 mm. (AVI) Figure S4 One-dimensional diffusion behavior of myosin V Wildtype on microtubules lacking the E-hook. (A and B) Data were obtained from single-molecule TIRFM experiments with 100 nM Cy3-labeled myo V Wildtype on subtilisin-treated microtu- bules (Atto488-labeled) in 25 mM KCl. (A) The distribution of the values for tA is plotted as histogram with an exponential curve fit (solid line), yielding a mean tA-value of 12.4 s (n=20). (B) In this graph, the displacement distribution of myo V Wildtype on S-microtubules is plotted as Box-Whisker Plot, where the top and bottom of the boxes indicate the 75 and 25 percentile, the whiskers indicate the 90 and 10 percentile, while the solid line within the box represents the median. As it was observed for myo V Wildtype on untreated microtubules (Figure S2A), also on S-microtubules myo V Wildtype exhibits no net displacement during diffusion and hence the median centers at zero. (TIF) Figure S4 One-dimensional diffusion behavior of myosin V Wildtype on microtubules lacking the E-hook. (A and B) Data were obtained from single-molecule TIRFM experiments with 100 nM Cy3-labeled myo V Wildtype on subtilisin-treated microtu- bules (Atto488-labeled) in 25 mM KCl. Acknowledgments We thank Thorsten Hugel, Gu¨nther Woehlke and Matthias Rief (Biophysics Department, TU Munich, Germany) for critical discussions; Thi-Hieu Ho and Renate Dombi for excellent technical assistance; GW for providing the TIRF microscope; and MR for his helpful critiques of our manuscript. Table S2 Summary of microtubule association and diffusion of various constructs at increasing salt-concen- trations. Values for microtubule association were calculated as mean 6 S.E.M. from the total count of microtubule-associated (stationary and diffusing) particles per unit length and time at the indicated salt-concentrations (left column). Among those, the diffusing fraction of motors was determined and calculated as mean 6 S.E.M. of the total number of diffusing motors per unit length and time. The portion of diffusing particles is expressed in % of the total References Cooper JR, Wordeman L (2009) The diffusive interaction of microtubule binding proteins. 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https://openalex.org/W2004279498	https://europepmc.org/articles/pmc3661875?pdf=render	English	null	What’s new for ESC Congress 2013? (Amsterdam 30 August – 4 September 2013)	Netherlands heart journal	2,013	cc-by	817	Neth Heart J (2013) 21:304–306 DOI 10.1007/s12471-013-0419-6 Neth Heart J (2013) 21:304–306 DOI 10.1007/s12471-013-0419-6 SPECIAL ARTICLE SPECIAL ARTICLE Keywords Heart The innovative Spotlight of the Congress is ‘The heart interacting with systemic organs’. For our patients, the interaction of cardiac conditions with other organs is funda- mentally important to outcome, to safety and to clinical management. Related speciality areas have much to learn from each other and the ESC Congress 2013 will attract specialists from other organ systems to help understand disease mecha- nisms and improve the management of our patients. What’s new for ESC Congress 2013? (Amsterdam 30 August – 4 September 2013) K. A. A. Fox Published online: 19 April 2013 Published online: 19 April 2013 Published online: 19 April 2013 # The Author(s) 2013. This article is published with open access at Springerlink.com # The Author(s) 2013. This article is published with open access at Springerlink.com Abstract The innovative Spotlight of the Congress is “The heart interacting with systemic organs”. For our patients, the interaction of cardiac conditions with other organs is funda- mentally important to outcome, to safety and to clinical management. Related specialty areas have much to learn from each other and the ESC Congress 2013 will attract specialists from other organ systems to help understand disease mechanisms and improve the management of our patients. experts and thought leaders. For the first time this year we have ‘rapid fire’ posters followed by interactive discussion around an electronic display featuring the key findings from each of the presented studies (Figs. 1, 2 and 3). Also for the first time, poster presentations will be stimulated by discus- sants who are experts in that field of investigation. Another innovation is ‘Meet the Legends in Cardiology’, where participants will be able to post questions in advance or during the session to individuals who have changed our understanding and helped determine the shape of cardiology in 2013. ‘Villages’ of linked topics There is huge and international enthusiasm for partici- pation in the congress with a record number of sub- missions for scientific planned sessions (more than 400 were selected) and the second highest ever number of abstracts were submitted (10490). To make the congress more manageable we have again arranged the congress into ‘villages’ of related topics. Thus, a village may include presentations in one room on heart failure and K. A. A. Fox () Centre for Cardiovascular Science, University of Edinburgh, 49 Little France Crescent, Edinburgh EH16 4SB, UK e-mail: k.a.a.fox@ed.ac.uk A personalised and interactive congress Fig. 1 (ESC 2351) Discussions around a poster at ESC Congress At ESC Congress 2013 we can each develop a personalised curriculum, depending on speciality interests and training needs. This is a truly international meeting - the largest number of abstract submissions were from Japan! We have joint sessions with many societies around the world and 16 linked sessions and direct opportunities to interact with K. A. A. Fox () Centre for Cardiovascular Science, University of Edinburgh, 49 Little France Crescent, Edinburgh EH16 4SB, UK e-mail: k.a.a.fox@ed.ac.uk Fig. 1 (ESC 2351) Discussions around a poster at ESC Congress Neth Heart J (2013) 21:304–306 305 Fig. 2 (ESC 3704) Round table discussions at ESC cardiac pacing, diabetes, and stable coronary artery disease. Linked to these will be dedicated scientific sessions, case- based Focus Sessions. Why should someone come and participate in the congress? Summaries of the Hotlines will appear online but to really understand the significance of the new research we need to discuss the findings with our colleagues. As National Society members we are all also members of the ESC! The ESC Congress provides the opportu- nity to decide what may influence understanding of innovations and practice at a national level. Editors of National Society Cardiovascular journals are Fig. 2 (ESC 3704) Round table discussions at ESC in an adjacent room on heart rhythm disturbances. Through the village concept the congress becomes more man- ageable and interactive. Important new guidelines will be presented at the ESC Congress 2013 on arterial hypertension, Fig. 3 Summary features of the ESC Congress 2013 Hub (ESC website) Fig. 3 Summary features of the ESC Congress 2013 Hub (ESC website) 306 Neth Heart J (2013) 21:304–306 the choice: be part of the frontiers in cardiology and prac- tice, or be an observer! the choice: be part of the frontiers in cardiology and prac- tice, or be an observer! provided with the Hotline presentations and the Hotline extracts can be translated and printed in local cardio- vascular journals. 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. The ESC Congress 2013 is the largest cardiovascular international congress and its true international nature pro- vides exciting and innovative opportunities to discuss and interact with colleagues from all around the world. You have
https://openalex.org/W4220972219	https://zenodo.org/record/6340275/files/estimationTBE.pdf	English	null	Autoregressive models for time series of random sums of positive variables: application to tree growth as a function of climate and insect outbreaks	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	9,951	Autoregressive models for time series of random sums of positive variables: application to tree growth as a function of climate and insect outbreaks Autoregressive models for time series of random sums of positive variables: application to tree growth as a function of climate and insect outbreaks Email: zinsou-max.debaly@ensai.fr, philippe.marchand@uqat.ca, miguel.montoro@uqat.ca Zinsou Max Debaly,a Philippe Marchandb and Miguel Montoro Gironab,c,d aCREST-ENSAI, UMR CNRS 9194, , , Campus de Ker-Lann, 51 rue Blaise Pascal, BP 37203, 35 Campus de Ker-Lann, 51 rue Blaise Pascal, BP 37203, 35172 Bruz cedex, bINSTITUT DE RECHERCHE SUR LES FORÊTS, 445, boul. de l’Université Rouyn-Noranda QC J9X 5E4 , Canada. cGROUPE DE RECHERCHE EN ECOLOGIE DE LA MRC-Abitibi (GREMA) 341, rue Principale Nord, Amos, QC, J9T 2L8, Canada13 cGROUPE DE RECHERCHE EN ECOLOGIE DE LA MRC-Abitibi (GREMA) 341, rue Principale Nord, Amos, QC, J9T 2L8, Canada13 cGROUPE DE RECHERCHE EN ECOLOGIE DE LA MRC-Abitibi (GREMA) 341, rue Principale Nord, Amos, QC, J9T 2L8, Canada13 dRESTORATION ECOLOGY RESEARCH GROUP, Department of Wildlife, Fish and En- vironmental Studies, Swedish University of Agricultural Sciences, Skogsmarksgränd, Umeå 907 36, Sweden dRESTORATION ECOLOGY RESEARCH GROUP, Department of Wildlife, Fish and En- vironmental Studies, Swedish University of Agricultural Sciences, Skogsmarksgränd, Umeå 907 36, Sweden Email: zinsou-max.debaly@ensai.fr, philippe.marchand@uqat.ca, miguel.montoro@uqat.ca Email: zinsou-max.debaly@ensai.fr, philippe.marchand@uqat.ca, miguel montoro@uqat ca Abstract. We present a broad class of semi-parametric models for time series of random sums of positive variables. Our methodology allows the number of terms inside the sum to be time-varying and is therefore well suited to many examples encountered in the natural sciences. We study the stability properties of the models and provide a valid statistical inference procedure to estimate the model parameters. It is shown that the proposed quasi- maximum likelihood estimator is consistent and asymptotically normally distributed. This work is complemented by simulation results and applied to annual growth rate time series of white spruce (Picea glauca) trees from a few dozen sites in Quebec spanning 41 years, including one major spruce budworm (Choristoneura fumiferana) outbreak from around 1968 to 1991. We found significant growth reductions due to budworm-induced by defoliation up to two years in the past. Our results also revealed positive effects of maximum temperature, precipitation and the climate moisture index in the summer, as well as negative effects of the climate moisture index in the spring and the maximum temperature in the previous summer. However, considering the interaction of climate and defoliation on growth did not improve the model’s performance on this dataset. Zinsou Max Debaly,a Philippe Marchandb and Miguel Montoro Gironab,c,d This study represent a major advances and our result represent an useful tool in the understanding of the combined effects of climate and insect defoliation on tree growth in the face of climate change, where the frequency and the severity of outbreaks, as well as an increase of temperature is expected. Keywords: dendrochronology, ecological modelling, natural disturbances, quasi-likelihood estimation, semi-parametric autoregressive models . 1 Introduction In many studies in ecology, we measure positive dependent variables on random numbers of statistical individuals sampled over time (Montoro Girona et al., 2019). One can find two main reasons why: first, researchers cannot observe the whole population; second, which individuals researchers can observe depends on their time-varying resources. Examples range from species behaviour to ecological services. For instance, in forestry, one can be interested in the time series of mass or size of certain species, taking a random sample of trees each year and observing the corresponding quantity (see Vourlitis et al. (2022) for example). Another – 2 – example consists in the occupied land area of colonies in relationship with the availables resources across time (Labrecque-Foy et al., 2020). In fisheries, scientists often investigate the temporal changes in fish catch weight, see Chan et al. (2020) for more details. In this paper, we contribute to the ongoing ecological study of the impact of climate change and insect outbreaks on tree growth, as measured by growth rings. Spruce budworm (Choristoneura fumiferana; SBW) outbreak is the most important defoliator of conifer trees in the North American boreal forest (Montoro Girona et al., 2018). Only in the province of Quebec (Canada), the forest surface affected by this species of Lepidoptera over the last century is twice the size of Ukrania (Navarro et al., 2018). At the epidemic stage, massive populations of larvae cause widespread damage to tree foliage: defoliation (Lavoie et al., 2019). SBW affect the main conifer boreal species in Canada, balsam fir (Abies balsamea), white spruce (Picea glauca) and black spruce (Picea Mariana). For this reason, SBW has a major impact in the regeneration and dynamics of boreal forest (Martin et al., 2020). However, SBW outbreaks periods, not only have major ecological impacts but also produce important economic consequences due to the loss of forest productivity. Previous works in this field have studied the changes of forests composition following insect outbreaks (see for instance Morin et al. (2021)), the response of SBW outbreaks to climate change (Fleming and Volney (1995) and Berguet et al. (2021)) and the demography i.e the rate of mortality of spruce during outbreaks (Gauthier et al., 2015). However, even the major implication in the face of climate change, there is limited knowledge regarding the combined effects of outbreaks and climate on tree growth. 1 Introduction Given that variations of temperature and precipitation affect organisms’ survival, reproduction cycles and spatial dispersion (Aber et al., 2001), it is critical to understand the links between past SBW outbreaks, climate and tree growth to understand how future climate change scenarios would impact forest productivity during outbreaks (Klapwijk et al., 2013). This is a major concern due to the increase in the severity of frequency excepted in the future for SBW outbreaks (Navarro et al., 2018; Seidl et al., 2017). We contribute to filling this gap by proposing a broad class of semi-parametric for positive-valued time series. Indeed, even though this type of data is common in forestry, the statistical approaches commonly used suffer from several drawbacks. These approaches range from descriptive exploratory techniques to linear mixed-effect models with time-varying vari- ables on transformed data (see Montoro Girona et al. (2016) and Boulanger and Arseneault (2004) for example) and correlated error terms (Girardin et al., 2016). Whereas the first one – 3 – (exploratory techniques) do not allow us to draw inferences, we can note at least two limits for the latter one. Firstly, as pointed out by several papers, see for example Chou et al. (2015), specifying a linear model on transformed data often leads to worst performances in prediction. Secondly, models with autocorrelated error terms do not take into account the complex dependency structure of tree-ring growth. The class of semi-parametric au- toregressive models we present here will be applied to investigate the relationship between climate, insect outbreaks and growth of white spruce. It also presents the advantage of accommodating the usual repeated measures design in ecology. Many previous works have focused on modelling non-Gaussian time series, such as positive-valued processes. Indeed, Gaussian processes can be represented as linear models, whereas time series of count or binary data are modelled by non-linear dynamics, see for example Sim (1990) and references therein. For positive-valued time series data, the range volatility model was proposed by Engle and Russell (1998) as an alternative for garch model in finance and its use has been rapidly expanding due to its various applications. We refer the interested reader to the review of Chou et al. (2015). Recently, Aknouche and Francq (2020) have considered a positive-valued time series whose conditional distribution has a time-varying mean that can depend on exogenous variables. 1 Introduction Our approach here is slightly different from theirs, since the positive process under consideration is itself the sum of a random number of other positive variables. It is strongly driven by the data we have to deal with. Indeed, these data consist of multiple time series collected over several ecological sites, where the number of individuals sampled changes over time as well as across sites. Hence, considering an aggregate value like the sum or the mean of growth rings leads to the loss of variability linked to the sampling schema. Moreover, one can note that in different fields like finance, some modeling strategies consisting in considering empirical quantities such as the realized volatility are employed. Historical returns of investment products within a defined time period are then analysed (see for example Allen et al. (2010)). However, unlike our framework and ecological studies in general, all transactions on investment products are recorded, i.e the whole statistical population is observed. The rest of this paper is organised as follows. In Section 2, we define the model under consideration throughout this paper and discuss our modelling choices. Time-series proper- ties of the models are also studied in that section. Maximum-likelihood based inference and its asymptotics properties are presented in Section 3. Section 4 contains a small simulation study and an application to empirical data on the growth of white spruce. All auxiliary – 4 – lemmas and mathematical proofs are contained in Section 5. lemmas and mathematical proofs are contained in Section 5. 2 Models and stability results We introduce here a generalized linear dynamic model for time series of random sum of pos- itive variables, motivated by the empirical application where we analyze the annual growth of spruce trees subject to climate variation and outbreaks of SBW. In this case, growth is measured by taking cores at 1.30 m heigh from the trunk of a sample of trees in a forest (Montoro Girona et al., 2017). The samples were prepared, measured and analyzed conform- ing to standard dendroecological protocol (Krause and Morin, 1995). Cores were air-dried, mounted on wood boards and sanded before tree rings were measured with WinDendro sys- tem (Guay et al., 1992) or a manual Henson micrometer with an accuracy of 0.01 mm. The tree-ring series measurements covered the last 41 years, and were cross-dated using TSAP-Wi (Rinntech, Heidelberg, Germany). We denote by Yk,t, t ∈Z, k = 1, . . . , K the time series of the total basal area increment related to the k−th observational site, i.e. the sum of the increases in trunk cross-sectional area for the nk,t trees sampled for site k on year t. We aim to model the dynamics of this process both in terms of its own past and m additional covariates Xk,t ∈Rm. In the empirical application presented in section 4, the covariate process encompasses climate variables such as temperature and precipitation, as well as the level of defoliation due to SBW in previous years. Our model is given by : Yk,t\|nk,t, Xk,t, n− k,t, Y − k,t = nk,t X l=1 ζl,k,t Yk,t\|nk,t, Xk,t, n− k,t, Y − k,t = nk,t X l=1 ζl,k,t (2.1) (2.1) where conditionally on nk,t, Xk,t, n− k,t = (nk,t−s, s ≥1) and Y − k,t = (Yk,t−s, s ≥1), the variables ζl,k,t, 1 ≤l ≤nk,t, representing the basal area increments of individual sampled trees, are independent and identically distributed like a random variable ζk,t of mean λk,t. Moreover, (nk,t)t∈Z is a sequence of i.i.d random variables and conditionally on n− k,t, the variable nk,t is independent from Xk,t and Y − k,t. The mean process is written φδ(λk,t) =: ηk,t = ωk + p X j=1 αj Yk,t−j nk,t−j + β⊤Xk,t, k = 1, . . . , K and t = 1, . . . , T, (2.2) (2.2) – 5 – ωk ∈R, αj ∈R, β = (β1, . . . 2 Models and stability results , βm) ∈Rm and φδ is a real-valued function defined on R+ that can depend on a parameter δ. It is worth mentioning, without loss of generality, that the covariate process considered at time t is included in the specification of λk,t since multiple lags of a given set of variables can be included by simply stacking them into a vector. It is for example the case of the defoliation level in our application, since growth can be affected by defoliation up to 5 years prior (from t −5 to t −1). The variables ζk,t will be referred to as the unity random variables. We do not make any assumption about the distribution of the variables ζk,t. Any distribution on (0, +∞) can be chosen. For example, an Exponential distribution with parameter 1/λk,t, log-Normal distribution with parameters log λk,t −σ2/2 and σ or a Gamma distribution with parameters αλk,t and α, to name a few. Whatever the distributions of unity random variables are, the conditional expectation of Yk,t is nk,tλk,t. However, under the assumption of the indenpen- dence of ζl,k,t, 1 ≤l ≤nk,t, if they are exponentially distributed, the conditional variance is nk,tλ2 k,t i.e a quadratic function of λk,t. For our example of Gamma-distributed unity ran- dom variables, the conditional variance is nk,tλk,t/α, i.e. a linear function of λk,t. But in the case of the log-Normal distribution, the conditional variance is nk,tλ4 k,t(exp σ2 −1). With our semi-parametric framework, we will only focus on the estimation of regression parameters θ = (δ, ω1, . . . , ωK, α1, . . . , αp, β⊤)⊤without the need to perform any distributional goodness of fit test. Notes 2.1 Copies of unity variables In our general set up, the copies ζl,k,t, 1 ≤l ≤nk,t of the unity random variables ζk,t are not required to be independent. In practice where for example ζl,k,t represents the measure of annual growth for a sampled tree, the general assumption of identical distribution can be thought as a local stationary condition inside the site k at time t. Notes 2.2 Marginal stationary distributions , p for some mapping h such that Eh(Yk,t−i/nk,t−i, λk,t−i) = 0 in (2.2) at the place of Yk,t−i/nk,t−i, i = 1, . . . , p. Indeed, with the latter two mentioned specifications, (2.1)-(2.2) define the so-called GLARMA model (see for example, Weiß (2018) for more details). In the present form (2.1)-(2.2) has some similarities with the well known ARCH model (Bollerslev (1986)). We leave the topic of GLARMA specification for furthers works. Notes 2.4 Contrast with the non-linear mixed model The model (2.1)-(2.2) has some similarities with the well-known mixed models. Indeed, as for mixed models, the ωk stands for the site fixed effect and the random effect in embedded is the distribution of unity variables. The simple example of ζl,k,t = λk,tϵl,k,t, where (ϵl,k,t)l≥1 is a sequence of identically distributed random variables of mean 1, fit with the so-called multiplicative form random effect models (Cameron and Trivedi, 2013). But more complex random effects can be handled. However, the model (2.1)-(2.2) is more general since it allows the individuals sampled over time to change. Indeed, as we will see in section 3, the individual measures are no longer needed when the sequence (Yk,t, nk,t) are available. Also, in terms of the application to resource management, it is often of interest to model and predict a population quantity like the sum of basal area growth in a forest. Notes 2.2 Marginal stationary distributions Notes 2.2 Marginal stationary distributions Note from equations (2.1)-(2.2), Yk,t = fθk(Xk,t−s, nk,t−s, ζℓ,k,t−s, s ≥0, ℓ≥1) for θk = (δ, ωk, α1, . . . , αp, β⊤)⊤. Then for 1 ≤ k1 ̸= k2 ≤K the distributions of (Yk1,0, nk1,0, Xk1,0) and (Yk2,0, nk1,0, Xk2,0) are not equal unless ωk1 = ωk2 and (Xk1,0, ζℓ,k1,0, ℓ≥1) is equal in distribution to (Xk2,0, ζℓ,k2,0, ℓ≥1). We will investigate in section 4 the consequences of the latter conditions on the proposed estimation procedure. Notes 2.3 Form of regression function (2.2) Note that ηk,t in (2.2) does not de- pend linearly on Yk,t−i, i = 1, . . . , p, but on Yk,t−i/nk,t−i, i = 1, . . . , p. In fact, through – 6 – (2.2), we make a link between the underlying mean process and the empirical estimate of the past mean process. Even for a constant size process, i.e. nk,t = nk, ∀t, since the regression parameter αi, i = 1, . . . , p is free of k, we still cannot yet express ηk,t as a lin- ear combination of Yk,t−i, i = 1, . . . , p. Moreover, one can expect Yk,t−i/nk,t−i −λk,t−i, i = 1, . . . , p or more generally h(Yk,t−i/nk,t−i, λk,t−i), i = 1, . . . , p for some mapping h such that Eh(Yk,t−i/nk,t−i, λk,t−i) = 0 in (2.2) at the place of Yk,t−i/nk,t−i, i = 1, . . . , p. Indeed, with the latter two mentioned specifications, (2.1)-(2.2) define the so-called GLARMA model (see for example, Weiß (2018) for more details). In the present form (2.1)-(2.2) has some similarities with the well known ARCH model (Bollerslev (1986)). We leave the topic of GLARMA specification for furthers works. (2.2), we make a link between the underlying mean process and the empirical estimate of the past mean process. Even for a constant size process, i.e. nk,t = nk, ∀t, since the regression parameter αi, i = 1, . . . , p is free of k, we still cannot yet express ηk,t as a lin- ear combination of Yk,t−i, i = 1, . . . , p. Moreover, one can expect Yk,t−i/nk,t−i −λk,t−i, i = 1, . . . , p or more generally h(Yk,t−i/nk,t−i, λk,t−i), i = 1, . . . Choice of the link function φ The logarithmic link function is often applied and coincides with the well known log-linear model, see for example Cameron and Trivedi (2013) for models for count data. This link function assumes a linear relationship between the logarithm of the mean process and the co- variates. However, there exist some other link functions that preserve the linear correlation at least on the positive part of R. Consider for example, the threshold mapping x 7→max(x, 0). This mapping is not smooth and most of time, one makes some restrictions on model pa- rameters to directly obtain the positiveness of the mean. Here, we will apply the inverse of the so-called softplus function as a link function. Indeed, the softplus function (see Glorot et al. (2011)) is interesting for two reasons. The first one related to modelling is that it pre- – 7 – serves the linearity on the positive part of real line. This is also pertinent for our biological application, as we expect a linear effect of covariates on growth above a certain threshold rep- resenting the minimal favorable conditions for growth. The minimum growth expected may not be exactly zero, which is why we will consider later a slightly different version of softplus that we will refer to as softplusδ for δ > 0 defined as softplusδ(x) = log(1 + δ + exp(x)). The second one and technical advantage is that the mapping softplusδ is infinitely differentiable. The Figure 1 in the Appendix shows the difference between the softplusδ link function and max(x, 0) where softplus stands for softplus0. One can note that softplusδ is lower bounded by log(1 + δ). Notes 2.5 Model Interpretation Obviously, with the softplusδ link function, the mean process increases with the j−th covariate process if βj > 0 and decreases with this one when βj < 0. Since softplusδ(x) ∼∞x, the mean process can be approximated, all other things remaining equal, by βjXj,t for large values of Xj,t and βj > 0 and then increases by βjα for increasing value α of Xj,t. Let us denote by RG(x, y), the relative rate of growth of the mean process between x and y i.e RG(x, y) = γδ(x)/γδ(y) where γδ is the derivative function of softplusδ. For β < 0, limx→∞RG(β(x + α), βx) = eβα. Therefore, the rate toward log(1 + δ) driven by Xj,t is given by eβjα when βj < 0. Choice of the link function φ Theorem 1 gives some stability conditions of model (2.1)-(2.2) with the inverse of the softplus function as the link whereas Lemma 1 in the section 5 stands for a general result for φ. Theorem 1. Under the assumptions (ST.1)-(ST.2) in section 5 and Pp j=1 \|αj\| < 1, there exists a unique set of K stationary, ergodic sequences (Yk,t, nk,t, Xk,t), k = 1, . . . , K that are the solution of equations (2.1)-(2.2) with E\|ηk,0\| < ∞, k = 1, . . . , K. Theorem 1. Under the assumptions (ST.1)-(ST.2) in section 5 and Pp j=1 \|αj\| < 1, there exists a unique set of K stationary, ergodic sequences (Yk,t, nk,t, Xk,t), k = 1, . . . , K that are the solution of equations (2.1)-(2.2) with E\|ηk,0\| < ∞, k = 1, . . . , K. 3 Estimation and asymptotics properties This section is devoted to the estimation of the conditional mean parameters by the Quasi- Maximum Likelihood Estimator (QMLE) based on a member of the exponential family. We consider the Exponential QMLE (EQMLE) because this estimator coincides with the Maxi- mum Likelihood Estimator (MLE) when the unity random variables follow the Exponential Γ(1, λ−1 k,t) distribution and the copies ζl,k,t, 1 ≤l ≤nk,t are independent. For our application of the model, the K time series are observed between the time points 1 and T. We provide an asymptotic theory for the estimated parameters and present the – 8 – results of a small simulation study investigating the finite-sample properties of the estimator. In the following, we will make λk,t depend on the parameter θ(∈Θ a compact set); that is log(exp ◦λk,t(θ)−1−δ) = ωk+ p X j=1 αj Yk,t−j nk,t−j +β⊤Xk,t =: ηk,t(θ), k = 1, . . . , K and t = 1, . . . , T, where δ ≥δ_ > 0. Let us denote the true, data-generating parameter value by θ0. The loss function from the Exponential quasi-maximum likelihood is given by : rT(θ) = K X k=1 T −1 T X t=1 Yk,t λk,t(θ) + nk,t log ◦λk,t(θ) =: K X k=1 T −1 T X t=1 ℓk,t(θ) =: K X k=1 ℓk(θ) (3.1) and ˆθT = argmin θ∈Θ rT(θ). (3.2) ˆθT = argmin θ∈Θ rT(θ). (3.2) The derivative of λk,t(θ) with respect to θ is given by : ∂λk,t(θ) ∂θ =: ˙λk,t(θ) = 1 1 + δ + eηk,t(θ), eηk,t(θ) 1 + δ + eηk,t(θ) ιk, Yk,t−1 nk,t−1 . . . Yk,t−p nk,t−p , X⊤ k,t ⊤ . where ιk is a vector of size K with 1 at the k−th position and 0 elsewhere. We will denote by ˙λk,t (resp. λk,t) the vector ˙λk,t(θ) (resp. λk,t(θ)) evaluated at the point θ = θ0. t (resp. λk,t) the vector ˙λk,t(θ) (resp. λk,t(θ)) evaluated at the point θ = θ0. We will study the asymptotic properties of the QMLE estimator (3.2). To do so, we employ Taniguchi and Kakizawa (2002) (Thm 3.2.23), which was extended in Klimko and Nelson (1978). The lemmas in the section 5 give the general result for the asymptotic prop- erties of QMLE (3.2). The following theorem stands for the consistency and the asymptotic normality of (3.2) for softplus −δ link function. 3 Estimation and asymptotics properties Let us set Vk = E " 1 λ2 k,0 nk,0 −Yk,0 λk,0 2 ˙λk,0 ˙λ⊤ k,0 # and Jk = E " nk,0 1 λ2 k,0 ˙λk,0 ˙λ⊤ k,0 # . Theorem 2. Suppose that the assumptions (C.1)-(C.4) in section 5 are met. Then, almost – 9 – surely, y lim T→∞ ˆθT = θ0. lim T→∞ ˆθT = θ0. lim T→∞ ˆθT = θ0. If in addition (AN.1)-(AN.3) hold true and θ0 is located in the interior of Θ, If in addition (AN.1)-(AN.3) hold true and θ0 is located in the interior of Θ, lim T→∞ √ T(ˆθT −θ0) = N(0, J−1V J−⊤) where J = PK k=1 Jk and V = PK k=1 Vk. 4.1 Simulation We examined the finite-sample performance of the QMLE presented in the previous section through a small simulation study. We present the result for QMLE under two different data generating processes referred to as scenario 1 and scenario 2. For the first one, the number of covariates is m = 10; Xk,t does not depend on k and is a sequence of i.i.d random variables distributed as exponential random variables of means λ1, . . . , λm. For the second one, for a fixed k, Xk,t is independently sampled from exponential distributions of mean 0.4kλ1, . . . , 0.4kλm. For the two data generating processes, for a fixed k, the process (nk,t)t≥1 is independently sampled from a Poisson distribution of mean τk. We sequentially choose K = 5, 10, 15, 20 and T = 50, 100, 200. The samples are nested, i.e the sample for the first scenario and K = 5, T = 50 is a subset of that of K = 5, T = 100. Indeed, we aim here to evaluate the consequences of increasing K and T on the performance of our estimator. For each sample, we compute the estimator (3.2) and the corresponding standard errors. The table 1 presents the simulation results. It appears that the model parameters are well estimated except for the ωk, k = 1, . . . , K when K is very small compared to T, which coincides here with K = 5, T = 50, 100, 200. It is worth noting that these results are partial since they are based on a one sampling schema. We leave deep simulation studies for further works. 4.2 Application to the white spruce growth series Dendrochronology, i.e. the studies of the time series of tree growth rings, is a powerful tool to reconstruct past natural and anthropic disturbances (Montoro Girona et al. (2016), Boulanger and Arseneault (2004) and Labrecque-Foy et al. (2020)). Tree-rings are hard disks – 10 – of information, able to record each environmental change, having thus a strong potential to understand complex phenomena such as disturbance ecology. Many previous studies used dendrochronological data to better understand insect outbreak dynamics (Navarro et al. (2018), Camarero et al. (2003) and Speer and Kulakowski (2017)). In this research, we used the dendroecological series from the study by Jardon et al. (2003), which includes annual tree-ring width measurements for 631 white spruce (Picea glauca) trees distributed across 45 sites in southwestern Quebec, Canada, with 1 to 23 trees per site. These time series comprise between 63 and 247 rings according to the tree’s age. We converted the ring width increments to basal area increments (BAI) using the full series, but due to covariate availability, we limit our analysis to the 1955-1995 time period (41 years) to study only one insect outbreak period (see fig 2 in Appendix). We interpolated climate variables at the study sites for the 41-year period using BioSIM (Régnière et al., 2014), a software package that interpolates daily climate station data based on latitudinal and elevational climate gradients, as well the spatial correlations estimated from 30-year climate normals. We computed the following climate summaries from daily data for the spring (April to June) and summer (July to September) seasons separately: mean of the daily maximum temperatures, total precipitation, and the climate moisture index (CMI) equal to the difference between precipitation and potential evapotranspiration (PET). Daily PET values were estimated by the Penman-Monteith equation as implemented in the SPEI package (Beguería and Vicente-Serrano, 2017) in R, based on BioSIM-interpolated values of the minimum and maximum temperature, wind speed at 2 m, solar radiation, dew point temperature and atmospheric pressure, using the "tall" crop model in SPEI. One major SBW outbreak occurred in Quebec during the study period, from 1967 to 1991. We obtained annual estimates of the severity of SBW outbreaks at the location of each study site from defoliation maps produced by the Quebec Ministry of Forests, Wildlife and Parks (MFFP). These maps are digitized versions of hand-drawn outlines of defoliated areas produced by aerial surveys of the affected regions. 4.2 Application to the white spruce growth series The defoliation level for each area is classified on a scale of 1 to 3 corresponding to a low (approx. 1 – 35%), moderate (36 – 70%) or high (71 – 100%) fraction of the year’s foliage defoliated by the SBW. We note that these defoliation levels mainly reflect the status of balsam fir (Abies balsamea) trees, which is the main SBW host and is generally more severely affected than white spruce. Therefore, these defoliation levels are a proxy for the outbreak severity, i.e. the potential herbivory pressure exerted by the budworm on spruce trees at the site. – 11 – Since tree growth and its vulnerability to both climate and defoliation depends on the tree-age, we split the dataset and separately fit our models for the following five age classes: < 75, 75 – 100, 100 – 125, 125 – 150 and > 150 years. We include as covariates the mean daily maximum of temperature, the total precipitation and the mean CMI for the current and previous spring and summer. Only one of precipitation and CMI appears in a given model version due to the correlation between those two variables. We also include as covariates the defoliation levels for the five previous years, a delay which estimates the time needed to fully regrow the lost foliage after an outbreak. Note that we do not expect defoliation to have a marked effect on the same year’s growth ring (Krause et al., 2003). Finally, we consider models with interaction effects of the previous year’s defoliation level and climate variables, representing the possibility that climate conditions can increase or decrease the tree’s sensitivity to SBW outbreaks. Data processing and analyses were performed in R Core Team (2021) with the package dplR (Bunn, 2008) used to process tree-ring data. We minimize the criterion (3.1) with the R command nlm (Dennis and Schnabel (1983)). All the developed software are made available under the Creative Commons (CC) license (see data availability statements). The model selection was carried out through the the QAIC criterion. The primary analysis based on partial autocorrelation plots leads us to select p = 1. According to the QAIC, the best models were those without an interaction between climate and defoliation. 4.2 Application to the white spruce growth series Our model results (Figures 3 and 4) reveal that higher defoliation levels leads to reduced tree-ring growth, but this effect vanishes after two years; however, note that while the direct effect vanishes, expected growth will remain lower in successive years due to the large estimated first-order autocorrelation coefficient (0.8 to 0.9, depending on age class). Moreover, there is no significant effect of defoliation on the next year’s growth for the youngest and oldest trees, even though it produces an effect two years following the defoliation. The result are quite different for middle aged trees, which are significantly affected one year following the defoliation but not in the second year. For the climate variables, high maximum temperatures in the summer produce increased growth, with up to 5.6 square centimetre increase in basal area from a 10 degree Celsius increase in summer maximum temperature. However, the previous summer’s temperature has a negative effect on growth. Finally, the spring CMI is negatively correlated with tree-ring growth whereas the summer CMI has a positive effect. However, both the CMI and precipitation in the previous spring increase tree-ring growth of the current year: an increase of 100 millimetres – 12 – n precipitation leads to at least a 6.8 square centimetre increase in basal area in precipitation leads to at least a 6.8 square centimetre increase in basal area growth. Proof of lemma 1 From (2.2), ηk,t = ωk + p X j=1 αj 1 nk,t−j nk,t−j X ℓ=1 ζk,t−j,ℓ(ϕδ(ηk,t−j), ζt−j,ℓ) + β⊤Xk,t. Then under the condition (ST.1), the processes (ηk,t = φδ(λk,t)t∈Z, k = 1, . . . , K obey some recursive stochastic equations Then under the condition (ST.1), the processes (ηk,t = φδ(λk,t)t∈Z, k = 1, . . . , K obey some recursive stochastic equations ηk,t = f(ηk,t−1, . . . , ηk,t−p; nk,t−1, . . . , nk,t−p, ζ∞ k,t−1, . . . , ζ∞ k,t−p, Xk,t). and with (A.1), for k = 1, . . . , K, (x, y) ∈R2p, E(\|f(x; nk,t−1, . . . , nk,t−p, ζ∞ k,t−1, . . . , ζ∞ k,t−p, Xk,t)−f(y; nk,t−1, . . . , nk,t−p, ζ∞ k,t−1, . . . , ζ∞ k,t−p, Xk,t)\|\|F ηk,t = f(ηk,t−1, . . . , ηk,t−p; nk,t−1, . . . , nk,t−p, ζ∞ k,t−1, . . . , ζ∞ k,t−p, Xk,t). and with (A.1), for k = 1, . . . , K, (x, y) ∈R2p, E(\|f(x; nk,t−1, . . . , nk,t−p, ζ∞ k,t−1, . . . , ζ∞ k,t−p, Xk,t)−f(y; nk,t−1, . . . , nk,t−p, ζ∞ k,t−1, . . . , ζ∞ k,t−p, Xk,t)\|\|Fk,t−1,n) ≤υα⊤\|x− with α = (α1, . . . , αp). Moreover E\|f(x; nk −1, . . . , nk −p, ζ∞ k 1, . . . , ζ∞ k p, Xk 0)\| < ∞. Then, with α = (α1, . . . , αp). Moreover E\|f(x; nk,−1, . . . , nk,−p, ζ∞ k,−1, . . . , ζ∞ k,−p, Xk,0)\| < ∞. Then, from Debaly and Truquet (2021) theorem 4, we get the stationary and ergodic solution with E\|ηk,0\| < ∞, k = 1, . . . , K.□ with α = (α1, . . . , αp). Moreover E\|f(x; nk,−1, . . . , nk,−p, ζ∞ k,−1, . . . , ζ∞ k,−p, Xk,0)\| < ∞. Then, from Debaly and Truquet (2021) theorem 4, we get the stationary and ergodic solution with E\|ηk,0\| < ∞, k = 1, . . . , K.□ The theorem 1 is a straight consequence of lemma 1 and follows the Lipschitz property of x 7→log(exp(x) + 1 + δ) for any δ > 0. For the asymptotic results for ˆθT, the following assumptions will be needed. 5 Proofs for the main results Throughout this section, we will denote by ζ∞ k,t = (ζk,t,l)l≥1, the sequence of copies of the unity random variables ζk,t. Moreover ζk,t can be decomposed into two components : its mean λk,t function of Xk,t and a free random variable ζt. For example, ζk,t = λk,tζt for a positive random variable ζt of mean 1. We will write ζk,t := ζk,t(λk,t, ζt) to denote the relationship between ζk,t and λk,t and ζt. Accordingly, ζk,t,l = λk,tζt,l with ζt,l, l ≥1 i.i.d with mean 1 or in general ζk,t,l := ζk,t,l(λk,t, ζt,l) with Eζk,t,l = λk,t. Let Fk,t denote the σ-algebra generated by ζs, Xk,s+1, s ≤t and Fk,t,n generated by nk,s, ζs, Xk,s+1, s ≤t. Finally, we will denote by ϕδ the inverse of φδ : ϕδ(x) = φ−1 δ (x). For stability, we will consider the following set of assumptions : (A.1) The function ϕδ is υ−Lipschitz and υ Pp i=1 \|αi\| < 1. (A.1) The function ϕδ is υ−Lipschitz and υ Pp i=1 \|αi\| < 1. (ST.1) For k = 1, . . . , K, (nk,t−1, ζ∞ k,t−1, Xk,t)t∈Z is stationary, ergodic, (nk,t, ζ∞ k,t) is independent from Fk,t−1,n and E\|Xk,0\|1 < ∞. (ST.1) For k = 1, . . . , K, (nk,t−1, ζ∞ k,t−1, Xk,t)t∈Z is stationary, ergodic, (nk,t, ζ∞ k,t) is independent from Fk,t−1,n and E\|Xk,0\|1 < ∞. (ST.2) For k = 1, . . . , K, E(\|ζk,t(λk,t, ζt) −ζk,t(λk,t, ζt)\|\|Fk,t−1,n) ≤\|λk,t −λk,t\|. It is worth noting that the example ζk,t = λk,tζt for a positive random variable ζk of mean 1 verifies the condition (ST.2). Lemma 1. Under the assumptions (A.1)-(ST.1), there exists a unique set of K stationary, ergodic sequences (Yk,t, nk,t, Xk,t), k = 1, . . . , K that are a solution of equations (2.1)-(2.2) with E\|ηk,0\| < ∞, k = 1, . . . , K. The proof of lemma 1 uses the techniques of iterated random maps. We refer the interested readers to Debaly and Truquet (2021) theorem 2 and 4 which investigated the problem of the solution of recursive stochastic equations with covariates or Debaly and Truquet (2019) in the case where no covariates are included in the dynamic. – 13 – Proof of lemma 1 From (2.2), Proof of lemma 1 From (2.2), Proof of lemma 1 From (2.2), (A.2) The conditons (A.1) and (ST.1) are met and θ0 verifies : υ Pp i=1 \|αi,0\| < 1. (A.3) For k = 1, . . . , K, Enk,0 < ∞and E sup θ ϕδ(ηk,0(θ0)) ϕδ(ηk,0(θ)) + \| log ◦ϕδ(ηk,0(θ))\| < ∞. (A.4) For (δ, δ) ∈[δ−, ∞)2, (η, η) ∈R2, ϕδ(η) = ϕδ(η) ⇒(δ = δ, η = η). (C.1) The conditons (ST.1) and (ST.2) are met and θ0 verifies : Pp i=1 \|αi,0\| < 1. (C.2) For k = 1, . . . , K, Enk,0 < ∞. (C.3) For k = 1, . . . , K, conditionally on Xk,0, the distribution of Yk,−1 nk−1 , · · · , Yk,−p nk−p is not supported by an hyperplan of Rp. (C.3) For k = 1, . . . , K, conditionally on Xk,0, the distribution of Yk,−1 nk−1 , · · · , Yk,−p nk−p is not supported by an hyperplan of Rp. – 14 – (C.4) For k = 1, . . . , K, the distribution of Xk,0 is not degenerate. For k = 1, . . . , K, the distribution of Xk,0 is not degenerate. (C.4) For k = 1, . . . , K, the distribution of Xk,0 is not degenerate. Lemma 2. Suppose that the assumptions (A.2)-(A.4) and (C.3)-(C.4) are met. Then, al- most surely, Lemma 2. Suppose that the assumptions (A.2)-(A.4) and (C.3)-(C.4) are met. Then, al- most surely, Lemma 2. Suppose that the assumptions (A.2)-(A.4) and (C.3)-(C.4) are met. Then, al- most surely, lim T→∞ ˆθT = θ0. We do not prove the lemma 2. Similar results for time-series models can be found in Diop and Kengne (2021), Aknouche and Francq (2020) or Debaly and Truquet (202x) among others. Proof of consistency part of theorem 2 We will check (A.2) to (A.4). Proof of consistency part of theorem 2 We will check (A.2) to (A.4). • (A.2) comes from (C.1). • (A.2) comes from (C.1). • One can note that here ϕδ(x) = log(1 + δ + exp(x)) and ϕδ(x) ≥log(1 + δ), ϕδ(x) ≤ κ1(θ)(1 + \|x\|) and \| log ◦ϕδ(x)\| ≤κ2(θ)(1 + \|x\|) + κ3(θ), where κi, i = 1, 2, 3 are contin- uous functions of θ. Then (A.3) holds since E supθ \|ηk,0(θ)\| < ∞. Indeed EYk,0/nk,0 = ϕδ(ηk,0) < ∞since E\|ηk,0\| < ∞. Proof of lemma 1 From (2.2), • One can note that here ϕδ(x) = log(1 + δ + exp(x)) and ϕδ(x) ≥log(1 + δ), ϕδ(x) ≤ κ1(θ)(1 + \|x\|) and \| log ◦ϕδ(x)\| ≤κ2(θ)(1 + \|x\|) + κ3(θ), where κi, i = 1, 2, 3 are contin- uous functions of θ. Then (A.3) holds since E supθ \|ηk,0(θ)\| < ∞. Indeed EYk,0/nk,0 = ϕδ(ηk,0) < ∞since E\|ηk,0\| < ∞. • For (A.4), we note that • For (A.4), we note that ϕδ(η) = ϕδ(η) ⇒δ −δ = exp η −exp η and 0 = limη→−∞,η→−∞exp η −exp η = δ −δ. Then δ = δ and η = η.□ Let us set σ2 k,0 = Var Yk,0 λk,0 \| Fk,−1,n ∨nk,0 , ∂δϕδ the derivative of ϕδ with respect to δ, θ−δ the vector of parameters without δ. We will consider the following assumptions for the asymptotic distribution of ˆθT. Let us set σ2 k,0 = Var Yk,0 λk,0 \| Fk,−1,n ∨nk,0 , ∂δϕδ the derivative of ϕδ with respect to δ, θ−δ the vector of parameters without δ. We will consider the following assumptions for the asymptotic distribution of ˆθT. (A.5) The function ϕδ is twice continuously differentiable and for k = 1, . . . , K, E σ2 k,0 ϕ2 δ(ηk,0) h ∂δϕδ(ηk,0(θ0))2 + ϕ′ δ(ηk,0(θ0))2∥∇θ−δηk,0(θ0)∥2 2 i < ∞and E 1 ϕ2 δ(ηk,0) h ∂δϕδ(ηk,0(θ0))2 + ϕ′ δ(ηk,0(θ0))2∥∇θ−δηk,0(θ0)∥2 2 i < ∞. E σ2 k,0 ϕ2 δ(ηk,0) h ∂δϕδ(ηk,0(θ0))2 + ϕ′ δ(ηk,0(θ0))2∥∇θ−δηk,0(θ0)∥2 2 i < ∞and E 1 ϕ2 δ(ηk,0) h ∂δϕδ(ηk,0(θ0))2 + ϕ′ δ(ηk,0(θ0))2∥∇θ−δηk,0(θ0)∥2 2 i < ∞. (A.6) For k = 1, . . . , K, the distribution of (∂δϕδ(ηk,0(θ0)), ϕ′ δ(ηk,0(θ0))∇θ−δηk,0(θ0)) is not degenarate. (A.6) For k = 1, . . . , K, the distribution of (∂δϕδ(ηk,0(θ0)), ϕ′ δ(ηk,0(θ0))∇θ−δηk,0(θ0)) is not degenarate. – 15 – (A.7) For k = 1, . . . , K, E supθ \|W i,j k,0(θ)\| < ∞, where W i,j k,0(θ) is one of the following quantities for all pairs i, j 1 ϕ2 δ(ηk,0(θ)) ϕδ(ηk,0(θ0)) ϕδ(ηk,0(θ)) + 1 ∂ϕδ(ηk,0(θ)) ∂θi ∂ϕδ(ηk,0(θ)) ∂θj 1 ϕ2 δ(ηk,0(θ)) ϕδ(ηk,0(θ0)) ϕδ(ηk,0(θ)) ∂ϕδ(ηk,0(θ)) ∂θi ∂ϕδ(ηk,0(θ)) ∂θj 1 ϕδ(ηk,0(θ)) ϕδ(ηk,0(θ0)) ϕδ(ηk,0(θ)) + 1 ∂2ϕδ(ηk,0(θ)) ∂θi∂θj . (AN.1) The K stationary sequences solution of (2.1)-(2.2) are independent of each other. (AN.1) The K stationary sequences solution of (2.1)-(2.2) are independent of each other. (AN.2) For k = 1, . . . Proof of lemma 1 From (2.2), , K, En2 k,0 < ∞ Eσ4 k,0 < ∞ (AN.3) For k = 1, . . . , K, E\|Xk,0\|4 1 < ∞and EY 4 k,0 < ∞. E\|Xk,0\|4 1 < ∞and EY 4 k,0 < ∞. E\|Xk,0\|4 1 < ∞and EY 4 k,0 < ∞. Lemma 3. Under the assumptions of lemma 1 and if (A.5)-(A.7) and (AN.1) hold, then Lemma 3. Under the assumptions of lemma 1 and if (A.5)-(A.7) and (AN.1) hold, then lim T→∞ √ T(ˆθT −θ0) = N(0, J−1V J−⊤) where J = PK k=1 Jk and V = PK k=1 Vk, Vk = E " 1 λ2 k,0 nk,0 −Yk,0 λk,0 2 ˙λk,0 ˙λ⊤ k,0 # , Jk = E " nk,0 1 λ2 k,0 ˙λk,0 ˙λ⊤ k,0 # and ˙λk,0 = (∂δϕδ(ηk,0(θ0)), ϕ′ δ(ηk,0(θ0))∇θ−δηk,0(θ0))⊤. where J = PK k=1 Jk and V = PK k=1 Vk, where J = PK k=1 Jk and V = PK k=1 Vk, Vk = E " 1 λ2 k,0 nk,0 −Yk,0 λk,0 2 ˙λk,0 ˙λ⊤ k,0 # , Jk = E " nk,0 1 λ2 k,0 ˙λk,0 ˙λ⊤ k,0 # and ˙λk 0 = (∂δϕδ(ηk 0(θ0)) ϕ′ δ(ηk 0(θ0))∇θ ηk 0(θ0))⊤ Vk = E " 1 λ2 k,0 nk,0 −Yk,0 λk,0 2 ˙λk,0 ˙λ⊤ k,0 # , Jk = E " nk,0 1 λ2 k,0 ˙λk,0 ˙λ⊤ k,0 # and ˙λk,0 = (∂δϕδ(ηk,0(θ0)), ϕ′ δ(ηk,0(θ0))∇θ−δηk,0(θ0))⊤. ˙λk,0 = (∂δϕδ(ηk,0(θ0)), ϕ′ δ(ηk,0(θ0))∇θ−δηk,0(θ0))⊤. As for lemma 2, we do not prove the lemma 3. We refer the interested reader to Diop and Kengne (2021), Aknouche and Francq (2020) or Debaly and Truquet (202x) among others. Proof of asymptotic normality part of theorem 2 For the proof of asymptotic nor- mality part of theorem 2, one can note that in the single framework (k = 1), assumptions (AN.2) yield the asymptotic normality of √ T∇ℓk(θ0) using the central limit theorem for difference martingale. Next, – 16 – ∂2ℓk,t(θ) ∂θi∂θj = 1 λ2 k,t(θ) Yk,t λk,t(θ) −nk,t ∂λk,t(θ) ∂θi ∂λk,t(θ) ∂θj + 1 λ2 k,t(θ) Yk,t λk,t(θ) ∂λk,t(θ) ∂θi ∂λk,t(θ) ∂θj − 1 λk,t(θ) Yk,t λk,t(θ) −nk,t ∂2λk,t(θ) ∂θi∂θj =: Ik,t(θ) + IIk,t(θ) + IIIk,t(θ). Proof of lemma 1 From (2.2), For the first term, For the first term, sup θ \|Ik,t(θ)\| ≤ni,t λi,t log(1 + δ_) + 1 sup θ 1 λ2 k,t(θ) ∂λk,t(θ) ∂θi ∂λk,t(θ) ∂θj and and 1 λk,t(θ) ˙λk,t(θ) ≼κδ_ 1, ιk, Yk,t−1 nk,t−1 . . . Yk,t−p nk,t−p , X⊤ k,t ⊤ where for x = (x1, . . . , xd), y = (y1, . . . , yd), x ≼y means xi ≤yi, i = 1, . . . , d and κδ_ a function of δ_. Then, E supθ \|Ik,t(θ)\| < ∞under the assumption (AN.3). It can be shown similarly that E supθ \|IIk,t(θ)\| < ∞and E supθ \|IIIk,t(θ)\| < ∞. By the Taylor expansion of rT(·) between ˆθT and θ, 0 = √ T∇rT(ˆθT) = K X k=1 √ T∇ℓk(ˆθT) = K X k=1 √ T∇ℓk(θ0) ! + K X k=1 ∇2ℓk(θ0) ! √ T(ˆθT −θ0) + oP(1) The independence condition on paths (AN.1), assumption (AN.2) and central limit theorem for difference martingale allow us to conclude PK k=1 √ T∇ℓk(θ0) converges in dis- tribution to central Gaussian vector of variance V as T tends to infinity. The assumption (AN.3) and ergodic theorem entails that PK k=1 ∇2ℓk(θ0) converges to J. Moreover, condition (AN.1), (C.3) and (C.4) ensure that the matrix J is invertible.□ (C.4) Acknowledgements Funding was provided by the Contrat de service de recherche forestière number 3329-2019-142332177 obtained by PM and MMG from the Ministère des Forêts, de la Faune et des Parcs (Quebec, Canada), the Doctoral scholarships from GENES (EN- – 17 – SAE/ENSAI) obtained by ZMD. We thank G. 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John Wiley & Sons, 2018. – 23 – Appendix Appendix Table 1: Results for simulations α1 β1 β2 β3 β4 β5 β6 β7 β8 β9 β10 K T Scenario 0.6 0 1 -1 0.5 -0.5 -1.5 1.5 -2 2 0 5 50 1 0.552 -0.162 0.731 -0.991 0.308 -0.626 -1.636 1.416 -1.382 1.871 0.046 0.049 0.095 0.137 0.196 0.192 0.287 0.333 0.249 0.281 0.210 0.073 2 0.596 0.110 0.852 -0.852 0.441 -0.658 -1.615 1.534 -1.476 1.881 0.053 0.045 0.064 0.107 0.128 0.168 0.320 0.262 0.260 0.202 0.203 0.056 100 1 0.502 0.109 0.876 -0.777 0.539 -0.732 -1.173 1.466 -1.769 1.622 -0.021 0.036 0.065 0.116 0.138 0.106 0.205 0.2240 0.130 0.121 0.132 0.060 2 0.512 0.064 0.917 -0.815 0.508 -0.626 -1.129 1.500 -1.773 1.778 -0.017 0.032 0.055 0.111 0.107 0.103 0.204 0.175 0.112 0.105 0.122 0.055 200 1 0.524 0.068 0.869 -0.625 0.601 -0.235 -1.249 1.300 -1.780 1.664 -0.056 0.028 0.041 0.089 0.122 0.095 0.151 0.165 0.107 0.115 0.102 0.046 2 0.558 0.096 0.935 -0.679 0.609 -0.202 -1.357 1.347 -1.854 1.780 -0.061 0.025 0.041 0.072 0.099 0.074 0.132 0.152 0.101 0.092 0.099 0.035 10 50 1 0.380 -0.030 0.659 -0.848 0.529 -0.420 -1.138 0.890 -1.678 1.470 -0.028 0.052 0.073 0.136 0.155 0.174 0.242 0.228 0.164 0.231 0.155 0.088 2 0.375 -0.139 0.555 -0.702 0.407 -0.141 -1.190 0.903 -1.618 1.389 0.078 0.055 0.065 0.125 0.181 0.165 0.246 0.299 0.180 0.242 0.146 0.119 100 1 0.410 -0.018 0.771 -0.702 0.478 -0.146 -1.638 1.184 -1.392 1.418 0.1176 0.035 0.070 0.106 0.129 0.129 0.189 0.194 0.132 0.162 0.124 0.062 2 0.35 -0.021 0.800 -0.770 0.384 -0.286 -1.721 1.102 -1.318 1.396 0.101 0.039 0.077 0.120 0.186 0.384 -0.286 -1.721 1.102 -1.318 1.396 0.101 200 1 0.429 -0.004 0.712 -0.830 0.219 -0.576 -1.135 1.005 -1.372 1.496 -0.028 0.027 0.037 0.086 0.111 0.082 0.135 0.135 0.088 0.123 0.089 0.043 2 0.399 -0.060 0.748 -0.802 0.184 -0.577 -1.010 1.101 -1.521 1.583 0.021 0.027 0.042 0.083 0.139 0.093 0.142 0.163 0.103 0.141 0.111 0.048 15 50 1 0.345 0.024 0.429 -1.004 0.569 -0.419 -0.981 0.896 -1.233 1.278 -0.037 0.034 0.055 0.113 0.169 0.152 0.144 0.153 0.117 0.127 0.129 0.0713 2 0.320 -0.013 0.363 -1.231 0.615 -0.728 -1.163 1.082 -0.882 1.280 0.008 0.047 0.101 0.156 0.291 0.223 0.344 0.270 0.222 0.310 0.209 0.125 100 1 0.367 0.006 0.617 -0.620 0.311 -0.241 -0.898 0.965 -1.214 1.314 -0.057 0.028 0.040 0.075 0.105 0.080 0.147 0.134 0.096 0.115 0.088 0.042 2 0.366 0.015 0.722 -0.580 0.095 -0.229 -0.978 0.902 -0.929 1.327 -0.002 0.033 0.067 0.113 0.202 0.116 0.213 0.188 0.122 0.187 0.123 0.068 200 1 0.414 -0.035 0.660 -0.549 0.235 -0.428 -0.797 0.938 -1.203 1.281 0.0511 0.019 0.030 0.060 0.069 0.058 0.104 0.102 0.064 0.070 0.063 0.028 2 0.400 -0.018 0.763 -0.585 0.258 -0.656 -0.765 1.093 -1.265 1.298 0.093 0.022 0.039 0.098 0.096 0.087 0.130 0.139 0.084 0.107 0.083 0.039 20 50 1 0.406 -0.076 0.502 -0.382 0.326 -0.215 -1.033 0.995 -0.993 1.069 -0.010 0.045 0.0591 0.091 0.138 0.113 0.228 0.198 0.132 0.171 0.121 0.048 2 0.371 -0.041 0.368 -0.388 0.249 -0.440 -1.426 1.047 -1.127 1.204 -0.022 0.050 0.097 0.158 0.172 0.143 0.128 0.226 0.227 0.172 0.159 0.066 100 1 0.299 -0.046 0.478 -0.462 0.226 -0.297 -0.993 0.786 -1.062 1.114 -0.048 0.032 0.048 0.074 0.108 0.119 0.152 0.124 0.088 0.114 0.095 0.050 2 0.275 -0.087 0.578 -0.385 0.089 -0.229 -0.978 0.876 -1.143 1.326 0.058 0.033 0.047 0.098 0.149 0.139 0.173 0.166 0.139 0.116 0.123 0.064 Table 1: Results for simulations – 24 – −10 −5 0 5 10 0 2 4 6 8 10 x softplus softplus−1 softplus−2 max(x,0) Figure 1: Comparaison between softplus and max(x,0) −10 −5 0 5 10 0 2 4 6 8 10 x softplus softplus−1 softplus−2 max(x,0) Figure 1: Comparaison between softplus and max(x,0) – 25 – Figure 2: Location of study sites from Jardon Project (2003) in Canadian boreal ecoregions. Appendix Figure 2: Location of study sites from Jardon Project (2003) in Canadian boreal ecoregions. – 26 – (a) (a) (b) (c) Figure 3: Model with Temperature + CMI + Defoliation. classes of age 1 : < 75, 2 : 75 – 100, 3 : 100 – 125, 4 : 125 – 150 and 5 : > 150 years (a) effects of maximum temperature in spring and summer in current and previous year; (b) effects of cmi index in spring and summer in current and previous year and (c) delayed effect of level of defoliation. The dashed horizontal line corresponds to zero. ( ) (b) ) (b) (c) (c) (c) Figure 3: Model with Temperature + CMI + Defoliation. classes of age 1 : < 75, 2 : 75 – 100, 3 : 100 – 125, 4 : 125 – 150 and 5 : > 150 years (a) effects of maximum temperature in spring and summer in current and previous year; (b) effects of cmi index in spring and summer in current and previous year and (c) delayed effect of level of defoliation. The dashed horizontal line corresponds to zero. – 27 – (a) (a) (b) (c) Figure 4: Model with Temperature + Precipitation + Defoliation. classes of age 1 : < 75, 2 : 75 – 100, 3 : 100 – 125, 4 : 125 – 150 and 5 : > 150 years (a) effects of maximum temperature in spring and summer in current and previous year; (b) effects of precipitation ndex in spring and summer in current and previous year and (c) delayed effect of level of defoliation. The dashed horizontal line corresponds to zero. (a) (b) (b) (c) (c) Figure 4: Model with Temperature + Precipitation + Defoliation. classes of age 1 : < 75, 2 : 75 – 100, 3 : 100 – 125, 4 : 125 – 150 and 5 : > 150 years (a) effects of maximum temperature in spring and summer in current and previous year; (b) effects of precipitation index in spring and summer in current and previous year and (c) delayed effect of level of defoliation. The dashed horizontal line corresponds to zero. – 28 –
https://openalex.org/W2936248173	https://www.repository.cam.ac.uk/bitstream/1810/297899/2/stz2412.pdf	English	null	Potential softening and eccentricity dynamics in razor-thin, nearly Keplerian discs	Monthly Notices of the Royal Astronomical Society	2,019	cc-by	23,153	ABSTRACT In many astrophysical problems involving discs (gaseous or particulate) orbiting a dominant central mass, gravitational potential of the disc plays an important dynamical role. Its impact on the motion of external objects, as well as on the dynamics of the disc itself, can usually be studied using secular approximation. This is often done using softened gravity to avoid singularities arising in calculation of the orbit-averaged potential – disturbing function – of a razor-thin disc using classical Laplace–Lagrange theory. We explore the performance of several softening formalisms proposed in the literature in reproducing the correct eccentricity dynamics in the disc potential. We identify softening models that, in the limit of zero softening, give results converging to the expected behaviour exactly, approximately or not converging at all. We also develop a general framework for computing secular disturbing function given an arbitrary softening prescription for a rather general form of the interaction potential. Our results demonstrate that numerical treatments of the secular disc dynamics, representing the disc as a collection of N gravitationally interacting annuli, are rather demanding: for a given value of the (dimensionless) softening parameter, ς ≪1, accurate representation of eccentricity dynamics requires N ∼Cς−χ ≫1, with C ∼O(10), 1.5 ≲χ ≲2. In discs with sharp edges a very small value of the softening parameter ς (≲10−3) is required to correctly reproduce eccentricity dynamics near the disc boundaries; this ﬁnding is relevant for modelling planetary rings. Key words: methods: analytical – celestial mechanics – planets and satellites: rings – planet– disc interactions. averaging of the fast-evolving dynamical variables over the orbits of particles under consideration (Murray & Dermott 1999). The orbit- averaging procedure, also known as Gauss’ method, is equivalent to calculating the time-averaged potential due to orbiting point masses by smearing them into massive elliptical ‘wires’ (having shape of their eccentric orbits) with non-uniform linear density proportional to the time spent by an object at a particular phase of its orbit. Such orbit-averaged potential, also known as secular disturbing function Rd, fully determines the secular dynamics of the system. ⋆E-mail: aas79@cam.ac.uk MNRAS 489, 4176–4195 (2019) doi:10.1093/mnras/stz2412 Advance Access publication 2019 September 2 MNRAS 489, 4176–4195 (2019) doi:10.1093/mnras/stz2412 Advance Access publication 2019 September 2 doi:10.1093/mnras/stz2412 Accepted 2019 August 15. Received 2019 August 14; in original form 2019 April 11 Accepted 2019 August 15. Received 2019 August 14; in original form 2019 April 11 Potential softening and eccentricity dynamics in razor-thin, nearly Keplerian discs Antranik A. Seﬁlian 1‹ and Roman R. Raﬁkov1,2 1Department of Applied Mathematics and Theoretical Physics, CMS, University of Cambridge, Wilberforce Road, Cambridge CB3 0WA, UK 2Institute of Advanced Study, Einstein Drive, Princeton, NJ 08540, USA Antranik A. Seﬁlian 1‹ and Roman R. Raﬁkov1,2 1Department of Applied Mathematics and Theoretical Physics, CMS, University of Cambridge, Wilberforce Road, Cambridge CB3 0WA, UK 2Institute of Advanced Study, Einstein Drive, Princeton, NJ 08540, USA Downloaded from https://academic.oup.com/mnras/article-abstract/489/3/4176/5558240 by University of Cambridge user on 17 October 2019 1 INTRODUCTION Astrophysical discs orbiting a central mass Mc are ubiquitous in a variety of contexts – galactic, stellar, and planetary (Latter, Ogilvie & Rein 2017). In many instances, masses of such discs Md are much less than the central object mass. Despite this fact, gravity of such discs can still play an important dynamical role in the orbital evolution of their constituent particles as well as the dynamics of external objects (e.g. Goldreich & Tremaine 1979; Heppenheimer 1980; Ward 1981; Kocsis & Tremaine 2011; Kazandjian & Touma 2013; Teyssandier, Terquem & Papaloizou 2013; Meschiari 2014; Silsbee & Raﬁkov 2015; Petrovich, Wu & Ali-Dib 2019; Seﬁlian & Touma 2019). Consequently, characterizing dynamical effects of disc gravity is important. For a test particle with semimajor axis ap, eccentricity ep, and apsidal angle ϖ p due to a co-planar point mass δmd orbiting with semimajor axis a, eccentricity ed, and apsidal angle ϖ d, upon smearing into elliptical rings, the secular disturbing function takes the form (Murray & Dermott 1999) Whenever Md ≪Mc, particles perturbed by the disc gravity move on nearly Keplerian orbits that evolve rather slowly. This justiﬁes the use of the so-called secular approximation which implies δR = Gδmdap a2 1 8b(1) 3/2 ap a e2 p −1 4b(2) 3/2 ap a eped cos(ϖp −ϖd) , (1) C⃝The Author(s) 2019 (1) ( ) Published by Oxford University Press on behalf of The Royal Astronomical Society. 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. Published by Oxford University Press on behalf of The Royal Astronomical Society. 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. Softened potentials of discs 4177 Unfortunately, by construction Heppenheimer’s method is in- applicable in situations where the disc eccentricity rapidly varies with semimajor axis, potentially resulting in orbit crossings (Davy- denkova & Raﬁkov 2018). An alternative approach, which avoids this problem, while at the same time alleviating the aforementioned singularity, is to use softened gravity by spatially smoothing the Newtonian point-mass potential in various ways – both analytically (e.g. 1 INTRODUCTION Com- plementary to this, we develop a general framework for computing the well-behaved secular disturbing function for a broad range of softened gravitational potentials. Rd = aout ain δR, Rd = aout ain δR, (3) (3) where ain and aout are the semimajor axes of the inner and outer disc edges. In this case, provided that δR is known as a function of a, only a single integration (over the semimajor axes of the rings) is needed, signiﬁcantly accelerating calculations.1 Our work is organized as follows. We describe the general analytical expressions governing the orbit-averaged potential due to a coplanar disc of arbitrary structure and arbitrary softening prescription in Section 2. Having provided a brief account of the different softened potentials under our probe and the unsoftened approach of Heppenheimer in Sections 2.1 and 2.2, respectively, we analyse the performance of softening formalisms in reproducing the correct secular dynamics for various disc models in Sections 3–5. We discuss and brieﬂy summarize our results in Sections 6 and 7, respectively. Technical details of our calculations can be found in appendices. Unfortunately, this straightforward procedure is ill-posed from the mathematical point of view. Indeed, it is well known that the Laplace coefﬁcients b(m) 3/2 featured in equation (1) diverge as b(m) 3/2(α) →(1 −α)−2 when α →1. This implies that the radial integration in equation (3) encounters an essential singularity at a = ap. As a result, for a co-planar particle orbiting inside a razor-thin disc, ain ≤ap ≤aout, this direct way of computing Rd does not converge to a ﬁnite value. This divergence, as well as the pressing need for having an efﬁcient way of computing Rd (via a one-dimensional integration over a only), have motivated the development of alternative analytic approaches for calculating Rd. These approaches can be generally grouped into two classes. Calculations of one kind are rooted in the derivation of the potential of an axisymmetric disc with power- law surface density proﬁle presented in Heppenheimer (1980), which does not suffer from the singularity of Laplace–Lagrange secular theory. A number of subsequent studies used this approach (Ward 1981) and extended it to the case of eccentric discs, both apsidally aligned (Silsbee & Raﬁkov 2015; Davydenkova & Raﬁkov 2018) and misaligned (Davydenkova & Raﬁkov, in preparation). Higher order (in eccentricity) extensions of this approach have also been developed (Seﬁlian & Touma 2019). 1 INTRODUCTION Tremaine 1998, 2001; Touma 2002; Hahn 2003; Touma & Sridhar 2012; Teyssandier & Ogilvie 2016) and numerically (e.g. Touma et al. 2009). In these models, the classical Laplace– Lagrange disturbing function (equation 1) is modiﬁed by softening the interaction potential in some way to circumvent the divergence of Rd as a →ap. In this method, orbit crossing does not lead to problems as long as the softening scale is ﬁnite. However, a physical justiﬁcation for a speciﬁc form of softening (absent in the Heppenheimer 1980 approach) often remains unclear, making the introduction of softening rather arbitrary. valid for a > ap as well as a < ap, as long as particle orbits do not cross. Here, b(m) s (α) is the Laplace coefﬁcient deﬁned by b(m) s (α) = 2 π π 0 cos(mθ)[1 + α2 −2α cos θ]−sdθ, (2) (2) which obeys b(m) s (α−1) = α2sb(m) s (α). Explicit time independence of δR guarantees that the semimajor axes of the secularly interacting objects stay ﬁxed. When considering gravitational effects of a razor-thin continuous disc with smooth distribution of surface density, a straightforward way to compute the secular disturbing function would be to orbit- average the disc potential (obtained by direct integration over its full surface) along the particle orbit. However, this procedure involves a triple integration (two-dimensional integral over the disc surface and orbit averaging) and is numerically challenging. A more efﬁcient approach lies in representing the disc as a collection of massive, nested, confocal elliptical ‘wires’ (also referred to as ‘annuli’ or ‘rings’ in this work) with ﬁxed semimajor axes (e.g. Touma, Tremaine & Kazandjian 2009; Batygin 2012). Due to the additive nature of gravity, the disturbing function due to a disc can be represented as a sum of individual contributions in the form (1) produced by all wires, which amounts to integration of δR (equation 1) over the radial extent of the disc: The primary goal of our present work is to assess how well the different calculations relying on potential softening reproduce secular dynamics driven by the gravity of a razor-thin disc. The main metric we use in this exercise is the convergence of the results of such calculations to the true secular evolution (represented by the unsoftened Heppenheimer method) in the limit of vanishing softening, when the limit of Newtonian gravity is recovered. 1 INTRODUCTION This framework for treating secular dynamics has been extensively veriﬁed using direct orbit integrations under different conditions (Silsbee & Raﬁkov 2015; Fontana & Marzari 2016; Davydenkova & Raﬁkov 2018). In this work, we refer to this type of calculation as the unsoftened Heppenheimer method. 2 DISTURBING FUNCTION DUE TO A DISC The coefﬁcients Ad and Bd in equation (4) are related to the disc mass (or surface density) and eccentricity proﬁles in the following fashion: Ad(ap) = 2G npa3 p × ap ain μd(a)φ22 a ap da + aout ap μd(a)ap a φ11 ap a da , (5) Bd(ap) = G npa3 p × ap ain μd(a)ed(a)φ12 a ap da + aout ap μd(a)ed(a)ap a φ12 ap a da , (6) (5) (6) where ed = ed(a)(cos ϖd(a), sin ϖd(a)) is the eccentricity vector for an annular disc element.2 Functions φij(α), with i, j = 1, 2, entering these expressions fully characterize the softened ring–ring secular interaction, see equation (11). They are unique for each potential softening pre- scription, with explicit forms for the models that we explore in this work speciﬁed in Table 1. This Table shows that coefﬁcients φij appearing in the literature are linear combinations of softened Laplace coefﬁcients B(m) s deﬁned by B(m) s (α, ϵ) = 2 π π 0 cos(mθ)[1 + α2 −2α cos θ + ϵ2(α)]−sdθ. (7) The softening parameter ϵ(α) appearing in this deﬁnition remains non-zero as α →1, thus preventing the divergence of the softened Laplace coefﬁcients B(m) s (α, ϵ) at α = 1 (unlike the classical b(m) s (α)). The explicit form of ϵ(α) is different for every softening method considered in this work, see Section 2.1 and Table 1. Appendix C collates some useful relations for softened Laplace coefﬁcients B(m) s (α, ϵ), as well as their approximate asymptotic behaviour and relationships to complete elliptic integrals. The softening parameter ϵ(α) appearing in this deﬁnition remains non-zero as α →1, thus preventing the divergence of the softened Laplace coefﬁcients B(m) s (α, ϵ) at α = 1 (unlike the classical b(m) s (α)). The explicit form of ϵ(α) is different for every softening method considered in this work, see Section 2.1 and Table 1. Appendix C collates some useful relations for softened Laplace coefﬁcients B(m) s (α, ϵ), as well as their approximate asymptotic behaviour and relationships to complete elliptic integrals. The mathematical structure of Rd given by equation (4) is similar to that of the classical Laplace–Lagrange planetary theory (Murray & Dermott 1999), see equation (1). 2 DISTURBING FUNCTION DUE TO A DISC The Laplace coefﬁcients that are softened by the introduction of a softening parameter ϵ2(α) are deﬁned in equation (7). Note that the expressions of φij reported in Touma (2002) have been corrected in a subsequent paper of Touma & Sridhar (2012). Formalism ϵ2(α) φ11 φ12 φ22 Laplace–Lagrange – 1 8 αb(1) 3/2 −1 4 αb(2) 3/2 φ11 Tremaine (1998) (Tr98) β2 c 1 8 (2α d dα + α2 d2 dα2 )B(0),Tr 1/2 1 4 (2 −2α d dα −α2 d2 dα2 )B(1),Tr 1/2 φ11 = 1 8 α[B(1),Tr 3/2 −3αβ2 c B(0),Tr 5/2 ] = −1 4 α[B(2),Tr 3/2 −3αβ2 c B(1),Tr 5/2 ] Touma (2002) (T02) β2 = b2 c/a2 > −5 8 αB(1),T 3/2 + 3 16 α2B(0),T 5/2 + 3 8 α(1 + α2)B(1),T 5/2 9 8 αB(0),T 3/2 + 1 8 αB(2),T 3/2 −9 8 α(1 + α2)B(0),T 5/2 −5 8 αB(1),T 3/2 + 3 16 α2B(0),T 5/2 + 3 8 α(1 + α2)B(1),T 5/2 −15 16 α2B(2),T 5/2 −3 8 αβ2(αB(0),T 5/2 −B(1),T 5/2 ) + 21 16 α2B(1),T 5/2 + 3 8 α(1 + α2)B(2),T 5/2 + 3 16 α2B(3),T 5/2 −15 16 α2B(2),T 5/2 −3 8 β2(B(0),T 5/2 −αB(1),T 5/2 ) Hahn (2003) (H03) H2(1 + α2) 1 8 α[B(1),H 3/2 −3αH 2(2 + H 2)B(0),H 5/2 ] −1 4 α[B(2),H 3/2 −3αH 2(2 + H 2)B(1),H 5/2 ] φ11 Teyssandier & Ogilvie (2016) (TO16) S2α 1 8 αB(1),TO 3/2 −1 4 αB(2),TO 3/2 φ11 Downloaded from https://academic.oup.com/mnras/article-abstract/489/3/4176/5558240 by University of Cambridge user on 17 October 2019 Rd = npa2 p 1 2Ad(ap)e2 p + Bd(ap) · ep . (4) (4) Downloaded from https://academic.oup.com/mnras/article-abstract/489/3/4176/5558240 by University of Cambridge user on 17 October 2019 Here, np is the test-particle mean motion (n2 p = GMc/a3 p), and we have introduced a two-component eccentricity vector for a test particle such that ep = ep(cos ϖp, sin ϖp). 2 DISTURBING FUNCTION DUE TO A DISC Prior to providing the details of different softening prescriptions examined in this work in Section 2.1, we brieﬂy summarize some of their common features. The ultimate goal of all these prescriptions is the calculation of the disturbing function Rd due to gravity of a (generally eccentric) disc comprised of massive objects (stars, planetesimals, ring particles) or ﬂuid elements (in gaseous discs) moving on Keplerian orbits. We consider the disc to be razor-thin and coplanar. Mass distribution of such a disc can be uniquely characterized by the mass density per unit semimajor axis μd(a), eccentricity ed(a), and apsidal angle ϖ d(a) of the trajectories of its constituent elements, as functions of the semimajor axis a. In practice, it is often convenient to use the surface density at periastron d(a) instead of μd(a); its relation to μd for arbitrary proﬁles of ed and ϖ d has been established in Statler (2001), Davydenkova & Raﬁkov (2018), and Davydenkova & Raﬁkov (in preparation). Constancy of semimajor axis in secular theory implies that μd(a) does not change in time. The same statement is true for d(a) to lowest order in ed since μd(a) ≈2πa d(a) + O(ed) (Davydenkova & Raﬁkov 2018). 1The Laplace coefﬁcients entering in δR can be easily evaluated, without relying on integration over θ in equation (2), by expressing them through elliptic integrals, see Appendix C3. Close inspection of the various softening methods for computing secular disc potential (Section 2.1) reveals that all of them arrive MNRAS 489, 4176–4195 (2019) 4178 A. A. Seﬁlian and R. R. Raﬁkov at the following general form of the disturbing function for a test particle moving on an orbit with semimajor axis ap, eccentricity ep, and apsidal angle ϖ p: Table 1. The coefﬁcients φij(α) of the secular disturbing function with softened gravity featured in equations (5) and (6), which govern the individual secular ring–ring interaction (equation 11), adopted from the literature (listed in the ﬁrst column). Here α is deﬁned such that α = a</a> where a> = max(a1, a2) and a< = min(a1, a2). The softened interactions under consideration are those of Tremaine (1998), Touma (2002), Hahn (2003), and Teyssandier & Ogilvie (2016) – see Section 2.1 for further details. For reference, the expressions of φLL ij corresponding to the (unsoftened) Newtonian ring–ring interaction (i.e. classical Laplace–Lagrange formalism) are also shown in the ﬁrst row. 2.1.2 Formalism of Touma (2002) – T02 (here i = 1, 2 and j ̸= i) is given by equations (A22)–(A24) in terms of B(m) s (α, F). In the above expression, we have deﬁned a> = max(a1, a2) and a< = min(a1, a2) such that 4 α = a</a>. Touma (2002) derived the orbit-averaged potential of a disc by assuming individual particles comprising the disc to interact via Plummer potential with a ﬁxed length scale bc (Binney & Tremaine 2008). Smearing particles into gravitating eccentric wires, Touma (2002, see also Touma & Sridhar 2012) derived the expressions (equations 6 of Touma 2002) for φij(α) in the form of linear combinations of softened Laplace coefﬁcients B(m),T s , similar to those of Tremaine (1998): Note that in equations (5) and (6) we split integration over a in two parts: over the part of the disc interior to ap, and exterior to it. We do this because for some softening functions F the coefﬁcients φij(α) do not obey certain symmetry properties when a/ap is replaced with ap/a, see equation (C4). Moreover, in general φ11 and φ22 are not necessarily identical as in classical Laplace–Lagrange theory (i.e. equation 8); see Table 1 and Appendix A for further details. B(m),T s = 2 π π 0 cos(mθ) 1 + α2 −2α cos θ + β2−s dθ. (13) (13) As to the physical meaning of Ad and Bd, we remind the reader that Ad represents the precession rate of the free eccentricity vector of a test particle in the disc potential, while Bd characterizes However, in Touma (2002) the softening parameter ϵ2(α) = β2 is no longer a constant but depends on the distance such that β = bc/max (a1, a2). Within this formalism, one can think of a disc as comprised of nested annuli with a constant thickness bc. 3Note that the interparticle force resulting from such potential does not, in general, obey Newton’s third law (as long as F(r1, r2) ̸= const). 4Here, we clarify that the deﬁnitions of φ11(α) and φ22(α), even when different (see Table 1 and Appendix A), are swapped upon interchanging a1 with a2 but keeping, by construction, α = a</a> < 1 – see equations (A22) and (A23) for details. 2 DISTURBING FUNCTION DUE TO A DISC Indeed, let us consider mass distribution of a point mass smeared along an elliptical orbit, μd(a) →mplδ(a −apl) (where δ(z) is the Dirac delta-function), and set softening to zero (so that B(m) s (α, ϵ →0) →b(m) s (α)). Then, one ﬁnds that Rd reduces to the unsoftened, orbit-averaged potential δR due to a planet with mass mpl and semimajor axis apl, with the unsoftened coefﬁcients φij in the form (Murray & Dermott 1999) φLL 11 (α) = φLL 22 (α) = 1 8αb(1) 3/2(α), (8) φLL 12 (α) = −1 4αb(2) 3/2(α), (9) φLL 11 (α) = φLL 22 (α) = 1 8αb(1) 3/2(α), (8) (9) 2We refer the reader to Heppenheimer (1980), Silsbee & Raﬁkov (2015), and Davydenkova & Raﬁkov (2018) for the expressions of Ad and Bd computed using the unsoftened Heppenheimer method for different disc models. 2We refer the reader to Heppenheimer (1980), Silsbee & Raﬁkov (2015), and Davydenkova & Raﬁkov (2018) for the expressions of Ad and Bd computed using the unsoftened Heppenheimer method for different disc models. MNRAS 489, 4176–4195 (2019) MNRAS 489, 4176–4195 (2019) Softened potentials of discs 4179 see equation (1). the torque exerted on the particle orbit by the non-axisymmetric component of the disc gravity. Corresponding forced eccentricity vector is ep,f = −Bd/Ad. In particular, test particles initiated on circular orbits experience eccentricity oscillations of maximum amplitude em p = 2\|ep,f \|. Accordingly, it is intuitive to think of equations (4)–(6) as the continuous version of classical Laplace–Lagrange planetary theory, modiﬁed by the introduction of non-zero softening parameter ϵ to avoid the mathematical divergence of the classical disturbing function as a →ap. p As Ad(ap) and Bd(ap) uniquely determine Rd for different forms of softening, comparison of their behaviour in the limit of ϵ →0 with that found in the unsoftened Heppenheimer (1980) approach (validated in Silsbee & Raﬁkov 2015; Fontana & Marzari 2016; Davydenkova & Raﬁkov 2018) is sufﬁcient to assess the validity of a particular softening model, see Section 3. We emphasize that the functional forms of φij are not simple replacements of b(m) s appearing in the unsoftened deﬁnitions (8)–(9) by B(m) s . This can be seen in Table 1 where we summarize some of the expressions for φij(α) proposed in the literature and analysed in this paper (see Section 2.1). 2.1.1 Formalism of Tremaine (1998) – Tr98 i(r1, r2) = −Gmj (r1 −r2)2 + F(r1, r2) −1/2, (10) (10) Tremaine (1998) suggested an expression for the secular disturbing function due to a continuous disc, which uses modiﬁed Laplace coefﬁcients in the form Tremaine (1998) suggested an expression for the secular disturbing function due to a continuous disc, which uses modiﬁed Laplace coefﬁcients in the form with i, j = 1, 2 and j ̸= i. Here F(r1, r2) represents an arbitrary softening function introduced to cushion the singularity which arises otherwise at null interparticle separations. Note that in general this potential may depend not only on the relative distance between the two masses r1 −r2, but also on their distances to the dominant central mass r1, r2. B(m),Tr s = 2 π π 0 cos(mθ) 1 + α2 −2α cos θ + β2 c −s dθ. (12) (12) Here, β2 c is the dimensionless softening parameter, treated as a constant, i.e. independent of distance. The physical interpretation of this manoeuvre is that βc, inhibiting the formal divergence of Rd as a →ap, can be viewed as the disc aspect ratio. Within this prescription, it is intuitive to think of the eccentric ‘wires’ that comprise the disc as having a distance-dependent radius b = βcmax (a1, a2). In Tremaine (1998), coefﬁcients φ ij(α) were expressed as derivatives of B(m),Tr 1/2 with respect to α; see equations 26 of Tremaine (1998). These expressions, along with their versions modiﬁed using the recursive relations for Laplace coefﬁcients (see Appendix C1), can be found in Table 1. Explicit demonstration of the connection between the potential (equation 10) and Rd given by equation (4) represents a stand- alone result of this work. In particular, our calculations in Ap- pendix A, which can be skipped at ﬁrst reading, show that the softening parameter ϵ featured in equation (7) is related to F via ϵ2 = [max(a1, a2)]−2F(a1, a2), where a1, 2 are the semimajor axes of the interacting particles (see equation A21). The most general expressions of φij entering the arbitrarily softened ring– ring disturbing function, Ri = Gmj a> φ11(α) e2 1 + φ22(α) e2 2 + φ12(α) e1e2 cos(ϖ1 −ϖ2) , Ri = Gmj a> φ11(α) e2 1 + φ22(α) e2 2 + φ12(α) e1e2 cos(ϖ1 −ϖ2) , (11) (11) 2 DISTURBING FUNCTION DUE TO A DISC Nevertheless, examination of these expressions shows that when ϵ2(α) →0, the coefﬁcients φij(α) do reduce to their unsoftened versions φLL ij (α) given by equations (8) and (9). 2.1 Summary of existing softening models In Appendix A, we show that the form of the disturbing function given by equations (4)–(6) is generic for a wide class of softening models (and not just the ones covered in Section 2.1), for which the interaction potential between the two masses m1 and m2 (mi ≪Mc) located at r1 and r2, correspondingly, relative to the central mass, has a form3 Here we provide a brief description of the four different softening prescriptions that have been previously proposed in the literature. Corresponding expressions for their softening parameters ϵ2(α) and coefﬁcients φij(α) are provided in Table 1. 2.1.1 Formalism of Tremaine (1998) – Tr98 3Note that the interparticle force resulting from such potential does not, in general, obey Newton’s third law (as long as F(r1, r2) ̸= const). 4Here, we clarify that the deﬁnitions of φ11(α) and φ22(α), even when different (see Table 1 and Appendix A), are swapped upon interchanging a1 with a2 but keeping, by construction, α = a</a> < 1 – see equations (A22) and (A23) for details. 3Note that the interparticle force resulting from such potential does not, in general, obey Newton’s third law (as long as F(r1, r2) ̸= const). 5Note that the order of these procedures is opposite to what is usual in the Laplace–Lagrange treatment (e.g. Murray & Dermott 1999). For further details, see e.g. Heppenheimer (1980). 2.2 The unsoftened Heppenheimer method A different approach to computing the disturbing function of a razor-thin disc has been developed by Heppenheimer (1980) without resorting to any form of softened gravity (see also Ward 1981). The essence of this method is in computing the potential by direct integration over the disc surface before expanding the integral limits (which involve instantaneous particle position r) in terms of small eccentricity of a test particle.5 This expansion is followed by time- averaging over the orbit of a test particle. ψ2 = 2 1 α1 α1−p−qφ12(α)dα + 2 1 α2 αp+q−2φ12(α)dα, (19) (19) with α1 = ain/ap and α2 = ap/aout. with α1 = ain/ap and α2 = ap/aout. The coefﬁcients ψ1 and ψ2 are functions of the power-law indices (p and q), any softening parameter involved (through φij), as well as the test-particle semimajor axis ap (through α1, 2). They are related to Ad and Bd via The outcome of this procedure is a set of expressions, akin to equations (4)–(6), which are convergent throughout the disc, in con- trast to the classical Laplace–Lagrange theory. Mathematically, this convergent behaviour is due to the fact that the emergent expressions contain Laplace coefﬁcients b(m) 1/2(α) – and not b(m) 3/2 – which diverge only weakly (logarithmically) as α →1: b(m) 1/2(α) ∝log(1 −α). As a result, upon integrating these expressions over the radial extent of the disc, one obtains a convergent and ﬁnite result for Rd. Physically, convergent expression is only natural since the calculation of the disc potential by direct two-dimensional integration over its surface is fully convergent at every point in the disc. The Heppenheimer method simply allows one to properly capture this property, unlike Ad(ap) = 2K npa2 p ψ1, Bd(ap) = K npa2 p ed(ap)ψ2. (20) (20) As shown in Appendix D, for certain ranges of power-law indices p and q both ψ1 and ψ2 converge to values depending only on p and q and any softening parameter used, provided that the test-particle orbit is well separated from the disc boundaries (i.e. in the limit α1, 2 →0). For p and q in these ranges (determined in Appendix D for each of the considered softened formalisms, similar to SR15), the coefﬁcients ψ1 and ψ2 are determined by the local behaviour of d(a) and ed(a) in the vicinity of test-particle semimajor axis. 2.1.4 Formalism of Teyssandier & Ogilvie (2016) – TO16 2.1.4 Formalism of Teyssandier & Ogilvie (2016) – TO16 Teyssandier & Ogilvie (2016) modiﬁed the unsoftened expressions (8), (9) for φLL ij (α) by simply replacing the usual Laplace coefﬁcients b(m) s with softened versions deﬁned such that We start this exercise using a model of apse-aligned (i.e. dϖ d/da = 0), truncated power-law (hereafter PL) disc as a simple example. We characterize surface density and eccentricity of such a disc by b(m) s with softened versions deﬁned such that B(m),TO s = 2 π π 0 cos(mθ) 1 + α2 −2α cos θ + S2α −s dθ. (15) (15) d(a) = 0 a0 a p , ed(a) = e0 a0 a q (16) (16) Thus, their softening parameter is ϵ2(α) = S2α, where S is a dimensionless constant. According to the authors, this substitution approximates the process of vertical averaging over the disc with constant aspect ratio S, and alleviates the classical singularity. The corresponding expressions for φij(α) are given by equations 7– 9 of Teyssandier & Ogilvie (2016). for ain ≤a ≤aout, where 0 and e0 are the pericentric surface density and eccentricity of the disc at some reference semimajor axis a0. Plugging this ansatz into equations (4)–(6), the secular disturbing function Rd due to PL discs can be simpliﬁed to (cf. Silsbee & Raﬁkov 2015) The aforementioned softening prescriptions have their softening parameters ϵ2(α) controlled by different constants — βc, bc, H, and S. For this reason, in what follows – with some abuse of notation – we will collectively refer to these constants as ‘softening parameters’ and denote them by ς. Rd = K ψ1e2 p + ψ2eped(ap) cos(ϖp −ϖd) , (17) (17) where K = πG 0ap 0 a1−p p and the dimensionless coefﬁcients ψ1 and ψ2 are given by 0 0 and ψ2 are given by ψ1 = 2 1 α1 α1−pφ22(α)dα + 2 1 α2 αp−2φ11(α)dα, (18) (18) 2.1.3 Formalism of Hahn (2003) – H03 Hahn (2003) computed the orbit-averaged interaction between two eccentric wires by accounting for their vertical thickness. The MNRAS 489, 4176–4195 (2019) MNRAS 489, 4176–4195 (2019) A. A. Seﬁlian and R. R. Raﬁkov 4180 the standard Laplace–Lagrange procedure (when applied to contin- uous discs). vertical extent h of a ring effectively softens its gravitational potential over a dimensionless scale H ∼h/a, which was assumed to be constant in that work (see also Ward 1989). Hahn (2003) demonstrated that the resultant φij(α) are functions of softened Laplace coefﬁcients In his pioneering calculation, Heppenheimer (1980) applied this method to axisymmetric power-law discs to recover the orbit- averaged disc potential to second order in eccentricities. This calculation has been subsequently extended to more general disc structures (Silsbee & Raﬁkov 2015; Davydenkova & Raﬁkov 2018, hereafter, SR15 and DR18 respectively), as well as to higher order in eccentricities (Seﬁlian & Touma 2019). This framework has been extensively veriﬁed for eccentric discs using direct integrations of test-particle orbits in actual disc potentials (e.g. SR15, Fontana & Marzari 2016, DR18), validating this approach. B(m),H s = 2 π π 0 cos(mθ) 1 + α2 −2α cos θ + H 2(1 + α2) −s dθ (14) (14) with constant H ≪1. In other words, the softening parameter is given by ϵ2(α) = H2(1 + α2) in that work. The explicit expressions for φij(α) in terms of B(m),H s are given by equations 17 of Hahn (2003). 3 COMPARISON: POWER-LAW DISCS Our goal is to examine the performance of different softening prescriptions outlined in Section 2.1 in comparison with the results obtained using the unsoftened Heppenheimer method (Section 2.2). 2.2 The unsoftened Heppenheimer method Given this, we ﬁrst focus on inﬁnitely extended (α1, 2 →0) PL discs with p and q within these ranges (we defer discussion of secular dynamics near the disc edges to Section 5). Then, ψ1 and ψ2 become independent of ap (i.e. functions of p, q, and ς only), making them useful as simple metrics for judging the validity of different models of softening. MNRAS 489, 4176–4195 (2019) Softened potentials of discs 4181 Softened potentials of discs 4181 100 101 102 103 C. 0.5/S 1 10-3 10-2 10-1 100 -103 -102 -100 D. -1/S -101 -10-1 10-1 2 TO16 p = - 0.5, q = 1.0 p = +1.0, q = 0.5 Softening 10-3 10-2 10-1 100 0.5 1 1.5 2 B. -1 -0.5 0 0.5 1 A. Softening SR15 H03 T02 Tr98 1 p = - 0.5, q = 1.0 p = +1.0, q = 0.5 2 Figure 1. Behaviour of the axisymmetric (ψ1, equation 18, top panels) and non-axisymmetric (ψ2, equation 19, bottom panels) components of the softened gravitational potential due to an inﬁnite power-law disc as a function of softening ς. The calculations assume two different disc structures speciﬁed by the values of p and q shown by different line types as explained in legend. For clarity, the results obtained by the softened formalisms of Tremaine (1998), Touma (2002), and Hahn (2003) are collated in the left-hand panels and those obtained by the softening method of Teyssandier & Ogilvie (2016) are shown in the right-hand panels. The left-hand panels also show the coefﬁcients ψ1 and ψ2 obtained by SR15 not assuming any softening (black horizontal lines). See the text (Section 3.1) for details. 10-3 10-2 10-1 100 0.5 1 1.5 2 B. -1 -0.5 0 0.5 1 A. Softening SR15 H03 T02 Tr98 1 p = - 0.5, q = 1.0 p = +1.0, q = 0.5 2 100 101 102 103 C. 0.5/S 1 10-3 10-2 10-1 100 -103 -102 -100 D. -1/S -101 -10-1 10-1 2 TO16 p = - 0.5, q = 1.0 p = +1.0, q = 0.5 Softening Downloaded from https://academic.oup.com/mnras/article-abstract/489/3/4176/5558240 by University of Cambridge user on 17 October 2019 Figure 1. Behaviour of the axisymmetric (ψ1, equation 18, top panels) and non-axisymmetric (ψ2, equation 19, bottom panels) components of the softened gravitational potential due to an inﬁnite power-law disc as a function of softening ς. 6The softening length bc present in the formulation of Touma (2002) is scaled by the test-particle semimajor axis ap in all the ﬁgures where we present results for inﬁnite PL discs. We do this to properly collate the results computed by different softening formalisms in one ﬁgure. 7Equations A37 and A38 in Silsbee & Raﬁkov (2015) provide analytic expressions for ψ1 and ψ2, respectively, for inﬁnite PL discs. 2.2 The unsoftened Heppenheimer method The calculations assume two different disc structures speciﬁed by the values of p and q shown by different line types as explained in legend. For clarity, the results obtained by the softened formalisms of Tremaine (1998), Touma (2002), and Hahn (2003) are collated in the left-hand panels and those obtained by the softening method of Teyssandier & Ogilvie (2016) are shown in the right-hand panels. The left-hand panels also show the coefﬁcients ψ1 and ψ2 obtained by SR15 not assuming any softening (black horizontal lines). See the text (Section 3.1) for details. 3.1 Behaviour with respect to variation of softening softening. Nevertheless, we generally8 ﬁnd that ς ≲10−3 guarantees the convergence of ψ1 and ψ2 to within few per cent of the correct values for all p and q as long as ain ≪ap ≪aout (see Fig. 4). Fig. 1 illustrates the behaviour of ψ1 and ψ2 predicted by each of the softening formalisms described in Section 2.1 for an inﬁnite PL disc, shown as a function of the corresponding ‘softening’6 ς for two different sets of p, q (indicated in panel B). For reference, black horizontal lines show the values of ψ1 and ψ2 expected from the calculations of SR15 using the unsoftened Heppenheimer approach.7 The same panels also indicate that ψ1(ς) and ψ2(ς) predicted by the softened formalism of Tremaine (1998, in green), while converging to ﬁnite values as ς = βc →0, do not reproduce the SR15 results exactly in this limit. Indeed, one can see that even for the smallest adopted value of βc = 10−3, the softening prescription of Tremaine (1998) yields ψ1 and ψ2 different by tens of per cent from SR15. It is easy to demonstrate that these quantitative differences do not vanish by further decreasing βc. For instance, when p = 1, the coefﬁcient ψ1 can be evaluated analytically as The left-hand panels of Fig. 1 illustrate the behaviour of the softening models of Tremaine (1998), Touma (2002), and Hahn (2003). They demonstrate that the latter two formalisms predict ψ1 and ψ2 in quantitative agreement with the unsoftened calculations of SR15: results of both Touma (2002, in blue) and Hahn (2003, in red) converge to the SR15 results as their corresponding softening ς approaches zero; both the amplitude and sign of ψ1 and ψ2 are reproduced. It is also evident that, depending on disc model, ψ1 and ψ2 converge to values given by SR15 at different values of ψTr98 1 = − 1 2 β2 c + 1 + E 2/ β2 c + 4 π β2 c + 4 = −1 2 + 1 2π + O(β2 c ) (21) ψTr98 1 = − 1 2 β2 c + 1 + E 2/ β2 c + 4 π β2 c + 4 = −1 2 + 1 2π + O(β2 c ) (21) (21) in agreement with Panel A (E(k) is the complete elliptic integral of a second kind). 3.1 Behaviour with respect to variation of softening 10-8 10-7 10-6 10-5 10-4 10-3 10-2 10-1 100 10-2 10-1 100 101 102 103 10-8 10-7 0.5 1 Distance separating a test-particle and its neighboring disc rings 0 ∞ x-1 p = -0.5 H03 T02 Tr98 TO16 DR18 Softening 10 -3 10 -2 10 -1 x = 1- m Right-hand panels of Fig. 1 show the behaviour of ψ1 (Panel C) and ψ2 (Panel D) as a function of ‘softening’, ς = S, resulting from the approach of Teyssandier & Ogilvie (2016). There are several features to note here. First, this model predicts ψ1 > 0 for all values of softening S and disc models (i.e. p and q), implying prograde free precession. This is in contrast with the other softening prescriptions, as well as SR15, which correctly capture retrograde free precession for p = 1 and prograde for p = −0.5 (see Panel A). Similarly, ψ2 is always negative, contrary to the expectations (see Panel B). Secondly, in the limit of S →0, both ψ1 and ψ2 attain values independent of the disc model, which is clearly inconsistent with the dependence on (p, q) seen in Figs 1(A) and (B). Thirdly, and most importantly, both ψ1 and ψ2 diverge as the softening S → 0. Indeed, it sufﬁces to employ the asymptotic expansion of the Laplace coefﬁcients B(m),TO 3/2 in the limit of α →1 (equation C7) to demonstrate that both ψ1 and ψ2 (equations 18 and 19) behave as ψTO16 1 ≈1 2S + O(S), ψTO16 2 ≈−1 S + O(S) (22) (22) Figure 3. Same as Fig. 2, but now for an axisymmetric power-law disc with p = −0.5. Note that for this disc model softened ˜ψ1(x) does not cross zero and converges to a positive value as x →0, in agreement with the results in Fig. 1(A). as S →0 for all values of p and q. The behaviour shown in Figs 1(C) and (D) agrees with these asymptotic expressions. as S →0 for all values of p and q. The behaviour shown in Figs 1(C) and (D) agrees with these asymptotic expressions. 3.1 Behaviour with respect to variation of softening Distance separating a test-particle and its neighboring disc rings 0 ∞ x = 1- m 10-8 10-7 10-6 10-5 10-4 10-3 10-2 10-1 100 -103 -102 -101 -100 -10-1 -10-2 -10-3 -x-1 Distance separating a test-particle and its neighboring disc rings 0 ∞ Distance separating a test-particle and its neighboring disc rings 0 ∞ Distance separating a test-particle and its neighboring disc rings 0 ∞ 10-3 10-2 10-1 100 101 102 103 p = 1 A. x-1 10-8 10-7 10-6 10-5 10-4 10-3 10-2 10-1 100 x = 1- m -103 -102 -101 -100 -10-1 -10-2 -10-3 10-8 10-7 -0.5 -0.4 -0.3 H03 T02 Tr98 TO16 DR18 Softening 10 -3 10 -2 10 -1 Downloaded from https://academic.oup.com/mnras/article-abstract/489/3/4176/5558240 by University of Cambridge user on 17 October 2 Downloaded from https://academic.oup.com/mnras/article-abstract/489/3/4176/5558240 by University of Cambridge user on 17 October 2019 Figure 2. Behaviour of the cumulative pre-factors ˜ψ1(x) (panel A) and ˜ψ2(x) (panel B) of the disturbing function due to a power-law disc (p = 1, q = 0.5 and ain →0, aout →∞) with softened gravity, shown as a function of x – relative separation between a given test-particle orbit and the nearest neighbouring disc rings. Formalisms of Hahn (2003), Touma (2002), Tremaine (1998), and Teyssandier & Ogilvie (2016) are shown by different colours as indicated in panel (A), for different values of softening (shown by different line types). The purple lines represent results obtained by the unsoftened expressions of Davydenkova & Raﬁkov (2018, DR18) based on the Heppenheimer method (see Section 6.3). Insets illustrate the behaviour as x →0 for the three convergent softened formalisms – see the text (Section 3.2) for more details. 10-8 10-7 10-6 10-5 10-4 10-3 10-2 10-1 100 10-2 10-1 100 101 102 103 10-8 10-7 0.5 1 Distance separating a test-particle and its neighboring disc rings 0 ∞ x-1 p = -0.5 H03 T02 Tr98 TO16 DR18 Softening 10 -3 10 -2 10 -1 x = 1- m Figure 3. Same as Fig. 2, but now for an axisymmetric power-law disc with p = −0.5. Note that for this disc model softened ˜ψ1(x) does not cross zero and converges to a positive value as x →0, in agreement with the results in Fig. 1(A). 3.1 Behaviour with respect to variation of softening At the same time, the unsoftened approach of SR15 predicts ψ1 = −1/2 for p = 1 disc. Moreover, close inspection of Figs 1(A) and (B) shows that, in the limit of βc →0, the ψ1 8For particles with orbits near sharp disc edges, we ﬁnd that smaller values of ς is required to recover the expected dynamics, see Section 5. MNRAS 489, 4176–4195 (2019) MNRAS 489, 4176–4195 (2019) 4182 82 A. A. Seﬁlian and R. R. Raﬁkov Distance separating a test-particle and its neighboring disc rings 0 ∞ 10-3 10-2 10-1 100 101 102 103 p = 1 A. x-1 10-8 10-7 10-6 10-5 10-4 10-3 10-2 10-1 100 x = 1- m -103 -102 -101 -100 -10-1 -10-2 -10-3 10-8 10-7 -0.5 -0.4 -0.3 H03 T02 Tr98 TO16 DR18 Softening 10 -3 10 -2 10 -1 10-3 10-2 10-1 100 101 102 103 10-8 10-7 1.4 1.6 1.8 2 p = 1.0 q = 0.5 B. Distance separating a test-particle and its neighboring disc rings 0 ∞ x = 1- m 10-8 10-7 10-6 10-5 10-4 10-3 10-2 10-1 100 -103 -102 -101 -100 -10-1 -10-2 -10-3 -x-1 Figure 2. Behaviour of the cumulative pre-factors ˜ψ1(x) (panel A) and ˜ψ2(x) (panel B) of the disturbing function due to a power-law disc (p = 1, q = 0.5 and ain →0, aout →∞) with softened gravity, shown as a function of x – relative separation between a given test-particle orbit and the nearest neighbouring disc rings. Formalisms of Hahn (2003), Touma (2002), Tremaine (1998), and Teyssandier & Ogilvie (2016) are shown by different colours as indicated in panel (A), for different values of softening (shown by different line types). The purple lines represent results obtained by the unsoftened expressions of Davydenkova & Raﬁkov (2018, DR18) based on the Heppenheimer method (see Section 6.3). Insets illustrate the behaviour as x →0 for the three convergent softened formalisms – see the text (Section 3.2) for more details. 10-3 10-2 10-1 100 101 102 103 10-8 10-7 1.4 1.6 1.8 2 p = 1.0 q = 0.5 B. 3.2 Details of convergence of different softening prescriptions To this goal, we compute the values of ψ1 and ψ2 in an inﬁnitely extended PL disc, like in Section 3.1, but now with a narrow clean gap (in semimajor axis) just around the test-particle orbit, and explore the effect of varying the width of this gap (Ward 1981). The inner and outer edges of the gap, in which d(a) is set to zero, are at ad, i = (1 −x)ap ≤ap and ad, o = (1 −x)−1ap ≥ap, respectively, with a single parameter x controlling the gap width. As x →0, the width of the gap goes to zero. We compute secular coefﬁcients in such a gapped disc denoted ˜ψ1(x) and ˜ψ2(x), by appropriately changing the upper integration limits in equations (18) and (19), i.e. from 1 to αm ≡1 −x. This eliminates gravitational effect of the disc annuli with ad, i(x) < a < ad, o(x). -0.5 0 0.5 1 1.5 2 2.5 3 3.5 p+q 0.5 1 1.5 2 B. 2 In Fig. 2, we display the behaviour of ˜ψ1(x) (Panel A) and ˜ψ2(x) (Panel B) as a function of x = 1 − ad,i/ad,o for various values of softening ς to highlight the effects of different softening prescriptions. The calculations assume a base PL disc model with p = 1 and q = 0.5 (recall that ψ1 depends on p, while ψ2 depends on p + q; equations 18and 19). There are several notable features in this ﬁgure. First, when the gap is wider than the characteristic softening length ςap, i.e. ς ≲x ≤1, the amplitudes of both ˜ψ1(x) and ˜ψ2(x) increase from zero at x = 1 (inﬁnitely wide gap) to their maximum values reached at x ∼ς. In all cases ψ1 is positive, meaning prograde precession of a test-particle orbit in a wide gap, in agreement with the unsoftened results of Ward (1981) and Davydenkova & Raﬁkov (2018) – secular effect of a collection of distant disc ‘wires’ conforms to expectations of the classical Laplace–Lagrange theory (i.e. prograde precession). -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 q 2 3 4 5 m ep / ed(a) p = 1 C. In the range ς ≲x ≪1, we ﬁnd that ˜ψ1(x) ∼\| ˜ψ2(x)\| ∼x−1, irrespective of the softening model used; their maximum values are always ∼ς−1. 3.2 Details of convergence of different softening prescriptions and ψ2 curves computed using softening model of Tremaine (1998) are offset vertically from the unsoftened calculations by 1/2π and −1/π, respectively, for any (p, q) – see also Fig. 4. We will analyse reasons for this quantitative discrepancy in Section 6.1. Different softening prescriptions explored in this work are designed to modify the behaviour of the integrand in equations (5) and (6) primarily in the vicinity of the test-particle orbit, i.e. as a →ap MNRAS 489, 4176–4195 (2019) MNRAS 489, 4176–4195 (2019) -0.5 0 0.5 1 1.5 2 2.5 3 3.5 p -0.5 0 0.5 1 SR15 H03 T02 Tr98 A. Softening = 0.001 1 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 q 2 3 4 5 m ep / ed(a) p = 1 -0.5 0 0.5 1 1.5 2 2.5 3 3.5 p+q 0.5 1 1.5 2 B. C. 2 Figure 4. Dependence of the coefﬁcients ψ1 (panel A) and ψ2 (panel B) on the power-law disc model represented by the indices p and p + q, respectively. Panel C shows the amplitude em p of eccentricity oscillations (normalized by disc eccentricity ed) induced by disc gravity. Results for softened formalisms of Hahn (2003, in red), Touma (2002, in blue) and Tremaine (1998, in green) are computed using softening ς = 10−3. Calculations assume inﬁnitely extended disc (i.e. no edge effects). For reference, open black circles show the proﬁles of ψ1, ψ2, and em p as computed by SR15: curves for Hahn (2003) and Touma (2002) fall on t f th hil th f T i (1998) h t t ff t i t Softened potentials of discs 4183 -0.5 0 0.5 1 1.5 2 2.5 3 3.5 p -0.5 0 0.5 1 SR15 H03 T02 Tr98 A. Softening = 0.001 1 -0.5 0 0.5 1 1.5 2 2.5 3 3.5 p+q 0.5 1 1.5 2 B. 2 -0.5 0 0.5 1 1.5 2 2.5 3 3.5 p -0.5 0 0.5 1 SR15 H03 T02 Tr98 A. Softening = 0.001 1 or α →1. For this reason, it is interesting to look in more detail on how this modiﬁcation actually allows each softening model to achieve (or not) the expected results. This exercise also illustrates the contribution of different parts of the disc to secular dynamics. 3.2 Details of convergence of different softening prescriptions This convergent behaviour is easy to understand since for ς ≲x the role of softening is negligible, B(m) s (α, ς) ≈b(m) s (α), and all φij effectively reduce to their classical counterparts φLL ij given by equations (8) and (9), which can be easily veriﬁed using the expressions listed in Table 1. The scaling of ˜ψ1(x) and \| ˜ψ2(x)\| with x is simply a result of asymptotic behaviour of b(m) 3/2(α) →(1 −α)−2 as α →1, upon radial integration in equations (18) and (19). Secondly, upon reaching their extrema at x ∼ς, amplitudes of ˜ψ1(x) and ˜ψ2(x) computed using softening prescriptions of Tr98, T02, and H03 start decreasing as x decreases. In the range of semimajor axes corresponding to x ≲ς, softening signiﬁcantly modiﬁes the behaviour of B(m) s (α, ς) away from the divergent behaviour of b(m) s (α). The modiﬁcation is such that the softened interaction with the disc annuli ≲ςap away from the test-particle orbit starts to dynamically counteract the contribution of the more distant annuli (with x ≈1). As a result of this compensation, ˜ψ1 and ˜ψ2 cross zero and change sign at some x = Cς2, where C ∼1 is a constant.9 Figure 4. Dependence of the coefﬁcients ψ1 (panel A) and ψ2 (panel B) on the power-law disc model represented by the indices p and p + q, respectively. Panel C shows the amplitude em p of eccentricity oscillations (normalized by disc eccentricity ed) induced by disc gravity. Results for softened formalisms of Hahn (2003, in red), Touma (2002, in blue) and Tremaine (1998, in green) are computed using softening ς = 10−3. Calculations assume inﬁnitely extended disc (i.e. no edge effects). For reference, open black circles show the proﬁles of ψ1, ψ2, and em p as computed by SR15: curves for Hahn (2003) and Touma (2002) fall on top of them, while those for Tremaine (1998) show constant offset in terms of both ψ1 and ψ2 (illustrated by scale bars in panels A and B) resulting in deviation between em p curves (panel C). Figure 4. Dependence of the coefﬁcients ψ1 (panel A) and ψ2 (panel B) on the power-law disc model represented by the indices p and p + q, respectively. Panel C shows the amplitude em p of eccentricity oscillations (normalized by disc eccentricity ed) induced by disc gravity. 9For p = 1, ˜ψ1 becomes analytic for the softened formalisms of both H03 and Tr98 allowing us to quantify the value of C. Performing the integral over dα in equations (18) and (19), we ﬁnd CTr98 = (π −1)/2 and CH03 = π; in agreement with Fig. 2. For other values of p and q, for which ψ1 < 0 (c.f. Fig. 4), we numerically ﬁnd that C varies by at most a factor of 10. 3.2 Details of convergence of different softening prescriptions Results for softened formalisms of Hahn (2003, in red), Touma (2002, in blue) and Tremaine (1998, in green) are computed using softening ς = 10−3. Calculations assume inﬁnitely extended disc (i.e. no edge effects). For reference, open black circles show the proﬁles of ψ1, ψ2, and em p as computed by SR15: curves for Hahn (2003) and Touma (2002) fall on top of them, while those for Tremaine (1998) show constant offset in terms of both ψ1 and ψ2 (illustrated by scale bars in panels A and B) resulting in deviation between em p curves (panel C). At the same time, ˜ψTO16 1 and ˜ψTO16 2 calculated according to Teyssandier & Ogilvie (2016) clearly show different behaviour. Instead of decreasing in amplitude as x ≲ς, they remain essentially MNRAS 489, 4176–4195 (2019) A. A. Seﬁlian and R. R. Raﬁkov 4184 by Silsbee & Raﬁkov (2015) using the unsoftened Heppenheimer approach. constant, having reached their saturated values ∼ς−1 at x ∼ς. This explains the lack of convergence with S obvious in Figs 1(C) and (D), since the values to which \| ˜ψTO16 1 \| and \| ˜ψTO16 2 \| converge keeps increasing as ς →0. Moreover, both coefﬁcients also never change sign, always predicting prograde precession ( ˜ψTO16 1 > 0). The origin of this difference with other smoothing prescriptions will be addressed in Section 6.2. It is clear that the softened formalisms of both Touma (2002) and Hahn (2003) perfectly reproduce the expected behaviour of the pre-factors ψ1 and ψ2 as a function of p and q (i.e. for various PL disc models). On the other hand, the prescription of Tremaine (1998) predicts a behaviour of ψ1 and ψ2 only in qualitative agreement with the expected results: the computed values of secular coefﬁcients deviate by tens of per cent from that of SR15. For all values of p and q, the formalism of Tremaine (1998) yields an additional positive contribution to ψ1 equal to 1/2π and a negative contribution to ψ2 equal to −1/π (these offsets are highlighted in Figs 4A and B by scale bars). 4.1 Quartic disc model We start by looking at the secular dynamics in the potential of a Quartic disc characterized by the surface density To summarize, Figs 2 and 3 indicate that secular dynamics in softened power-law discs is dictated by the delicate balance of the opposing contributions due to nearby (i.e. with x ≲ς) and distant disc annuli (i.e. with x ≳ς), in qualitative agreement with the unsoftened results of Ward (1981). These ﬁgures also demonstrate that the softening prescription of TO16 yields inaccurate results due to its inability to capture the dynamical effects of disc annuli adjacent to the test-particle orbit (those with x ≲ς), see Section 6.2. We will discuss additional implications of these calculations in Section 6.3. d(a) = ˜ 0 (aout −a)2(ain −a)2 (aout −ain)4 , (23) (23) and linear eccentricity proﬁle in the form ed(a) = ˜e0 1 + aout −a aout −ain (24) (24) for ain ≤a ≤aout (with ain = 0.1 au, aout = 5 au), where ˜ 0 = 1153 g cm−2 and ˜e0 = 0.01 are normalization constants (one of the models in DR18). Fig. 5 summarizes the salient features of secular dynamics in the potential of such a disc adopting a softening value of ς = 10−3. It shows the excellent agreement between the radial proﬁles of Ad, Bd, and em p computed using the unsoftened calculations of Davydenkova & Raﬁkov (2018) and those computed using softening prescriptions of Touma (2002) and Hahn (2003). Similar to the case of PL discs, we ﬁnd that the softening prescription of Tremaine (1998) yields results that agree qualitatively with the expected results but differ quantitatively. Deviations of Ad and Bd computed using this model from Davydenkova & Raﬁkov (2018), in particular, modify the locations at which Ad and Bd become zero. This explains the slight shift in the semimajor axes at which em p = 2Bd/Ad goes through zero or diverges, see Fig. 5. 4 COMPARISON: NON-POWER-LAW DISCS We now turn our attention to the performance of the different softening prescriptions for more general discs. Namely, we focus on two apse-aligned, non-PL disc models previously studied by Davy- denkova & Raﬁkov (2018) based on the unsoftened Heppenheimer method. The dynamics in such non-PL discs, according to DR18, differ from the PL discs in a very important way: the free-precession of test particles can naturally change from retrograde to prograde (and vice versa) within such discs. Furthermore, an important feature of the models considered below is that d smoothly goes to zero at ﬁnite radii in a manner that does not give rise to the edge effects, see DR18 and Section 5. In Fig. 3, we show calculations for ˜ψ1(x) similar to those in Fig. 2(A) but for a different disc model – axisymmetric PL disc with p = −0.5. In this case unsoftened calculations (e.g. SR15) predict that disc gravity should drive prograde precession of a test particle in a smooth disc. One can clearly see that many of the features present in Fig. 2 are reproduced for this model as well: discrepancy between the TO16 model and others, ˜ψ1(x) ∼x−1 scaling for ς ≲ x ≪1, decay of ˜ψ1(x) for ς2 ≲x ≲ς, and ultimate convergence to ψ1 in a disc with no gap. The only obvious difference is the fact that ˜ψ1 does not cross zero10 for this disc model with p = −0.5. 3.2 Details of convergence of different softening prescriptions Although these differences are not very signiﬁcant, they lead to (1) predicting a wrong sign for the test-particle free- precession rate for p ≈0 or p ≈3 (for which SR15 yields ψ1 ≈0), and (2) a mismatch of tens of per cent between the disc-driven forced eccentricity oscillations, em p /ed(a) = \|ψ2/ψ1\|, and the expectations based on SR15. The latter point is illustrated in Fig. 4(C). Upon further decrease of x below ς2, both ˜ψ1 and ˜ψ2 computed using models of Tr98, T02, and H03 ultimately converge to their corresponding values obtained for a continuous disc (i.e. for x = 0, see Fig. 1) independent of the assumed value of ς. We note that the opposite contributions to e.g. ψ1 produced by the distant (x ≳ς, positive) and nearby (i.e. with x ≲ς, negative) disc annuli is not unique to softened gravity. Indeed, both Ward (1981) and Davydenkova & Raﬁkov (2018), using the unsoftened Heppenheimer method, found that a particle orbit fully embedded in a p = 1 disc has negative precession rate, whereas a particle orbiting fully in the gap precesses in the positive sense (and at high rate if the gap is narrow). As the gap width is reduced, a smooth transition between the two regimes must occur as the test-particle orbit starts crossing the gap edge (i.e. for x ≲ep), with the disc annuli crossing the particle orbit giving rise to a negative contribution to ˜ψ1. Eventually, the shrinking of the gap brings ˜ψ1 to a ﬁnite negative value (for p = 1 disc) as x →0. This sequence is very similar to the behaviour we ﬁnd with softened gravity for x ≲ς. 10This is the case for all power-law disc models with p < 0 or p > 3 for which the expected free precession rate is positive, see Fig. 4. 11We do not present results obtained by the method of Teyssandier & Ogilvie (2016). proﬁle proﬁle d(a) = ˜ 0 exp 4 −[(a/ac) + (ac/a)]2 wc (25) (25) centred around ac = 1.5 au with width wc = 0.18 and surface density ˜ 0 = 100 g cm−2 at ac. The eccentricity proﬁle is still given by equation (24). In Fig. 6, we plot the behaviour of the corresponding Ad, Bd, and em p for the three (convergent) softened formalisms with ς = 10−3, together with those of unsoftened Heppenheimer method (DR18, in black). Once again, the results obtained using the formalisms of Touma (2002) and Hahn (2003) fall on top of the expecta- tions. However, for this disc model the formalism of Tremaine (1998) reproduces the unsoftened calculations of Davydenkova & Raﬁkov (2018) quite well: the relative deviations are always less than 10 per cent. This improvement will be discussed further in Section 6.1. quantitative differences between the studies of Tremaine (2001) and Gulati, Saini & Sridhar (2012), who analysed the slow (m = 1) modes supported by softened Kuzmin discs with softening prescriptions b∝r and b = const, respectively. 3.3 Variation of disc model – p and q We now examine the dependence of ψ1 and ψ2 on the speciﬁcs of the disc model reﬂected in power-law indices p and q. Figs 4(A) and (B) illustrate the results based on different softening prescriptions11 assuming a softening value of ς = 10−3 (for which Figs 1(A) and (B) suggest good convergence of ψ1 and ψ2). For reference, black open circles show the expected behaviour of ψ1 and ψ2 computed 10This is the case for all power-law disc models with p < 0 or p > 3 for which the expected free precession rate is positive, see Fig. 4. 11We do not present results obtained by the method of Teyssandier & Ogilvie (2016). 10This is the case for all power-law disc models with p < 0 or p > 3 for which the expected free precession rate is positive, see Fig. 4. The difference between the Tremaine (1998) and Touma (2002) calculations illustrated here could be relevant for understanding the 11We do not present results obtained by the method of Teyssandier & Ogilvie (2016). MNRAS 489, 4176–4195 (2019) Softened potentials of discs Softened potentials of discs 4185 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 ep m 1.4 1.6 1.8 0.037 0.039 0.041 A. Gaussian -1.5 -1 -0.5 0 0.5 Ad, 10-3yr-1 1.4 1.6 1.8 -1.4 -1.2 -1 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 -1 -0.5 0 0.5 1 1.5 2 2.5 Bd, 10-5yr-1 1.4 1.6 1.8 1 1.5 2 2.5 p a , AU B. C. DR18 H03 T02 Tr98 Figure 6. Same as Fig. 5, but now for a Gaussian disc with d(a) and ed(a) given by equations (25) and (24), respectively. Note that for this disc model the formalism of Tremaine (1998, in green) shows quite good agreement with the unsoftened results, even at the quantitative level. See the text (Section 4.2) for details. 4185 f p f -0.6 -0.4 -0.2 0 0.2 0.4 Ad, 10-3yr-1 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 p a , AU -0.5 0 0.5 1 Bd, 10-5yr-1 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 ep m 4.3 4.4 4.5 0 0.05 B. C. A. DR18 H03 T02 Tr98 Quartic Figure 5. Performance of different softening formalisms (different colours) with softening parameter ς = 10−3 in the potential of a Quartic disc, see equation (23), with the eccentricity proﬁle (24). 3.3 Variation of disc model – p and q The disc extends from ain = 0.1 au to aout = 5 au. Shown as a function of semimajor axis ap are the proﬁles of (A) the amplitude em p of the disc-induced eccentricity oscillations, (B) the rate of disc-driven free precession Ad, and (C) the coefﬁcient Bd appearing in the non-axisymmetric part of the disturbing function (4). The black lines represent the expected unsoftened results as computed by Davydenkova & Raﬁkov (2018). Curves for Hahn (2003) and Touma (2002) fall on top of the unsoftened results, while the softening method of Tremaine (1998) shows only qualitative agreement. 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 ep m 1.4 1.6 1.8 0.037 0.039 0.041 A. Gaussian -1.5 -1 -0.5 0 0.5 Ad, 10-3yr-1 1.4 1.6 1.8 -1.4 -1.2 -1 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 -1 -0.5 0 0.5 1 1.5 2 2.5 Bd, 10-5yr-1 1.4 1.6 1.8 1 1.5 2 2.5 p a , AU B. C. DR18 H03 T02 Tr98 Figure 6. Same as Fig. 5, but now for a Gaussian disc with d(a) and ed(a) given by equations (25) and (24), respectively. Note that for this disc model the formalism of Tremaine (1998, in green) shows quite good agreement with the unsoftened results, even at the quantitative level. See the text (Section 4.2) for details. proﬁle d(a) = ˜ 0 exp 4 −[(a/ac) + (ac/a)]2 wc (25) centred around ac = 1.5 au with width wc = 0.18 and surface density ˜ 0 = 100 g cm−2 at ac. The eccentricity proﬁle is still Downloaded from https://academic.oup.com/mnras/article-abstract/489/3/4176/5558240 by University of Cambridge user on 17 Octob -0.6 -0.4 -0.2 0 0.2 0.4 Ad, 10-3yr-1 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 p a , AU -0.5 0 0.5 1 Bd, 10-5yr-1 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 ep m 4.3 4.4 4.5 0 0.05 B. C. A. DR18 H03 T02 Tr98 Quartic 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 ep m 1.4 1.6 1.8 0.037 0.039 0.041 A. Gaussian -1.5 -1 -0.5 0 0.5 Ad, 10-3yr-1 1.4 1.6 1.8 -1.4 -1.2 -1 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 -1 -0.5 0 0.5 1 1.5 2 2.5 Bd, 10-5yr-1 1.4 1.6 1.8 1 1.5 2 2.5 p a , AU B. C. 3.3 Variation of disc model – p and q DR18 H03 T02 Tr98 Downloaded from https://academic.oup.com/mnras/article-abstract/489/3/4176/5558240 by University of Cambridge user on 17 October 2019 Figure 6. Same as Fig. 5, but now for a Gaussian disc with d(a) and ed(a) given by equations (25) and (24), respectively. Note that for this disc model the formalism of Tremaine (1998, in green) shows quite good agreement with the unsoftened results, even at the quantitative level. See the text (Section 4.2) for details. Figure 5. Performance of different softening formalisms (different colours) with softening parameter ς = 10−3 in the potential of a Quartic disc, see equation (23), with the eccentricity proﬁle (24). The disc extends from ain = 0.1 au to aout = 5 au. Shown as a function of semimajor axis ap are the proﬁles of (A) the amplitude em p of the disc-induced eccentricity oscillations, (B) the rate of disc-driven free precession Ad, and (C) the coefﬁcient Bd appearing in the non-axisymmetric part of the disturbing function (4). The black lines represent the expected unsoftened results as computed by Davydenkova & Raﬁkov (2018). Curves for Hahn (2003) and Touma (2002) fall on top of the unsoftened results, while the softening method of Tremaine (1998) shows only qualitative agreement. proﬁle 6 DISCUSSION So far the disc models that we explored were either inﬁnitely extended (Section 3) or had surface density smoothly petering out to zero at ﬁnite radii (Section 4). This allowed us to not worry about the effects of sharp disc edges – discontinuous drops of the surface density – on secular dynamics, which are known to be important (Silsbee & Raﬁkov 2015; Davydenkova & Raﬁkov 2018). Results of previous sections reveal a diversity of outcomes when different softening models are applied. Two models – those of Hahn (2003) and Touma (2002) – successfully reproduce the unsoftened calculations based on the Heppenheimer method in the limit of zero softening. In the same limit, the formalism of Tremaine (1998) yields convergent results which are, however, different from the unsoftened calculations, typically by tens of per cent. Finally, the softening method of Teyssandier & Ogilvie (2016) does not lead to convergent results in the limit of vanishing softening parameter. Interestingly, the two successful models (Touma 2002; Hahn 2003) have been derived using rather different underlying assumptions (see Sections 2.1.2 and 2.1.3), producing different mathematical expressions for φij (see Table 1), and yet their results are consistent with the unsoftened calculations as ς →0. We now relax this assumption and examine the performance of different softening models in the vicinity of a sharp edge of the disc, where surface density drops discontinuously from a ﬁnite value to zero at a ﬁnite semimajor axis a = aedge. To that effect we analyse the behaviour of secular coefﬁcient Ad computed using the formalism of Hahn (2003) for different values of softening (results for B d are very similar) near the disc edge (we veriﬁed that softening prescriptions of Touma 2002 and Tremaine 1998 give very similar results in the limit ς →0). Fig. 7 shows the run of Ad near the inner edge ain of the disc for test particles both inside ( ap < ain) and within ( ap > ain) the disc as predicted by the formalism of Hahn (2003). The calculation assumes circular PL disc with p = 1 and 0 = 100 g cm−2 extending between ain = 1 au to aout = 10 au, where we have set a0 = aout (equation 16). 4.2 Gaussian rings Similar results can be obtained by the softened formalisms of both Touma (2002) and Tremaine (1998). Figure 7. The behaviour of the free precession rate Ad near the inner edge ain = 1 au of a circular power-law disc with surface density d(a) = 100 g cm−2 (10 au/a) (equation 16). One can see that the expected divergent behaviour of Ad near the disc edge is reproduced by the softening prescription of Hahn (2003) in the limit ς →0. However, very near the sharp edge of the disc ς has to be very small for quantitative accuracy to be attained. Similar results can be obtained by the softened formalisms of both Touma (2002) and Tremaine (1998). Finally, we note that the dynamics of particles orbiting outside the disc (where d(a) = 0) is successfully reproduced by the classical Laplace–Lagrange theory without adopting any softening prescrip- tion (e.g. see Petrovich et al. 2019). Indeed, outside the radial extent of the disc semimajor axis overlap (i.e. ap = a) is naturally excluded thus avoiding the classical singularity. Outside the disc the unsoft- ened calculations based on the Heppenheimer method (e.g. SR15, DR18) reduce to the classical Laplace–Lagrange theory exactly. 4.2 Gaussian rings Next we investigate secular dynamics in the potential of another disc model from DR18 – a Gaussian ring with the surface density MNRAS 489, 4176–4195 (2019) A. A. Seﬁlian and R. R. Raﬁkov 4186 Figure 7. The behaviour of the free precession rate Ad near the inne edge ain = 1 au of a circular power-law disc with surface density d(a = 100 g cm−2 (10 au/a) (equation 16). One can see that the expected divergent behaviour of Ad near the disc edge is reproduced by the softening prescription of Hahn (2003) in the limit ς →0. However, very near the sharp edge of the disc ς has to be very small for quantitative accuracy to be attained. Similar results can be obtained by the softened formalisms of both Touma (2002) and Tremaine (1998). edge by more than an order of magnitude. Thus, accurately capturing secular dynamics near the sharp edges of discs/rings requires using very small values of softening.12 This ﬁnding could be problematic, for instance, for numerical modelling of planetary rings, often found to have very sharp edges (Graps et al. 1995; Tiscareno 2013). Note that in Fig. 7 softened Ad passes through zero exactly at ain, showing two sharp peaks of opposite signs just around this radius. Similar behaviour was found by Davydenkova & Raﬁkov (2018) for zero-thickness discs with d dropping sharply but continuously near the edge, demonstrating that variation of the sharpness of the edge is akin to softening gravity. In the case of truly zero-thickness disc and no softening (e.g. SR15) the segment of Ad curve connecting the two peaks turns into a vertical line at ain. Similar divergent behaviour of Ad (and Bd) arises also at the outer edge of the disc considered in Fig. 7 and, in general, at any radius within a disc where d(a) exhibits a discontinuity. Figure 7. The behaviour of the free precession rate Ad near the inner edge ain = 1 au of a circular power-law disc with surface density d(a) = 100 g cm−2 (10 au/a) (equation 16). One can see that the expected divergent behaviour of Ad near the disc edge is reproduced by the softening prescription of Hahn (2003) in the limit ς →0. However, very near the sharp edge of the disc ς has to be very small for quantitative accuracy to be attained. 6.1 On the softening prescription of Tremaine (1998) Results of Sections 3 and 4 indicate that the softening prescription of Tremaine (1998) – unlike that of Touma (2002) and Hahn (2003) – leads to quantitative differences when compared to the unsoftened calculations. We now demonstrate where these differences come from. Downloaded from https://academic.oup.com/mnras/article-abstract/489/3/4176/5558 Downloaded from https://academic.oup.com/mnras/a The form of the softened Laplace coefﬁcient B(m),Tr s deﬁned by equation (12) suggests interaction potential (10) with F(r1, r2) = β2 c max(r2 1, r2 2) for the softening model of Tremaine (1998). In Appendix B, we show that propagating this form of F(r1, r2) through our general framework results in the following expressions for the coefﬁcients φij: φ11 = φ22 = α 8 B(1),Tr 3/2 −3αβ2 c B(0),Tr 5/2 −δ(α −1)β2 c B(0),Tr 3/2 , (26) φ12 = −α 4 B(2),Tr 3/2 −3αβ2 c B(1),Tr 5/2 −δ(α −1)β2 c B(1),Tr 3/2 . (27) φ11 = φ22 = α 8 B(1),Tr 3/2 −3αβ2 c B(0),Tr 5/2 −δ(α −1)β2 c B(0),Tr 3/2 , (26) (26) φ12 = −α 4 B(2),Tr 3/2 −3αβ2 c B(1),Tr 5/2 −δ(α −1)β2 c B(1),Tr 3/2 . (27) (27) These expressions are different from the entries in Table 1 for Tremaine (1998) in a single but very important way – presence of terms involving Dirac delta-function. Such terms arise because the form of F(r1, r2) adopted in Tremaine (1998) is not sufﬁciently smooth – its ﬁrst derivative is discontinuous at r1 = r2, while the calculation of φij involves second-order derivatives of F, see equa- tions (A25)–(A27), as well as equation (A28). Such singular terms do not arise in other types of softening prescriptions examined in our work since they all use inﬁnitely differentiable versions of F(r1, r2). Thus, these terms should not be interpreted as representing some kind of ‘self-interaction’ within the disc, they merely reﬂect the mathematical smoothness properties of F used in Tremaine (1998). Figure 8. Similar to Fig. 1, but now using the expressions for φij given by equations (26) and (27) and equations (32) and (33) obtained by propagating F(r1, r2) = ς2max(r2 1, r2 2) of Tremaine (1998) and F(r1, r2) = ς2r1r2 of Teyssandier & Ogilvie (2016), respectively, through the general framework outlined in Appendix A. Shown as a function of softening ς are ψ1 (panel A) and ψ2 (panel B) for two PL disc models speciﬁed by p and q indicated in panel A. 6 DISCUSSION To understand this variation of outcomes, we developed a general framework for computing secular coefﬁcients φij (thus fully determining the softened secular model via equations 4–6) given an arbitrary softened two-point interaction potential in the form (10). This procedure involves orbit-averaging the softened potential along the particle trajectories; its details are presented in Appendix A. There is also an alternative approach, sketched in Appendix A4, which assumes the disc to be a continuous entity from the start. Both of them arrive at the same expressions for Rd. The unsoftened calculations based on Heppenheimer (1980) invariably predict that the free eccentricity precession rate Ad, as well as Bd, should diverge as the sharp edge of the disc is approached (e.g. SR15, DR18). Tremaine (2001) also found precession rate to diverge near the edge of a Jacobs–Sellwod ring (Jacobs & Sellwood 2001). This is indeed the case as shown by the dashed curve computed using SR15. Using these results we show in Appendix B that the expressions for φij found by Touma (2002) and Hahn (2003) can be recovered exactly using this general framework if we set F(r1, r2) = b2 c and F(r1, r2) = H 2(r2 1 + r2 2), respectively, in expression (10) for the two-point potential. This approach also allows us to address some The softened calculation using Hahn (2003) does largely repro- duce this behaviour. However, we ﬁnd that very close to the ring edge (at \|a −ain\|/ain ∼10−3) the agreement is achieved only for ς ≤10−4, which is considerably smaller than the values (ς ∼10−2) required to reproduce the dynamics of particles far from the disc edges, ain ≪ap ≪aout, see Fig. 1. For ς = 10−2 the softened calculation predicts Ad different from the SR15 results near the disc 12On the other hand, this condition is relaxed when the edge is not exactly sharp but rather has a ﬁnite width r over which the disc surface density smoothly peters out to zero; in this case ς only needs to be ≲r/r. MNRAS 489, 4176–4195 (2019) Softened potentials of discs 4187 4187 Softening -0.9 -0.8 -0.7 -0.6 -0.5 -0.4 -0.3 1 A. p = +1.0, q = 0.5 p = +0.5, q = 0.0 SR15 TO16* Tr98 * 10-3 10-2 10-1 100 0.5 1 1.5 2 2 B. Figure 8. Similar to Fig. 6.1 On the softening prescription of Tremaine (1998) Black lines represent the expectations based on Silsbee & Raﬁkov (2015), to which the new expressions for ψ1 and ψ2 successfully converge as ς →0. Presence of these terms in equations (26)–(27) introduces correc- tions to coefﬁcients Ad and Bd (equations 5 and 6) in apse-aligned discs in the form see equation (20). These corrections exactly match the offsets seen in Fig. 4 between the calculations of Tremaine (1998) and the unsoftened calculations, thus explaining the origin of these uniform shifts. We also conﬁrmed this explanation in Fig. 8, where we show the convergence of modiﬁed Tremaine (1998) coefﬁcients to the correct unsoftened values as softening is varied for two values of p and q. δAd(ap) = −πG 2npap β2 c d(ap)B(0),Tr 3/2 α=1 , (28) δBd(ap) = + πG 2npap β2 c d(ap)ed(ap)B(1),Tr 3/2 α=1 . (29) δAd(ap) = −πG 2npap β2 c d(ap)B(0),Tr 3/2 α=1 , (28) (28) δBd(ap) = + πG 2npap β2 c d(ap)ed(ap)B(1),Tr 3/2 α=1 . (29) (29) To summarize, equations (26) and (27) should replace the expressions given by equation 26 of Tremaine (1998) in applications to continuous discs. However, when considering the interaction of two individual annuli with different semimajor axes (like in the classical Laplace–Lagrange theory), one has α ̸= 1 and terms in equations (26) and (27) containing Dirac delta-function naturally vanish, reducing ψ1 and ψ2 back to the expressions quoted in Tremaine (1998). Accounting for these corrections, we conﬁrmed that the correct (un- softened) behaviour of the coefﬁcients of Rd can be reproduced for the non-PL discs – Quartic and Gaussian models, see Section 4. Note that δAd(ap) and δBd(ap) are proportional to the local disc surface density d(ap) and B(m),Tr 3/2 (α = 1) ∼β−2 c , see equation (C7). This likely explains the improved agreement between the calculations of Tremaine (1998) and Davydenkova & Raﬁkov (2018) for Gaussian rings (see Fig. 6), which feature mass concentration in a narrow range of radii (in contrast to the Quartic model, see Fig. 5). For PL discs the terms proportional to Dirac delta-function in equations (26) and (27) give rise to the following modiﬁcations of the coefﬁcients ψ1 and ψ2 deﬁned by equations (18) and (19): 6 DISCUSSION 1, but now using the expressions for φij given by equations (26) and (27) and equations (32) and (33) obtained by propagating F(r1, r2) = ς2max(r2 1, r2 2) of Tremaine (1998) and F(r1, r2) = ς2r1r2 of Teyssandier & Ogilvie (2016), respectively, through the general framework outlined in Appendix A. Shown as a function of softening ς are ψ1 (panel A) and ψ2 (panel B) for two PL disc models speciﬁed by p and q indicated in panel A. Black lines represent the expectations based on Silsbee & Raﬁkov (2015), to which the new expressions for ψ1 and ψ2 successfully converge as ς →0. of the questions raised above, which we do in Sections 6.1 and 6.2 below. Softening -0.9 -0.8 -0.7 -0.6 -0.5 -0.4 -0.3 1 A. p = +1.0, q = 0.5 p = +0.5, q = 0.0 SR15 TO16* Tr98 * 10-3 10-2 10-1 100 0.5 1 1.5 2 2 B. Downloaded from https://academic.oup.com/mnras/article-abstract/489/3/4176/5558240 by University of Cambridge user on 17 October 2019 6.2 On the softening prescription of Teyssandier & Ogilvie (2016) We now turn our attention to the model of Teyssandier & Ogilvie (2016) trying to understand its distinct (divergent) behaviour. From the expression for B(m),TO s in equation (15) one infers that this model features softening parameter in the form ϵ2(α) = S2α. To soften secular interaction, Teyssandier & Ogilvie (2016) directly substituted b(m) 3/2 in the classical expressions (8) and (9) for φLL ij with δψ1 = −1 4β2 c B(0),Tr 3/2 α=1 = −1 2π + O β2 c , (30) δψ2 = 1 2β2 c B(1),Tr 3/2 α=1 = 1 π + O β2 c , (31) δψ1 = −1 4β2 c B(0),Tr 3/2 α=1 = −1 2π + O β2 c , (30) (30) δψ2 = 1 2β2 c B(1),Tr 3/2 α=1 = 1 π + O β2 c , (31) (31) MNRAS 489, 4176–4195 (2019) 4188 A. A. Seﬁlian and R. R. Raﬁkov This is typically not a problem for the unsoftened calculations, which converge to the expected full disc result even with a relatively coarse radial sampling of the integral contribution to e.g. the free precession rate. Indeed, purple curves in Figs 2 and 3 demonstrate this by showing the unsoftened ˜ψ1(x) and ˜ψ2(x) computed without accounting14 for the contributions from ad, i < ap < ad, o (see Section 3.2) to the integral terms in the unsoftened expressions of Davydenkova & Raﬁkov (2018). These curves converge to the correct full disc result without exhibiting large variations in ˜ψ1(x) and ˜ψ2(x), typical for softened cases. B(m),TO 3/2 , see Section 2.1.4; this simple swap of Laplace coefﬁcients has not been justiﬁed rigorously. On the other hand, in Appendix B we show that softening parameter in the form ϵ2(α) = ς2α corresponds to softening function F(r1, r2) = ς2r1r2 in the two-point potential (10), see equation (A21). Propagating such a form of F(r1, r2) through our general framework in Appendix A, we ﬁnd the following expressions for the coefﬁcients φij with ς = S (Appendix B): φ11 = φ22 = α 8 B(1),TO 3/2 + 1 2S2B(0),TO 3/2 −3 4S2(2 + 2α2 + S2α)B(0),TO 5/2 , (32) φ12 = −α 4 B(2),TO 3/2 + 1 2S2B(1),TO 3/2 −3 4S2(2 + 2α2 + S2α)B(1),TO 5/2 . 6.2 On the softening prescription of Teyssandier & Ogilvie (2016) (33) On the contrary, the results for the softened gravity presented in Section 3.2 do elicit concern about the number of rings N that is needed to accurately capture the eccentricity dynamics of continuous razor-thin discs. Indeed, Figs 2 and 3 reveal that the expected secular dynamics can be recovered using various softened gravity prescriptions only when one properly accounts for the gravitational effects of all disc annuli, including those very close to the orbit of particle under consideration. Indeed, we demonstrated that to reproduce both the magnitude and the sign of e.g. the free precession rate, the distance a separating a given test-particle orbit from nearest neighbouring inner and outer disc rings should be quite small, a/ap ≲0.1ς2. Only then does the delicate cancellation of large (in magnitude) contributions produced by different parts of the disc recovers the expected (unsoftened) result. Thus, the separation between the modelled disc rings has to be substantially lower than the softening length itself (ςap), meaning that N has to be very large, N ≳10ς−2. This could easily make numerical studies of the secular eccentricity dynamics in discs very challenging. (32) Approach of Teyssandier & Ogilvie (2016) accounts for only the ﬁrst terms in equations (32) and (33), with coefﬁcients which are O(S0), see Table 1. However, as we show below, the correct behaviour of φij as S →0 is guaranteed only when all the terms present in the above expressions are taken into account. To demonstrate this, in Fig. 8 we repeat the same convergence study as in Section 3.1 but with the modiﬁed φij given by equa- tions (32) and (33). One can see that the correct implementation of the softening ϵ2(α) = S2α proposed by TO16 leads to the recovery of the expected test-particle dynamics in inﬁnite PL discs; this is very different from the divergent behaviour obvious in Figs 1(C) and (D). Similar to Hahn (2003) and Touma (2002), both ψ1 and ψ2 smoothly converge to their expected unsoftened values in the limit of S →0 for various PL disc models (i.e. p and q). Further tests using other disc models, looking at the edge effects, etc. reinforce this conclusion. We further conﬁrmed this expectation by studying the conver- gence of disc-driven free precession rate in numerically discretized softened discs to the precession rate Ad computed exactly for continuous softened discs (equations 5 and 18). 6.2 On the softening prescription of Teyssandier & Ogilvie (2016) To this end, we represented a given disc model as a collection of N logarithmically spaced rings, and measured the agreement between the radial proﬁles of theoretical and numerical results for Ad (or ψ1 for PL discs) by using the following global metric:15 This discussion strongly suggests that for any adopted form of softening, the expansion of the secular disturbing function must be performed following a certain rigorous procedure 13 as done, for instance, in Appendix A. In other words, a direct replacement of the classical Laplace coefﬁcients b(m) 3/2 in equation (1) with their softened analogues is, evidently, not sufﬁcient for obtaining a well-behaved softened version of Laplace–Lagrange theory for co-planar discs. M(f ) = aout ain [ftheor(a) −fnum(a)]2da aout ain f 2 theor(a)da . (34) (34) Here, fnum(ai) is the value of the metric basis (e.g. precession rate Ad) evaluated at the position ai of ith ring by summing up the contributions of all other rings in the disc, while ftheor(ai) is the analogous quantity computed in the limit of a continuous disc, i.e. as N →∞(it is given by the non-discretized version of equation (5) if f = Ad, or equation (18) if f = ψ1). Repeating this calculation for various combinations of (N, ς), we can determine the smallest number of rings N(ς) that ensures the desired convergence to within, e.g. ∼10 per cent (i.e. M(f ) ∼0.1), for a given value of softening ς. Here, fnum(ai) is the value of the metric basis (e.g. precession rate Ad) evaluated at the position ai of ith ring by summing up the contributions of all other rings in the disc, while ftheor(ai) is the analogous quantity computed in the limit of a continuous disc, i.e. as N →∞(it is given by the non-discretized version of equation (5) if f = Ad, or equation (18) if f = ψ1). Repeating this calculation for various combinations of (N, ς), we can determine the smallest number of rings N(ς) that ensures the desired convergence to within, e.g. ∼10 per cent (i.e. M(f ) ∼0.1), for a given value of softening ς. 14Note that, technically, in the unsoftened case this mathematical procedure is not equivalent to introducing an actual physical gap in the disc, as the latter would result in additional boundary terms. 15For PL discs, we neglect rings within 10 per cent of disc edges when computing M(ψ1). 13An analogous method is to modify the literal expansion of disturbing function (see Murray & Dermott 1999, Ch. 6) to account for softened interactions (e.g. Tr98, Lee, Dempsey & Lithwick 2019, H03). This could be done by replacing b(m) 1/2 with B(m) 1/2 in equation 7.1 of Murray & Dermott (1999) before applying the derivatives with respect to α. We note that this procedure could apply for all F(r1, r2) with continuous ﬁrst derivatives satisfying D1 + D2 = −1; see Appendix A. 6.4 Further generalizations and extensions All calculations in this work are based on the expansion of the secular disturbing function Rd due to a coplanar disc – softened and unsoftened – to second order in eccentricities. This approximation may yield inaccurate results when the disc or particle eccentricities are high, e.g. in the vicinity of secular resonances where Ad(ap) = 0 (Davydenkova & Raﬁkov 2018), see Figs 5 and 6. Such situations may necessitate a higher order extension of the disc potential. Figure 9. Scaling of number of softened annuli (rings) N with softening parameter ς to ensure convergence of disc-driven free precession Ad (or ψ1) in discretized discs to the expected results in continuous softened discs (equations 5 and 18). Calculations assume axisymmetric disc models extending from ain = 0.1 au to aout = 5 au: two PL discs (speciﬁed by p), a Quartic disc (same as Fig. 5), and a Gaussian ring (same as Fig. 6). We have used the softening methods of Hahn (2003), Tremaine (1998), and (corrected) Teyssandier & Ogilvie (2016), as speciﬁed in the panel. Convergence is measured using the metric M(f ) deﬁned by equation (34). One can see that, when ς ≲0.1, N ∼Cς−χ, with C ∼10 and 1.5 ≲χ ≲ 2. Similar results can be obtained for eccentric discs, and other softening prescriptions. See the text (Section 6.3) for details. Such an exercise was pursued recently by Seﬁlian & Touma (2019), who presented a calculation of Rd to fourth order in eccen- tricities based on the unsoftened method of Heppenheimer (1980). The general framework for calculating Rd with arbitrary softening prescriptions presented in Appendix A can also be extended to higher order in eccentricities in similar way,18 see e.g. Touma & Sridhar (2012). We expect that conclusions similar to those drawn from our analysis in Sections 3–5 will also apply to the higher order expansions. metric disc models as indicated in the legend.16 Fig. 9 shows that as ς →0, the number of rings scales as N ∼Cς−χ with17 C ∼10 and χ ≈(1.8−1.9). The only notable exception is the Gaussian ring, for which convergence is faster (i.e. N∝ς−1.5), probably because of mass concentration in a narrow range of radii. Additionally, although we only analysed coplanar conﬁgurations in this work, the general framework presented in Appendix A may be extended to account for non-coplanar conﬁgurations and study the inclination dynamics. 6.3 Implications for numerical applications In numerical studies of secular dynamics, self-gravitating discs are often treated as a collection of N eccentric annuli (rings), with prescribed spacing (justiﬁed by the constancy of the semimajor axis), interacting gravitationally with each other (e.g. Touma et al. 2009; Batygin 2012). This representation approximates a continu- ous particulate or ﬂuid disc in the limit of N →∞. Fig. 9 depicts a sample of the results obtained using the soft- ening methods of Hahn (2003), Tremaine (1998), and (rectiﬁed) Teyssandier & Ogilvie (2016); see section 6.2, for various axisym- Computational cost associated with the evaluation of mutual ring–ring interactions in this setup, going as O(N2), imposes limitations on the number of rings that can be used in practice. 13An analogous method is to modify the literal expansion of disturbing function (see Murray & Dermott 1999, Ch. 6) to account for softened interactions (e.g. Tr98, Lee, Dempsey & Lithwick 2019, H03). This could be done by replacing b(m) 1/2 with B(m) 1/2 in equation 7.1 of Murray & Dermott (1999) before applying the derivatives with respect to α. We note that this procedure could apply for all F(r1, r2) with continuous ﬁrst derivatives satisfying D1 + D2 = −1; see Appendix A. MNRAS 489, 4176–4195 (2019) Softened potentials of discs 4189 0.03 0.05 0.07 0.1 0.2 0.3 0.4 102 103 104 * Figure 9. Scaling of number of softened annuli (rings) N with softening parameter ς to ensure convergence of disc-driven free precession Ad (or ψ1) in discretized discs to the expected results in continuous softened discs (equations 5 and 18). Calculations assume axisymmetric disc models extending from ain = 0.1 au to aout = 5 au: two PL discs (speciﬁed by p), a Quartic disc (same as Fig. 5), and a Gaussian ring (same as Fig. 6). We have used the softening methods of Hahn (2003), Tremaine (1998), and (corrected) Teyssandier & Ogilvie (2016), as speciﬁed in the panel. Convergence is measured using the metric M(f ) deﬁned by equation (34). One can see that, when ς ≲0.1, N ∼Cς−χ, with C ∼10 and 1.5 ≲χ ≲ 2. Similar results can be obtained for eccentric discs, and other softening prescriptions. See the text (Section 6.3) for details. 0.03 0.05 0.07 0.1 0.2 0.3 0.4 102 103 104 * Fig. 6.4 Further generalizations and extensions We note that the proportionality constant C in the N(ς) relation is not perfectly deﬁned in the sense that it depends on the (i) desired accuracy (roughly inversely proportional to M(f )), (ii) adopted metric of accuracy (mild dependence), and (iii) softening prescription used – Fig. 9 shows that discretized calculations using softening model of Hahn (2003) require substantially lower (by ∼2) number of annuli than those using the models of Teyssandier & Ogilvie (2016) and Tremaine (1998). Nevertheless, these results further reinforce the requirement of large number of rings, with N ∼ ς−2, to capture the expected secular eccentricity dynamics in nearly Keplerian discs. 6.3 Implications for numerical applications 9, indicate that accurate representation of eccentricity dynamics in a cold, razor-thin disc requires a very large number of rings N whenever small values of the softening parameter are used. As we have shown in Section 5, very small values of softening ς ≲10−3 are, in fact, necessary to accurately capture eccentricity dynamics near the sharp edges of thin discs. This suggests that N has to be prohibitively large when softened gravity is applied e.g. to study the dynamics of planetary ring (Goldreich & Tremaine 1979; Chiang & Goldreich 2000; Pan & Wu 2016), which are known to have sharp edges. 16We exclude the softening method of Touma (2002) from this analysis as it introduces additional complexity due to the nature of softening parameter; ϵ2 = b2/max(a2 1, a2 2), see Section 2.1.2. 17For example, the curve computed using the (corrected) model of Teyssandier & Ogilvie (2016) has C = 10.9 and χ = 1.91, while the one for Quartic disc has C = 7.2 and χ = 1.75. 17For example, the curve computed using the (corrected) model of Teyssandier & Ogilvie (2016) has C = 10.9 and χ = 1.91, while the one for Quartic disc has C = 7.2 and χ = 1.75. 7 SUMMARY I., Goldreich P., 2000, ApJ, 540, 1084 Davydenkova I., Raﬁkov R. R., 2018, ApJ, 864, 74 Fontana A., Marzari F., 2016, A&A, 589, A133 Goldreich P., Tremaine S., 1979, AJ, 84, 1638 Goldreich P., Tremaine S., 1979, AJ, 84, 1638 (v) We developed a general analytical framework for computing the secular disturbing function between two co-planar rings with arbitrary interaction potential of rather general form (equation 10). This framework accurately reproduces the orbit-averaged razor-thin disc potential as ς →0 for a wide class of softened gravity models. Goldreich P., Tremaine S., 1980, ApJ, 241, 425 Graps A. L., Showalter M. R., Lissauer J. J., Kary D. M., 1995, AJ, 109, 2262 Gulati M., Saini T. D., Sridhar S., 2012, MNRAS, 424, 348 Hahn J. M., 2003, ApJ, 595, 531 (vi) Using this general framework, we demonstrated that an accurate implementation of the softened potentials suggested in both Tremaine (1998) and Teyssandier & Ogilvie (2016) leads to the recovery of the expected dynamical behaviour in the limit of small softening. Heppenheimer T. A., 1980, Icarus, 41, 76 Jacobs V., Sellwood J. A., 2001, ApJ, 555, L25 Kazandjian M. V., Touma J. R., 2013, MNRAS, 430, 2732 Kocsis B., Tremaine S., 2011, MNRAS, 412, 187 Latter H. N., Ogilvie G. I., Rein H., 2017, in Tiscareno M. S., Murray C. D., eds, Planetary Ring Systems. Properties, Structure, and Evolution. Cambridge Univ. Press, Cambridge, p. 549 (vii) Our results suggest that the numerical treatments of the secular eccentricity dynamics in softened, nearly Keplerian discs must obey important constraints. Namely, a ﬁne numerical sampling (i.e. large number N of discrete annuli representing the disc, with N ∼Cς−χ, C ∼O(10), 1.5 ≲χ ≲2) is required to ensure that the correct secular behaviour is properly captured by such calculations when ς is small. This ﬁnding has important ramiﬁcations for numerical treatments of planetary rings with sharp edges. Lee W.-K., Dempsey A. M., Lithwick Y., 2019, ApJ, 872, 184 Meschiari S., 2014, ApJ, 790, 41 Meschiari S., 2014, ApJ, 790, 41 Murray C. D., Dermott S. F., 1999, Solar System Dynamics. Cambridge Univ. Press, Cambridge Pan M., Wu Y., 2016, ApJ, 821, 18 Petrovich C., Wu Y., Ali-Dib M., 2019, AJ, 157, 5 In the future our results for the disc-driven eccentricity dynamics may be extended to higher order in eccentricity, as well as general- ized for treating inclination dynamics. Plummer H. C. 7 SUMMARY K., 1918, An Introductory Treatise on Dynamical Astron- omy. Cambridge Univ. Press, Cambridge Press W. H., Teukolsky S. A., Vetterling W. T., Flannery B. P., 2002, Numerical recipes in C++ : the art of scientiﬁc computing. Cambridge Univ. Press, Cambridge ACKNOWLEDGEMENTS Seﬁlian A. A., Touma J. R., 2019, AJ, 157, 59 Silsbee K., Raﬁkov R. R., 2015, ApJ, 798, 71 We express our gratitude to Scott Tremaine and Jihad Touma for a number of stimulating discussions, which have led to substantial improvements of the manuscript. We are also grateful to Gordon Ogilvie, Jean Teyssandier, Yoram Lithwick, and Cristobal Petrovich for useful discussions, and an anonymous referee for constructive comments. AAS acknowledges a scholarship by the Gates Cam- bridge Trust (OPP1144), while RRR was supported by NASA via grant 15-XRP15-2-0139. Open Access for this article was funded by the Bill & Melinda Gates Foundation. Statler T. S., 2001, AJ, 122, 2257 Teyssandier J., Ogilvie G. I., 2016, MNRAS, 458, 3221 Teyssandier J., Terquem C., Papaloizou J. C. B., 2013, MNRAS, 428, 658 Tiscareno M. S., 2013, in Oswalt T. D., French L. M., Kalas P., eds, Planets, Stars and Stellar Systems. Springer-Verlag, Berlin, p. 309 Touma J. R., 2002, MNRAS, 333, 583 Touma J. R., Sridhar S., 2012, MNRAS, 423, 2083 Touma J. R., Tremaine S., Kazandjian M. V., 2009, MNRAS, 394, 1085 7 SUMMARY In this work, we investigated the applicability of softened gravity for computing the orbit-averaged potential of razor-thin eccentric discs. We compared disc-driven secular dynamics of coplanar test particles computed using softening prescriptions available in the literature with the calculations based on the unsoftened method of Heppenheimer (1980). Our ﬁndings are summarized below. (i) We conﬁrmed that the softening methods of both Touma (2002) and Hahn (2003) correctly reproduce eccentricity dynamics of razor-thin discs in the limit of vanishing softening parameter ς for all disc models. Qualitatively similar results were stated in Hahn (2003), who showed that the secular effects of a continuous disc can be recovered only when the disc rings are sufﬁciently numerous that their radial separation is below the softening length. Although, interestingly, Hahn (2003) and Lee et al. (2019) claimed good convergence of the precession rate to the expected value already for N ∼O(ς−1) [however, note that Lee et al. (2019) also included effects of gas pressure in their calculations, in addition to disc gravity]. In our case, the condition on the separation between disc rings motivated by Figs 2 and 3 (i.e. a/ap ≲0.1ς2), along with the results presented in (ii) The softening prescription proposed in Tremaine (1998) yields convergent results as ς →0. However, quantitative dif- ferences of up to ∼(20 −30) per cent from the unsoftened cal- culations are observed. We demonstrate that these differences arise because of the insufﬁcient smoothness of the interparticle interaction assumed in Tremaine (1998). (iii) The softening formalism suggested in Teyssandier & Ogilvie (2016) does not result in convergent results in the limit of zero softening. 16We exclude the softening method of Touma (2002) from this analysis as it introduces additional complexity due to the nature of softening parameter; ϵ2 = b2/max(a2 1, a2 2), see Section 2.1.2. 18Another way to calculate the softened disturbing function for arbitrarily high eccentricities is to numerically compute the ring–ring interaction potential, as was done by Touma et al. (2009). 17For example, the curve computed using the (corrected) model of Teyssandier & Ogilvie (2016) has C = 10.9 and χ = 1.91, while the one for Quartic disc has C = 7.2 and χ = 1.75. MNRAS 489, 4176–4195 (2019) 4190 A. A. Seﬁlian and R. R. Raﬁkov (iv) Very small values of the (dimensionless) softening parameter are required for correctly reproducing secular eccentricity dynamics near sharp edges of discs/rings. Chiang E. Batygin K., 2012, Nature, 491, 418 Batygin K., 2012, Nature, 491, 418 Ward W. R., 1981, Icarus, 47, 234 ty o Ward W. R., 1981, Icarus, 47, 234 Binney J., Tremaine S., 2008, Galactic Dynamics: Second Edition. Princeton Univ. Press, Princeton, NJ Ward W. R., 1989, ApJ, 336, 526 APPENDIX A: CALCULATION OF THE SECULAR RING–RING INTERACTION REFERENCES Tremaine S., 1998, AJ, 116, 2015 Tremaine S., 2001, AJ, 121, 1776 Tremaine S., 2001, AJ, 121, 1776 19Note that we do not deal with the indirect part of the potential – which is left unsoftened – as it contains only periodic terms and does not affect the secular dynamics (Murray & Dermott 1999). A1 Expansion of the interaction function around small eccentricities Following the classical techniques of celestial mechanics (see, Plummer 1918, Ch. XVI), we start by expanding around circular orbits. Using Taylor expansion we write 3 + (f2 −M2)D4 0 ≡ T0 (A2) = exp log r1 a1 D1 + log r2 a2 D2 + (f1 −M1)D3 + (f2 −M2)D4 0 ≡ T0 = exp log r1 a1 D1 + log r2 a2 D2 + (f1 −M1)D3 + (f2 −M2)D4 0 ≡ T0 (A2) (A2) with 0 = a2 1 + a2 2 −2a1a2 cos θ + F(a1, a2) −1/2 , (A3) 0 = a2 1 + a2 2 −2a1a2 cos θ + F(a1, a2) −1/2 , (A3) where θ = M1 −M2 + ϖ 1 −ϖ 2, Mi represents the mean anomaly of the ith ring characterized with semimajor axis ai, and the linear operators Dk are given by (Plummer 1918) where θ = M1 −M2 + ϖ 1 −ϖ 2, Mi represents the mean anomaly of the ith ring characterized with semimajor axis ai, and the linear operators Dk are given by (Plummer 1918) D1 = a1 ∂ ∂a1 ≡a1∂1, D2 = a2 ∂ ∂a2 ≡a2∂2, and D3 = −D4 = ∂ ∂θ . (A4) (A4) Note that this expansion, as well as subsequent steps, is completely symmetric with respect to interchanging the particle indices. A1 Expansion of the interaction function around small eccentricities (A9) (A9) We have used the fact that cos (M1 −M2) = cos θcos (ϖ 1 −ϖ 2) and sin (M1 −M2) = sin θcos (ϖ 1 −ϖ 2) in the secular regime (Plummer 1918). We have used the fact that cos (M1 −M2) = cos θcos (ϖ 1 −ϖ 2) and sin (M1 −M2) = sin θcos (ϖ 1 −ϖ 2) in the secular regime (Plummer 1918). APPENDIX A: CALCULATION OF THE SECULAR RING–RING INTERACTION MNRAS 489, 4176–4195 (2019) MNRAS 489, 4176–4195 (2019) Softened potentials of discs 4191 Softened potentials of discs 4191 A1 Expansion of the interaction function around small eccentricities APPENDIX A: CALCULATION OF THE SECULAR RING–RING INTERACTION Here, we present a calculation of the secular disturbing function due to two co-planar rings interacting with each other via softened gravity in the form (10). We do not assume any speciﬁc form for the softening function F apart from requiring it to be a function of the instantaneous positions of interacting particles with respect to the centre of the system. We ﬁrst write the ring–ring interaction function as19 2 = r2 1 + r2 2 −2r1r2 cos(f1 −f2 + ϖ1 −ϖ2) + F(r1, r2) −1/2 , (A1) −r2)2 + F(r1, r2)]−1/2 = r2 1 + r2 2 −2r1r2 cos(f1 −f2 + ϖ1 −ϖ2) + F(r1, r2) −1/2 , = [(r1 −r2)2 + F(r1, r2)]−1/2 = r2 1 + r2 2 −2r1r2 cos(f1 −f2 + ϖ1 −ϖ2) + F(r1, r2) −1/2 , (A1) where F(r1, r2) is an arbitrary softening function introduced to cushion the singularity which arises otherwise at null interparticle separations. In the above expression, fi is the true anomaly of the ith ring, ϖ i is its longitude of periapse and ri is its instantaneous position, i = 1, 2. Our goal is to obtain the orbit-averaged expansion of to second order in eccentricities ei valid for arbitrary F(r1, r2). where F(r1, r2) is an arbitrary softening function introduced to cushion the singularity which arises otherwise at null interparticle separations. In the above expression, fi is the true anomaly of the ith ring, ϖ i is its longitude of periapse and ri is its instantaneous position, i = 1, 2. Our goal is to obtain the orbit-averaged expansion of to second order in eccentricities ei valid for arbitrary F(r1, r2). p f y g g p p p btain the orbit-averaged expansion of to second order in eccentricities ei valid for arbitrary F(r1, r2). 19Note that we do not deal with the indirect part of the potential – which is left unsoftened – as it contains only periodic terms and does not affect the secular dynamics (Murray & Dermott 1999). 19Note that we do not deal with the indirect part of the potential – which is left unsoftened – as it contains only periodic terms and does not affect the secular dynamics (Murray & Dermott 1999). A2 Computation of the action of relevant operators Equipped with expression (A7) for , we proceed to compute the action of operator T on 0 prior to orbit-averaging the resultant expression. With this in mind, we compute the action of several operators appearing in the deﬁnitions of A, B, and C on 0 and list them below: Equipped with expression (A7) for , we proceed to compute the action of operator T on 0 prior to orbit-averaging the resultant expression. A1 Expansion of the interaction function around small eccentricities Next, in order to calculate the action of the operator T deﬁned by equation (A2) on the disturbing function of circular softened rings 0, we make use of the elliptical expansions of r/a and f −M, 1 2e2[1 −cos(2M)] · D + 1 4e2[1 + cos(2M)] · D(D −1) + O(e3), (A5) (a−1r)D = 1 −e cos M · D + 1 2e2[1 −cos(2M)] · D + 1 4e2[1 + cos(2M)] · D(D −1) + O(e3), (a−1r)D = 1 −e cos M · D + 1 2e2[1 −cos(2M)] · D + 1 4e2[1 + cos(2M)] · D(D −1) + O(e3), (A5) (A5) = 1 + 2e sin M · D + 5 4e2 sin(2M) · D + e2[1 −cos(2M)] · D2 + O(e3) exp{(f −M)D} = 1 + 2e sin M · D + 5 4e2 sin(2M) · D + e2[1 −cos(2M)] · D2 + O(e3) exp{(f −M)D} = 1 + 2e sin M · D + 5 4e2 sin(2M) · D + e2[1 −cos(2M)] · D2 + O(e3) (A6) (A6) to multiply individual terms appearing in T, keep the ones up to second order in eccentricities, and drop all terms that do not contain the difference of mean anomalies, k(M1 −M2), as they are evidently periodic and vanish upon orbit-averaging. Performing this procedure and dropping an irrelevant constant term, one can demonstrate that reduces to to multiply individual terms appearing in T, keep the ones up to second order in eccentricities, and drop all terms that do not contain the difference of mean anomalies, k(M1 −M2), as they are evidently periodic and vanish upon orbit-averaging. Performing this procedure and dropping an irrelevant constant term, one can demonstrate that reduces to 0 ≡A0 e2 1 + B0 e2 2 + C0 e1e2 cos(ϖ1 −ϖ2), (A7) operators A, B, and C acting on 0 are deﬁned as + 1 4D1(D1 + 1), B ≡D2 4 + 1 4D2(D2 + 1), (A8) = T0 ≡A0 e2 1 + B0 e2 2 + C0 e1e2 cos(ϖ1 −ϖ2), (A7) where the operators A, B, and C acting on 0 are deﬁned as A ≡D2 3 + 1 4D1(D1 + 1), B ≡D2 4 + 1 4D2(D2 + 1), C ≡cos θ 2D3D4 + 1 2D1D2 −sin θ(D2D3 −D1D4). C ≡cos θ 2D3D4 + 1 2D1D2 −sin θ(D2D3 −D1D4). (A9) −sin θ(D2D3 −D1D4). A3 Orbit-averaging the interaction function Expressions (A10)–(A17) allow the computation of = T0, which needs to be time-averaged in order to recover the secular disturbing function. We do not show the cumbersome collated expression for T0 and proceed to the ﬁnal step of orbit-averaging, which will conclude our derivation. In short, our goal is to compute ⟨⟩= ⟨T0⟩= 1 2π 2π 0 T0 dθ, (A18) which essentially reduces to computing the individual terms ⟨A0⟩, ⟨B0⟩and ⟨C0⟩. At the outset, it is important to note that each of the terms appearing in T0 (through A0, B0, and C0, or the operators they entail) are proportional to cos(mθ)2s 0 . By making use of α = a</a>, where a < = min(a1, a2) and a> = max(a1, a2), this combination can be reduced to cos(mθ)2s 0 = a−2s > cos(mθ) 1 + α2 −2α cos θ + a−2 > F(a1, a2) −s . (A19) mθ) 1 + α2 −2α cos θ + a−2 > F(a1, a2) −s . ( cos(mθ)2s 0 = a−2s > cos(mθ) 1 + α2 −2α cos θ + a−2 > F(a1, a2) −s . (A19) For that reason, calculation of the orbit-averaged (by integrating over dθ) yields integrals of the form For that reason, calculation of the orbit-averaged (by integrating over dθ) yields integrals of the form For that reason, calculation of the orbit-averaged (by integrating over dθ) yields integrals of the form B(m) s (α) ≡2 π π 0 cos(mθ) 1 + α2 −2α cos θ + ϵ2(α) −sdθ, B(m) s (α) ≡2 π π 0 cos(mθ) 1 + α2 −2α cos θ + ϵ2(α) −sdθ, (A20) (A20) which is the generalization of the classical Laplace coefﬁcients b(m) s (recovered when F(a1, a2) = 0, see equation 2) with the dimensionless softening parameter which is the generalization of the classical Laplace coefﬁcients b(m) s (recovered when F(a1, a2) = 0, see equation 2) with the dimensionless softening parameter ϵ2(α) ≡a−2 > F(a1, a2), ϵ2(α) ≡a−2 > F(a1, a2), (A21) see equation (7). Employing this notation, we present the simpliﬁed expressions of ⟨A0⟩, ⟨B0⟩, and ⟨C0⟩obtained as a result of orbit-averaging: see equation (7). A2 Computation of the action of relevant operators With this in mind, we compute the action of several operators appearing in the deﬁnitions of A, B, and C on 0 and list them below: D2 3 0 = D2 4 0 = 3a2 1a2 2 sin2 θ 5 0 −a1a2 cos θ 3 0, (A10) sin2 θ 5 0 −a1a2 cos θ 3 0, (A10) D2 3 0 = D2 4 0 = 3a2 1a2 2 sin2 θ 5 0 −a1a2 cos θ 3 0, (A10) D1D2 0 = a1a2 cos θ −1 2∂1∂2F 3 0 + 3 a2 2 −a1a2 cos θ + a2 2 ∂2F a2 1 −a1a2 cos θ + a1 2 ∂1F 5 0, (A11) D2D3 0 = −a1a2 sin θ 3 0 + 3a1a2 sin θ a2 2 −a1a2 cos θ + a2 2 ∂2F 5 0, (A12) D1D4 0 = a1a2 sin θ 3 0 −3a1a2 sin θ a2 1 −a1a2 cos θ + a1 2 ∂1F 5 0, (A13) D1 3 0 = −3 a2 1 −a1a2 cos θ + a1 2 ∂1F 5 0, (A14) D2 3 0 = −3 a2 2 −a1a2 cos θ + a2 2 ∂2F 5 0, (A15) D1D2 0 = a1a2 cos θ −1 2∂1∂2F 3 0 + 3 a2 2 −a1a2 cos θ + a2 2 ∂2F a2 1 −a1a2 cos θ + a1 2 ∂1F 5 0, (A11) D2D3 0 = −a1a2 sin θ 3 0 + 3a1a2 sin θ a2 2 −a1a2 cos θ + a2 2 ∂2F 5 0, (A12) D1D4 0 = a1a2 sin θ 3 0 −3a1a2 sin θ a2 1 −a1a2 cos θ + a1 2 ∂1F 5 0, (A13) a2 2 ∂2F 5 0, (A12) D1 3 0 = −3 a2 1 −a1a2 cos θ + a1 2 ∂1F 5 0, D2 3 0 = −3 a2 2 −a1a2 cos θ + a2 2 ∂2F 5 0, (A15) MNRAS 489, 4176–4195 (2019) MNRAS 489, 4176–4195 (2019) MNRAS 489, 4176–4195 (2019) 4192 A. A. Seﬁlian and R. R. Raﬁkov where for conciseness we have written F instead of F(a1, a2). A2 Computation of the action of relevant operators Here, it is worthwhile to mention that, as far as the expansion technique is concerned, the terms ∂iF (with i = 1, 2) appearing in the above expressions are the only difference brought upon by softening the Newtonian point–mass interaction (equation A1). Another set of operators useful in computing T0 is the following: where for conciseness we have written F instead of F(a1, a2). Here, it is worthwhile to mention that, as far as the expansion technique is concerned, the terms ∂iF (with i = 1, 2) appearing in the above expressions are the only difference brought upon by softening the Newtonian point–mass interaction (equation A1). Another set of operators useful in computing T0 is the following: D1(D1 + 1) 0 = −D1D2 0 + 1 2D1 (2F −a1∂1F −a2∂2F) 3 0 , D2(D2 + 1) 0 = −D1D2 0 + 1 2D2 (2F −a1∂1F −a2∂2F) 3 0 , (A17) which can be obtained by making use of the identity (D1 + D2 + 1)0 = 1 2(2F −a1∂1F −a2∂2F)3 0. Here, we note that for all softening functions F for which 2F −a1∂1F −a2∂2F = 0, one ﬁnds D1 + D2 = −1. Consequently, in such cases, the operators D1(D1 + 1) and D2(D2 + 1) become identical rendering A0 = B0 (since D2 3 = D2 4, see equations A8 and A10). As a result, the resultant orbit-averaged disturbing function (A7) is symmetric in e1 and e2, similar to the case of classical Laplace–Lagrange theory. This is not true in general, for instance, when F(r1, r2) = const ̸= 0. A3 Orbit-averaging the interaction function The functional forms of the coefﬁcients Ti(α) given by equations (A25)–(A27) appearing in the orbit-averaged disturbing function due to two coplanar (arbitrarily) softened rings (equations A22–A24) such that α ≡a</a> ≤1. The ﬁrst column lists the softening prescriptions analysed in this work (see Section 2.1), while the second column shows the speciﬁc forms of the softening function F(r1, r2) in equation (A1). The corresponding expressions for the dimensionless softening parameters ϵ2(α) = a−2 > F(a1, a2) (equation A21) entering in the deﬁnition of softened Laplace coefﬁcients (equation A20) are also shown. Here, (x) represents the Heaviside step function and δ(x) = d(x)/dx stands for Dirac delta-function. Method F(r1, r2) ϵ2(α) T1(α) T2(α) T3(α) T4(α) T5(α) T6(α) T7(α) T8(α) H03 H 2(r2 1 + r2 2) H2(1 + α2) 0 2H2(1 + α2) 0 0 4αH2(2 + H2) 0 2H 2 a1 a2 2H 2 a2 a1 T02 b2 c β2 = (bc/a>)2 2β2 0 0 0 0 0 0 0 Tr98 β2 c max(r2 1, r2 2) β2 c 0 2β2 c −2β2 c δ(α −1) 0 4αβ2 c 0 2β2c α (a1 −a2) 2β2c α (a2 −a1) TO16 S2r1r2 S2α 0 2αS2 S2 0 S2(S2α + 2α2 + 2) 0 S2 S2 T6 = a2 a2 > ∂2[2F −a1∂1F −a2∂2F], T7 = a−1 2 ∂1F, T8 = a−1 1 ∂2F, (A27) where, as before, F ≡F(a1, a2), α = a</a>, and ∂i ≡∂/∂ai. Note that the expressions for φ11 and φ22 swap deﬁnitions upon replacing a1 by a2, whilst keeping α < 1 by construction. This can be understood by ﬁrst noting that functions Ti with i = 1, 2, 3, and 5 are invariant under a1 ⇋a2 while, at the same time, T4 and T7 (appearing in the second line of equation A22) translate to T6 and T8 (appearing in the second line of equation A23); and vice versa. where, as before, F ≡F(a1, a2), α = a</a>, and ∂i ≡∂/∂ai. Note that the expressions for φ11 and φ22 swap deﬁnitions upon replacing a1 by a2, whilst keeping α < 1 by construction. This can be understood by ﬁrst noting that functions Ti with i = 1, 2, 3, and 5 are invariant under a1 ⇋a2 while, at the same time, T4 and T7 (appearing in the second line of equation A22) translate to T6 and T8 (appearing in the second line of equation A23); and vice versa. A4 Alternative calculation: secular disc–particle interaction F′ = ∂F/∂ap), a> = max (ap, a), α = min (ap, a)/max (ap, a) and integration is done over the semimajor axis a of the disc elements. Calculation of the non-axisymmetric part of Rd resulting from non-zero disc eccentricity (i.e. Bd) is somewhat more tedious but can nevertheless be done similar to Davydenkova & Raﬁkov (2018). A3 Orbit-averaging the interaction function These identities, when combined, yield the desired expression of ⟨⟩= ⟨T0⟩; see equations (A7)–(A9). Subsequently, the softened ring–ring disturbing function in form (11) is recovered, with the coefﬁcients φij deﬁned by equations (A22)–(A24). This completes our calculation of the secular ring–ring interaction between two softened coplanar rings, up to second order in eccentricity and valid for arbitrary softening functions F(r1, r2). Note that in the absence of softening (i.e. F(r1, r2) = 0) Ti = 0 for all i and the classical expressions for φLL 11 , φLL 22 and φLL 12 – equations (8) and (9) – are recovered. Finally, we mention that the expansion technique exploited here can be used to recover the orbit-averaged disturbing function valid to arbitrary order in eccentricity, as well as inclinations. A3 Orbit-averaging the interaction function Employing this notation, we present the simpliﬁed expressions of ⟨A0⟩, ⟨B0⟩, and ⟨C0⟩obtained as a result of orbit-averaging: a> ⟨A0⟩(α) ≡φ11(α) = α 2 −5 4B(1) 3/2 + 3 8αB(0) 5/2 + 3 4(1 + α2)B(1) 5/2 −15 8 αB(2) 5/2 + 3 8T2B(1) 5/2 −3 16T5B(0) 5/2 + 1 8 T3 + α−1T4 B(0) 3/2 −3 8T1 a1 a2 B(0) 5/2 −B(1) 5/2 + 1 2T7B(0) 5/2 , (A22) α) = α 2 −5 4B(1) 3/2 + 3 8αB(0) 5/2 + 3 4(1 + α2)B(1) 5/2 −15 8 αB(2) 5/2 + 3 8T2B(1) 5/2 −3 16T5B(0) 5/2 + 1 8 T3 + α−1T6 B(0) 3/2 −3 8T1 a2 a1 B(0) 5/2 −B(1) 5/2 + 1 2T8B(0) 5/2 , a> ⟨B0⟩(α) ≡φ22(α) = α 2 −5 4B(1) 3/2 + 3 8αB(0) 5/2 + 3 4(1 + α2)B(1) 5/2 −15 8 αB(2) 5/2 + 3 8T2B(1) 5/2 −3 16T5B(0) 5/2 + 1 8 T3 + α−1T6 B(0) 3/2 −3 8T1 a2 a1 B(0) 5/2 −B(1) 5/2 + 1 2T8B(0) 5/2 , (A23) (A23) a> ⟨C0⟩(α) ≡φ12(α) = α 2 9 4B(0) 3/2 + 1 4B(2) 3/2 + 3 8αB(3) 5/2 + 21 8 αB(1) 5/2 + 3 4(1 + α2)B(2) 5/2 −9 4(1 + α2)B(0) 5/2 −1 4T3B(1) 3/2 −9 8T2B(0) 5/2 + 3 8T5B(1) 5/2 + 3 8T2B(2) 5/2 . (A24) In equations (A22)–(A24), we have deﬁned the dimensionless functions Ti(α) such that In equations (A22)–(A24), we have deﬁned the dimensionless functions Ti(α) such that T1 = a−2 > 2F −a1∂1F −a2∂2F , T2 = α ∂1F a2 + ∂2F a1 , T3 = ∂1∂2F, T4 = a1 a2 > ∂1[2F −a1∂1F −a2∂2F], T5 = α 2∂1F a1 + 2∂2F a2 + ∂1F a1 ∂2F a2 , T1 = a−2 > 2F −a1∂1F −a2∂2F , T2 = α ∂1F a2 + ∂2F a1 , T3 = ∂1∂2F, T4 = a1 a2 > ∂1[2F −a1∂1F −a2∂2F], T5 = α 2∂1F a1 + 2∂2F a2 + ∂1F a1 ∂2F a2 , = α 2∂1F a1 + 2∂2F a2 + ∂1F a1 ∂2F a2 , (A26) MNRAS 489, 4176–4195 (2019) MNRAS 489, 4176–4195 (2019) 4193 Softened potentials of discs Table B1. A4 Alternative calculation: secular disc–particle interaction Calculations presented above describe the orbit-averaged coupling between the two individual annuli, which subsequently need to be integrated over the semimajor axes of the disc elements to represent the effect of a continuous disc. In principle, one can also arrive at expressions (4) by assuming a continuous mass distribution in the disc from the start and performing a calculation similar to that in Davydenkova & Raﬁkov (2018). Namely, one would need to compute Rd = ⟨G S (rd)(rd, rp)dS⟩, where is the interaction potential given by equation (10), angle brackets indicate averaging over the orbit of the test particle given by rp and integration is carried out over the full surface of the disc S with rd denoting the location of a disc element. To obtain the expression for Rd accurate to second order in eccentricities one would need to expand (rd, rp) to second order in particle and disc eccentricities by e.g. writing rp = ap(1 −epcos Ep), where Ep is the eccentric anomaly of the particle orbit. This expansion should explicitly account for the dependence of F on rd and rp. Averaging the resulting expressions over Ep, one would arrive at the proper expression for Rd in the form (4). In particular, after a lengthy but straightforward calculation this method gives the following expression for t hy but straightforward calculation this method gives the following expression for the disc-driven precession rate Ad = πG 2npa2 p a (a)da a> 1 4 3apF′ F′ + 4ap −2 2F′ + apF′′ a2 p + a2 + F −12apF apB(0) 5/2(α) a4 > + αB(1) 3/2(α) − F′ −apF′′ apαB(1) 5/2(α) a2 > , (A28) + αB(1) 3/2(α) − F′ −apF′′ apαB(1) 5/2(α) a2 > , (A28) where prime denotes differentiation with respect to ap (e.g. F′ = ∂F/∂ap), a> = max (ap, a), α = min (ap, a)/max (ap, a) and integration is done over the semimajor axis a of the disc elements. Calculation of the non-axisymmetric part of Rd resulting from non-zero disc eccentricity (i.e. Bd) is somewhat more tedious but can nevertheless be done similar to Davydenkova & Raﬁkov (2018). where prime denotes differentiation with respect to ap (e.g. APPENDIX C: GENERALIZED LAPLACE COEFFICIENTS As demonstrated in Appendix A, softening the Newtonian point-mass potential by an arbitrary function F(r1, r2) modiﬁes the deﬁnition of the Laplace coefﬁcients as shown by equations (7) and (A20) by the introduction of a softening parameter ϵ2(α) = a−2 > F(a1, a2) (equation A21), 0 ≤α = a</a> ≤1. Here, we present some useful recursive relationships amongst different generalized Laplace coefﬁcients B(m) s (α), along with their asymptotic behaviour in the limits of α →0, 1 as well as their relationship to complete elliptic integrals. C1 Recursive relations Generalizing the results for the usual (unsoftened) Laplace coefﬁcients b(m) s (e.g. Plummer 1918, p. 159), the following relationships can be easily obtained for the generalized Laplace coefﬁcients deﬁned by equations (7) and (A20): mB(m) s = sαB(m−1) s+1 −sαB(m+1) s+1 , (C1) m(1 + α2 + ϵ2)B(m) s = α(m + 1 −s)B(m+1) s + α(m + s −1)B(m−1) s , (m + s)B(m) s = s(1 + α2 + ϵ2)B(m) s+1 −2sαB(m+1) s+1 . (m + s)B(m) s = s(1 + α2 + ϵ2)B(m) s+1 −2sαB(m+1) s+1 . (C3) The difference with the classical recursive relations for b(m) s amounts to substituting the combination 1 + α2 appearing in the case of ordinary Laplace coefﬁcients with 1 + α2 + ϵ2(α). Another useful expression relating the generalized Laplace coefﬁcients of arguments α and α−1 is Another useful expression relating the generalized Laplace coefﬁcients of arguments α and α−1 is B(m) s (α−1) = α2sB(m) s (α). (C4) Note that the above relationship is valid only as long as the softening parameter satisﬁes α2ϵ2(1/α) = ϵ2(α). For instance, this condition is violated when the softening parameter ϵ has no dependence on α (e.g. that of Tremaine 1998, see Table 1). Note that the above relationship is valid only as long as the softening parameter satisﬁes α2ϵ2(1/α) = ϵ2(α) violated when the softening parameter ϵ has no dependence on α (e.g. that of Tremaine 1998, see Table 1). Note that the above relationship is valid only as long as the softening parameter satisﬁes α2ϵ2(1/α) = ϵ2(α). For instance, this condition is violated when the softening parameter ϵ has no dependence on α (e.g. that of Tremaine 1998, see Table 1). APPENDIX B: SPECIFIC CASES OF F(r1, r2) Indeed, equation (C2) with m = 1 and s = 5/2 and equation (C1) with m = 1 and s = 3/2 read 3(1 + α2 + S2α)B(1),TO 5/2 = −3α 2 B(2),TO 5/2 + 15 2 αB(0),TO 5/2 , (B2) −6B(1),TO 3/2 = 9α B(2),TO 5/2 −B(0),TO 5/2 , (B3) respectively. Inserting the above two identities in equation (B1) one arrives at equation (32). Similarly, the expression of φ12 (equation A24) can be simpliﬁed with the aid of equation (C3) (with m = 0, s = 3/2), equation (C2) (with m = 2, s = 5/2) and equation (C1) (with m = 2, s = 3/2) resulting in equation (33) after some algebra. As discussed in Section 6.2, the terms in equations (32) and (33) explicitly proportional to S2 are absent in the original formulation of Teyssandier & Ogilvie (2016); see Table 1. 2 2 2 respectively. Inserting the above two identities in equation (B1) one arrives at equation (32). Similarly, the expression of φ12 (equation A24) can be simpliﬁed with the aid of equation (C3) (with m = 0, s = 3/2), equation (C2) (with m = 2, s = 5/2) and equation (C1) (with m = 2, s = 3/2) resulting in equation (33) after some algebra. As discussed in Section 6.2, the terms in equations (32) and (33) explicitly proportional to S2 are absent in the original formulation of Teyssandier & Ogilvie (2016); see Table 1. Similarly, for the formalism of Tremaine (1998), propagating their functional form of F(r1, r2) = β2 c max(r2 1, r2 2) through our general framework, we arrive at the expressions for φij(α) differing from those reported in Tremaine (1998) in a very special way: we ﬁnd φij to contain additional terms proportional to T3(α) ∼δ(α −1), where δ(x) is the Dirac delta-function. Such terms are absent in the original formulation of Tremaine (1998); see Tables 1 and B1. Emergence of these terms can be easily demonstrated by ﬁrst noting that in this case φ 11 = φ22 (as T1 = T4 = T6 = 0), employing the recursive relationships for Laplace coefﬁcients (in a similar order as done above for TO16) to simplify the general expressions of φ11(= φ22) and φ12, and ﬁnally arriving at equations (26) and (27). The ramiﬁcations of this ﬁnding is discussed in Section 6.1. APPENDIX B: SPECIFIC CASES OF F(r1, r2) The general framework developed in Appendix A allows us to recover the expressions of φij arrived at by Touma (2002) and Hahn (2003) upon specifying certain functional forms of F(r1, r2). Indeed, Touma (2002) performed the same calculations as presented in Appendix A for the case of Plummer potential – F(r1, r2) = b2 c – to second order in eccentricities, and later to fourth order in eccentricities (Touma & Sridhar 2012). Furthermore, we ﬁnd that the results obtained by Hahn (2003) can be recovered from our general framework by setting F(r1, r2) = H 2(r2 1 + r2 2). For reference, the functional forms of Ti for these forms of F(r1, r2), along with their softening parameters ϵ2(α), are summarized in Table B1, which can be used to show that equations (A22)–(A24) reduce to those in Table 1 after some algebra with the aid of the recursive relationships for B(m) s presented in Appendix C. s As to the formalism of Teyssandier & Ogilvie (2016), we ﬁnd, using their softening prescription of F(r1, r2) = S2r1r2, that our general framework yields φij expressions different from those reported by Teyssandier & Ogilvie (2016). Indeed, we ﬁrst note that in this case, T1 = MNRAS 489, 4176–4195 (2019) 4194 A. A. Seﬁlian and R. R. Raﬁkov T4 = T6 = 0 (Table B1) rendering the expressions of φ11 and φ22 identical such that T4 = T6 = 0 (Table B1) rendering the expressions of φ11 and φ22 identical such that T4 = T6 = 0 (Table B1) rendering the expressions of φ11 and φ22 identical such that T4 = T6 = 0 (Table B1) rendering the expressions of φ11 and φ22 identical such that T4 = T6 = 0 (Table B1) rendering the expressions of φ11 and φ22 identical such that φ11 = φ22 = α 8 −5B(1),TO 3/2 + 3 2αB(0),TO 5/2 + 3(1 + α2 + S2α)B(1),TO 5/2 −15 2 αB(2),TO 5/2 −3 4S2(S2α + 2α2 + 2)B(0),TO 5/2 + 1 2S2B(0),TO 3/2 (B1) (B1) Using the recursive relationships listed in Appendix C1, the above expression can be simpliﬁed further. Indeed, equation (C2) with m = 1 and s = 5/2 and equation (C1) with m = 1 and s = 3/2 read Using the recursive relationships listed in Appendix C1, the above expression can be simpliﬁed further. C3 Relationship to elliptic integrals Here, we express the generalized Laplace coefﬁcients B(m) s in terms of complete elliptic integrals. These expressions can be used for rapid numerical evaluation of the generalized Laplace coefﬁcients without relying on numerical integration of equation (A20) (or equation 7). Let us write, as before, 2αγ = 1 + α2 + ϵ2(α) and deﬁne χ = √2/(γ + 1) such that, for any general softening parameter ϵ2(α), we have 0 ≤χ ≤1 and γ ≥1. Now let us express B(m) s in terms of γ to write B(m) s = 21−s παs π 0 cos(mθ) (γ −cos θ)s dθ. B(m) s = 21−s παs π 0 cos(mθ) (γ −cos θ)s dθ. C2 Asymptotic behaviour ve approximate expressions for B(m) s in the asymptotic limits; for α →0 and α →1. Here, we derive approximate expressions for B(m) s in the asymptotic limits; for α →0 and α →1. Case 1: In the limit of α ≈0, one can factor out the term 1 + α2 + ϵ2(α) from the integrand of B(m) s to expand the denominator around γ −1 ≈0, where γ = (2α)−1[1 + α2 + ϵ2(α)]. This allows us to approximate B(m) s as B(m) s (α) ≈ 2 π(2αγ )s π 0 cos(mθ) × 1 + s γ cos θ + s(s + 1) 2γ 2 cos2 θ + s(s + 1)(s + 2) 6γ 3 cos3 θ dθ. (C5) B(m) s (α) ≈ 2 π(2αγ )s π 0 cos(mθ) × 1 + s γ cos θ + s(s + 1) 2γ 2 cos2 θ + s(s + 1)(s + 2) 6γ 3 cos3 θ dθ. (C5) B(m) s (α) ≈ 2 π(2αγ )s π 0 cos(mθ) × 1 + s γ cos θ + s(s + 1) 2γ 2 cos2 θ + s(s + 1)(s + 2) 6γ 3 cos3 θ dθ. (C5) MNRAS 489, 4176–4195 (2019) 4195 Softened potentials of discs 4195 Softened potentials of discs Using the orthogonality of the cosine functions, it is straightforward to show that Using the orthogonality of the cosine functions, it is straightforward to show that B(m) s ≈ αmFm (2αγ )s+m as α →0, where Fm = ⎧ ⎪ ⎪ ⎨ ⎪ ⎪ ⎩ 2 if m = 0 2s if m = 1 s(s + 1) if m = 2 1 3s(s + 1)(s + 2) if m = 3 (C6) B(m) s ≈ αmFm (2αγ )s+m as α →0, where Fm = ⎧ ⎪ ⎪ ⎨ ⎪ ⎪ ⎩ 2 if m = 0 2s if m = 1 s(s + 1) if m = 2 1 3s(s + 1)(s + 2) if m = 3 (C6) (C6) Case 2: In the opposite limit of x = 1 −α ≈0, the dominant contribution to B(m) s comes from θ ≪1 (Goldreich & Tremaine 1980). Thus, one can set cos (mθ) →1 in the numerator, approximate cos θ ≈1 −θ2/2 in the denominator and extend the integration limit to inﬁnity. Furthermore, setting α = 1 (i.e. C2 Asymptotic behaviour x = 0) everywhere except when it appears in the combination 1 −α, the generalized Laplace coefﬁcient can be approximated as B(m) s ≈2 π ∞ 0 dθ x2 + θ2 + ϵ2 α=1 s = 2 π (x2 + ϵ2 α=1)−1 if s = 3/2 2 3(x2 + ϵ2 α=1)−2 if s = 5/2 B(m) s ≈2 π ∞ 0 dθ x2 + θ2 + ϵ2 α=1 s = 2 π (x2 + ϵ2 α=1)−1 if s = 3/2 2 3(x2 + ϵ2 α=1)−2 if s = 5/2 (C7) B(m) s ≈2 π ∞ 0 dθ x2 + θ2 + ϵ2 α=1 s = 2 π (x2 + ϵ2 α=1)−1 if s = 3/2 2 3(x2 + ϵ2 α=1)−2 if s = 5/2 (C7) (C7) where ϵ2 α=1 is the softening parameter evaluated at α = 1. C3 Relationship to elliptic integrals (C8) (C8) ) = π/2 0 (1 −χ2 sin2 φ)−1/2dφ and E(χ) = π/2 0 1 −χ2 sin2 φ 1/2 dφ, we ﬁnd that B(1) 3/2 = 2[−(γ −1)K(χ) + γ E(χ)] πα(γ −1)√2α(γ + 1) , (C9) Introducing complete elliptic integrals K(χ) = π/2 0 (1 −χ2 sin2 φ)−1/2dφ and E(χ) = π/2 0 1 −χ2 sin2 φ 1/2 dφ, we ﬁnd that B(0) 3/2 = 2E(χ) πα(γ −1)√2α(γ + 1), B(1) 3/2 = 2[−(γ −1)K(χ) + γ E(χ)] πα(γ −1)√2α(γ + 1) , (C9) Introducing complete elliptic integrals K(χ) = π/2 0 (1 −χ2 sin2 φ)−1/2dφ and E(χ) = π/2 0 1 −χ2 Introducing complete elliptic integrals K(χ) = π/2 0 (1 −χ2 sin2 φ)−1/2dφ and E(χ) = π/2 0 1 −χ2 sin2 φ 1/2 dφ, we ﬁnd that Introducing complete elliptic integrals K(χ) = / 0 (1 −χ2 sin2 φ) 1/2dφ and E(χ) = / 0 1 −χ2 sin2 φ / dφ, we ﬁnd that B(0) 3/2 = 2E(χ) πα(γ −1)√2α(γ + 1), B(1) 3/2 = 2[−(γ −1)K(χ) + γ E(χ)] πα(γ −1)√2α(γ + 1) , (C9) B(2) 3/2 = 2[−4γ (γ −1)K(χ) + (4γ 2 −3)E(χ)] πα(γ −1)√2α(γ + 1) , B(3) 3/2 = 2 3 [−(γ −1)(32γ 2 −5)K(χ) + γ (32γ 2 −29)E(χ)] πα(γ −1)√2α(γ + 1) , (C10) B(0) 5/2 = 4[−(γ −1)K(χ) + 4γ E(χ)] 3π(2α)5/2(γ + 1)3/2(γ −1)2 , B(1) 5/2 = 4[−γ (γ −1)K(χ) + (γ 2 + 3)E(χ)] 3π(2α)5/2(γ + 1)3/2(γ −1)2 , (C11) B(2) 5/2 = 4[(γ −1)(4γ 2 −5)K(χ) −4γ (γ 2 −2)E(χ)] 3π(2α)5/2(γ + 1)3/2(γ −1)2 , B(3) 5/2 = 4[γ (γ −1)(32γ 2 −33)K(χ) −(32γ 4 −57γ 2 + 21)E(χ)] 3π(2α)5/2(γ + 1)3/2(γ −1)2 . (C12) B(0) 3/2 = 2E(χ) πα(γ −1)√2α(γ + 1), B(3) 3/2 = 2 3 [−(γ −1)(32γ 2 −5)K(χ) + γ (32γ 2 −29)E(χ)] πα(γ −1)√2α(γ + 1) , (C10) B(1) 5/2 = 4[−γ (γ −1)K(χ) + (γ 2 + 3)E(χ)] 3π(2α)5/2(γ + 1)3/2(γ −1)2 , (C11) B(2) 5/2 = 4[(γ −1)(4γ 2 −5)K(χ) −4γ (γ 2 −2)E(χ)] 3π(2α)5/2(γ + 1)3/2(γ −1)2 , B(3) 5/2 = 4[γ (γ −1)(32γ 2 −33)K(χ) −(32γ 4 −57γ 2 + 21)E(χ)] 3π(2α)5/2(γ + 1)3/2(γ −1)2 . This paper has been typeset from a TEX/LATEX ﬁle prepared by the author. PPENDIX D: CONVERGENCE CRITERION FOR THE PRE-FACTORS OF POWE Astrophysical discs often extend over a few orders of magnitude in radius so that aout/ain ≫1. In such situations, far from the disc edges one can take the limit of both α1 = ain/ap and α2 = ap/aout going to zero, provided that the gravitational potential of a power-law disc is insensitive to the locations of the disc boundaries (see equations 18 and 19). Then the pre-factors ψ1 and ψ2 of the disturbing function converge to values depending only on the power-law indices p and p + q, respectively, as well as on the adopted softening prescription. The conditions on the values of p and q which guarantee this convergence can be determined by expanding the coefﬁcients φij(α), which appear in the integrands of each of ψ1 and ψ2, in the limit of α ≈0. Using the Taylor expansions of softened Laplace coefﬁcients B(m) s , we determined that both ψ1 and ψ2 calculated using the softening methods of Hahn (2003) and Tremaine (1998), as well as its rectiﬁed version, are convergent as long as −1 < p < 4 and −2 < p + q < 5, respectively, for all values of softening (i.e. H, βc). This follows from the fact that for both Hahn (2003) and Tremaine (1998) we have φ11 = φ22 ∼α2 and φ12 ∼α3 to lowest order in α. These ranges of p and p + q are in line with the ﬁndings of Silsbee & Raﬁkov (2015). As to the (rectiﬁed) softening model of Teyssandier & Ogilvie (2016), a similar exercise yields that φ11 = φ22 ≈−1 4S2α + 3 8(1 + 3 2S4)α2 and φ12 ≈3 2S2α2 −15 16(1 + 5S4)α3 which, in the limit of S →0, translate to the same ranges for ψ1 and ψ2 convergence as Silsbee & Raﬁkov (2015). However, when S is relatively large, it is trivial to show that ψ1 and ψ2 are convergent over limited ranges of 0 < p < 3 and −1 < p + q < 4, respectively. A similar analysis for the softening method of Touma (2002) reveals that the ranges for ψ1 and ψ2 convergence are in line with the ﬁndings of Silsbee & Raﬁkov (2015) when the corresponding softening parameter bc →0. However, when bc is non-zero, the ranges are narrowed down to −1 < p < 2 and −2 < p + q < 3, respectively. C3 Relationship to elliptic integrals (C12) (C12) These expressions permit efﬁcient numerical evaluation of arbitrarily softened Laplace coefﬁcients as functions of α, since effective algorithms for computing K and E exist (e.g. Press et al. 2002). These expressions permit efﬁcient numerical evaluation of arbitrarily softened Laplace coefﬁcients as function for computing K and E exist (e.g. Press et al. 2002). PPENDIX D: CONVERGENCE CRITERION FOR THE PRE-FACTORS OF POWE This paper has been typeset from a TEX/LATEX ﬁle prepared by the author. MNRAS 489, 4176–4195 (2019)
https://openalex.org/W2597508560	https://revistas.ucm.es/index.php/RPUB/article/download/54897/50038	es		Teoría y práctica de las Relaciones Internacionales en la encrucijada: la (im)posibilidad del cosmopolitismo	Res publica	2,017	cc-by	4,459	NOTAS CRÍTICAS Res Publica. Revista de Historia de las Ideas Políticas ISSN: 1576-4184 http://dx.doi.org/10.5209/RPUB.54897 Teoría y práctica de las Relaciones Internacionales en la encrucijada: la (im)posibilidad del cosmopolitismo A propósito de Cristina García Pascual, Norma mundi. La lucha por el Derecho Internacional, Prólogo de Javier de Lucas, Madrid, Trotta, 2015, 270 pp. Laila Yousef Sandoval* La mayoría de los libros dedicados al pensamiento de las Relaciones Internacionales comienza constatando el mismo factum: la aporía interna que sufre la disciplina al constituirse como una pléyade de teorías, a veces complementarias y otras contrapuestas. No hay acuerdo en el ámbito de la teoría internacional, del mismo modo que parece no haberlo en la práctica de la política internacional. Tal como explica Javier de Lucas en el prólogo de este libro, esta situación genera una especie de desasosiego ante el Derecho Internacional, el cual puede llegar a ser visto como un “rara avis”: “incluso peor: una disciplina, más que singular, peligrosa: por fatua, por generar unas expectativas que conducen a la frustración de la confianza en el derecho por parte de los millones de ciudadanos”1. Pese a la variedad y dispersión de teorías internacionales y los valiosos matices que ofrece cada una de ellas, se puede considerar que la disputa teórica final queda reducida a la dicotomía clásica, presente en todos los niveles de la reflexión política, entre una concepción que cree que es posible realizar la paz en la tierra a través del derecho, y el pensamiento de que únicamente la violencia y el poder político pueden crear ley. Pero aquello que da una especificidad y problematicidad propias a la materia de las Relaciones Internacionales es que en la extensión del debate entre validez y facticidad al plano internacional lo que se juega es de alcance universal, esto es, la posibilidad o no de instituciones globales y cosmopolitas: “…problemática y discutida sigue siendo su entidad [la del Derecho Internacional], sus características, su objeto y su naturaleza jurídica, pero, sobre todo, en la medida en que constituye la plataforma necesaria sobre la que edificar cualquier proyecto de cosmopolitismo jurídico”2. El “problema del derecho internacional”3, tal y como lo denomina la autora García Pascual, no sólo refiere a la dificultad que implica el ejercicio de la política interestatal sino a * 1 2 3 Universidad Complutense de Madrid lyousef@ucm.es J. de Lucas, Prólogo a C. García Pascual, Norma mundi. La lucha por el Derecho Internacional, Madrid, Trotta, 2015, p. 13. C. García Pascual, Norma mundi. La lucha por el Derecho Internacional, op.cit., p. 17. Ibidem, p. 17. “Las dificultades de dar un fundamento objetivo al derecho internacional parecen [….] ciertamente insuperables”. Ibidem, pp. 49-50. Res publica (Madrid), 20(1) 2017: 165-172 165 166 Yousef Sandoval, L. Res publica (Madrid), 20(1) 2017: 165-172 la complejidad de la teoría misma que se encarga de ello, que por sus pretensiones totales, carece de la unidad y cohesión que se pide a toda materia teórica. El punto arquimédico a partir del cual García Pascual reconstruye y engarza los distintos relatos que ha ofrecido la filosofía acerca de las Relaciones Internacionales es “el concepto y el fundamento del orden jurídico”. Esto sirve de base y punto de partida para lograr un estudio común y unificado de las diferentes teorías, ya que la concepción que cada una de ellas tenga del propio derecho permitirá explicar sus diferencias específicas. Este recorrido está articulado en torno a cinco grandes apartados que componen el índice del libro: el origen de la idea cosmopolita en la Modernidad, la defensa del derecho internacional; su rechazo; la posible viabilidad del cosmopolitismo; y las deprimentes expectativas que augura para el futuro el Derecho Internacional del siglo XX. En el primer bloque del libro, titulado Tradición y modernidad del cosmopolitismo jurídico, se discuten los orígenes mismos del derecho internacional y de la intención cosmopolita. Es fundamental resaltar la importancia de Francisco de Vitoria como uno de los inauguradores del cosmopolitismo en los albores de la Modernidad4. Según Vitoria, el orden jurídico que funda esta comunidad universal debiera asentarse en el vínculo creado por la condición de ser seres humanos que comparten todos los individuos. Esto ofrece a los hombres una primacía jurídica, la de ser ellos, en vez de los estados, los verdaderos sujetos del derecho. Esta idea de comunidad global anticipada por Vitoria se ve continuada por la teoría del gobierno mundial de Kant que, desarrollada en pleno contexto westfaliano, constituye otro hito del cosmopolitismo. La radicalidad del proyecto kantiano estriba en que la paz mundial tiene una condición jurídica fundamental: la paz interna entendida como republicanismo: “Si la república es la mejor constitución para proteger la libertad de los individuos dentro del Estado, también constituye la mejor garantía de que esa libertad sea disfrutada en una convivencia pacífica entre los Estados”5. No hay que olvidar que para Kant la posibilidad de dicha comunidad cosmopolita no es alcanzable en el ámbito de la experiencia, sino que es la culminación de la idea de paz, pensable de manera necesaria en el campo del deber ser, imposible de completar en el ámbito del ser6, pero susceptible de ser realizada de manera aproximada. Una vez explicados los orígenes fundamentales del cosmopolitismo, García Pascual procede a trazar un estudio de aquellas doctrinas que han apostado por la posibilidad del cosmopolitismo y las que, por el contrario, han considerado que es una empresa inalcanzable, en los capítulos titulados La lucha por el derecho internacional. La teoría pura y los retos de la paz perpetua y La negación del derecho internacional. Uno de los mayores defensores de la primera tesis es Hans Kelsen. Su normativismo se enmarca en el “monismo jurídico”, esto es, en la doctrina que concibe el derecho internacional y el interno como dos sistemas incardinados, por contraste con las teorías dualistas, que reconocen que el derecho externo e interno son dos órdenes jurídicos completamente diferentes e incomparables. El monismo de 4 5 6 Ibidem, pp. 22-23 Ibidem, p. 41. Véase el Primer artículo definitivo para la paz perpetua: “La constitución civil de todo Estado debe ser republicana”. I. Kant, Sobre la paz perpetua, en: Ensayos sobre la paz, el progreso y el ideal cosmopolita, Madrid, Cátedra, 2005, p. 148. Trad. Joaquín Abellán. “La paz perpetua (el ﬁn último del derecho de gentes en su totalidad) es ciertamente una idea irrealizable”. I. Kant, Metafísica de las costumbres, Madrid, Tecnos, 2008. Estudio preliminar de Adela Cortina. Trad. Adela Cortina y Jesús Conill, §61, pp. 190-191. Yousef Sandoval, L. Res publica (Madrid), 20(1) 2017: 165-172 167 Kelsen entiende el derecho internacional del mismo modo que el interno, como un sistema de normas que es capaz de imponer una sanción y que posee el monopolio de la fuerza: “en ese sentido la búsqueda de la paz […] no constituye tanto una tarea política (en ningún caso moral) cuanto técnica”7. Partiendo de esa igualdad de estatus entre los dos derechos, Kelsen defiende la primacía del derecho internacional sobre el derecho interno. Esto funciona en el ámbito teórico, sin embargo, el propio Kelsen reconoce que en la práctica, el derecho internacional está en una fase primitiva de desarrollo y menos perfeccionado que el estatal8. Esto es así, en parte, porque no hay una autoridad central que pueda ejercer su derecho soberano de decisión, pues aunque haya instituciones internacionales, éstas no son capaces de ejecutar sus directrices directamente. Lo que le falta a las instituciones internacionales es un tribunal de justicia que se encargue de aplicar las sanciones conforme a derecho. Esto, junto con la noción de derecho que defiende Kelsen, entendido como edificio de normas sin contenido hasta el punto que “no puede haber legislador sin juez, aunque puede haber muy bien juez sin legislador”9, hace surgir varios interrogantes, tal y como señala García Pascual: quién decide cuándo aplicar una sanción y dónde situar fuera del derecho la piedra de toque normativa, ya que según Kelsen fuera del derecho no hay nada. La autora destaca que el aspecto positivo de esta concepción jurídica es que, en la medida en que las instituciones quedan libres de las influencias políticas, se aleja cualquier fantasma ideológico o imperial ya que “lo político se disuelve en lo jurídico y todo se convierte en derecho”.10 Como para Kelsen el problema radica en que no hay una autoridad jurídica suprema que medie imparcialmente cuando hay disputas entre los estados, idea como solución un gobierno mundial o “comunidad judicial” en la que la guerra quedaría terminantemente prohibida y que, al estar regido por derecho, jamás sería imperial. Si la SDN ha fracasado es, según Kelsen, porque no estuvo dirigida por un tribunal de justicia, sino por el Consejo, un órgano gubernamental11. Hay varios aspectos controvertidos en la teoría de Kelsen. Según García Pascual, “se muestra limitada en su capacidad propositiva o, si se quiere, vacía a la hora de ofrecer un fundamento fuerte del orden internacional”12. Otra crítica, la de Martti Koskenniemi, que desarrolla García Pascual a lo largo del libro, se centra en la reducción de la política al derecho que parece estar a la base de la teoría kelnesiana13. Todo parece indicar que la absoluta juridificación de la política es imposible y que la separación entre derecho y moral que Kelsen toma como punto de partida, acaba siendo por él mismo refutado en favor de un Derecho que no puede abandonar el horizonte normativo. Pues, el carácter de la Grundnorm, la norma fundamental del Derecho Internacional, origen y fundamento de las demás, no queda del todo claro ya que esa norma tiene que ser anterior al pacto contingente entre los estados. A ello 7 8 9 10 11 12 13 C. García Pascual, Norma mundi. La lucha por el Derecho Internacional, op.cit., p. 54 Ibidem, p. 62 H. Kelsen, op.cit., p. 54 apud C. García Pascual, Norma mundi. La lucha por el Derecho Internacional, op.cit., p. 72. C. García Pascual, Norma mundi. La lucha por el Derecho Internacional, op.cit., p. 77. H. Kelsen, La paz por medio del derecho, Madrid, Trotta, 2008, p. 75. C. García Pascual, Norma mundi. La lucha por el Derecho Internacional, op.cit., p. 78. “Al empujar la política fuera del ámbito de la ciencia y de la teoría, Kelsen disminuyó su importancia en las luchas diplomáticas y constitucionales de la época”. M. Koskenniemi, The Gentle Civilizer of Nations. The Rise and Fall of International Law 1870-1960 (Hersch Lauterpacht Memorial Lectures), Cambridge, Cambridge University Press, 2004, p. 247. Traducción personal. 168 Yousef Sandoval, L. Res publica (Madrid), 20(1) 2017: 165-172 se suma la incertidumbre acerca de qué mecanismos de cooperación harían de ese Derecho Internacional una instancia imparcial sin la sombra de los instintos hegemónicos de los estados, que se antojan inextirpables. Como contrapunto a estas doctrinas reafirmadoras del derecho, García Pascual dedica al negacionismo jurídico una sección del libro, que inicia con la siguiente pregunta: “¿existe algo que podamos denominar con propiedad derecho internacional?”14. El repaso a las teorías de estos negacionistas de la coexistencia cosmopolita entre estados está centrado, entre otros autores, en el pensamiento de Carl Schmitt15. Al jurista lo que le interesa es conservar el equilibrio internacional y, según él, para ello es necesario que cada estado soberano mantenga su capacidad de decisión sin injerencias de terceros estados. Por eso para Schmitt un gobierno mundial o un tribunal internacional resultan indeseables, porque supondría aceptar una homogeneidad entre los estados que iría en contra de la pluralidad que es propia del sistema mundial. Implicaría además que un estado ejerciera su hegemonía sobre los demás, ya que Schmitt no entiende, como pueda hacerlo Kant, que un gobierno mundial pueda estar basado en la idea de republicanismo. Para comprender la importancia que Schmitt otorga al estado soberano y el decisionismo que está a la base de su pensamiento, es fundamental comprender el carácter existencial de su política, muy visible en su teoría de amigo/enemigo y en su concepto de nomos. El nomos, entendido como “la forma inmediata en la que se hace visible, en cuanto al espacio, la ordenación política y social de un pueblo”16 viene a expresar cómo la ligazón existencial con la tierra, la concreción del suelo, genera derecho, más allá de las normas jurídicas concretas que luego se establezcan: “podríamos denominar el momento constitutivo por encima del estudio y la reflexión en torno al momento ya constituido (el derecho positivo o la ordenación existente), la positividad concreta y originaria del nomos por encima de la positividad abstracta de la norma”17. Como señala García Pascual, no se trata solo de una cuestión geográfica, sino sobre todo de cómo esa distribución geográfica determina la estructura política18. Pues bien, el nomos de la tierra, la configuración espacial del globo que configura el desarrollo político internacional, está atravesado por la pluralidad y la heterogeneidad, por la peculiar manera 14 15 16 17 18 C. García Pascual, Norma mundi. La lucha por el Derecho Internacional, op.cit., p. 89. A parte de Schmitt, García Pascual desarrolla extensamente un análisis sobre la tradición anglosajona de Austin y Hardt. Esta tradición entiende el derecho internacional como “uso social normativo” (Ibidem, p. 92), considera las leyes como un mero producto positivo fruto de la costumbre, como es el caso de Austin (“Para el profesor inglés, las reglas del derecho de gentes forman parte de las leyes morales positivas, como tales emanan de la opinión pública, más o menos difusa y, por tanto, son análogas a las reglas del honor, de la moda, de la cortesía y diversas de las leyes jurídicas positivas”, Ibidem, p. 93), sin algo que podamos llamar centro decisorio soberano, véase la teoría de Hart (“Lo que llamamos derecho internacional, nos dice Hart, se asemeja formalmente a un sistema simple de derecho consuetudinario o primitivo, y materialmente, es decir, en cuanto a los contenidos de sus norma, a cualquier sistema de derecho estatal”, Ibidem, p. 106). García Pascual no queda satisfecha con estas argumentaciones porque no terminan de dar respuestas al problema del derecho internacional. La autora también incluye un comentario a la teoría de Hermann Heller, para quien la soberanía constituye la condición de la existencia del derecho internacional. “En Heller, el derecho aparece como producto del Estado, es decir, de la instancia con capacidad decisoria que hace positivo lo que hasta entonces permanecía en el ámbito de los principios o normas éticas aceptados por la comunidad”, Ibidem, p. 140. C. Schmitt, El Nomos de la Tierra en el Derecho de Gentes del Ius publicum europaeum (1950), Granada, Comares, 2002, pp. 35-36. C. García Pascual, Norma mundi. La lucha por el Derecho Internacional, op.cit., p. 117. Ibidem, p. 118. Yousef Sandoval, L. Res publica (Madrid), 20(1) 2017: 165-172 169 que cada estado soberano tiene de relacionarse con el espacio sobre el que decide. Eso se manifiesta, en el contexto del derecho de la Modernidad, en la importancia del estado, cuyo “efecto racionalizador”19 marca el fin del contexto teológico y el inicio del Ius publicum europaeum20. Y si uno de sus principales adversarios teóricos21 es Hans Kelsen es porque para Schmitt el derecho es política y ésta no puede ser subsumida bajo condiciones jurídicas: al derecho hay que reconocerle su rasgo político constituyente y no meramente su carácter constituido como mera aplicación técnica de las leyes. Su animadversión al pensamiento de Kelsen aumenta en el ámbito de la teoría acerca de la guerra. Schmitt es un gran crítico de las guerras basadas en causas justas, típicas de la Edad Media y reimplantadas en el siglo XX, pues en base a una argumentación moralizadora consideran al adversario, no como un enemigo justo, sino como un criminal injusto. Por eso está en contra de cualquier tipo de tribunal internacional, más aún viendo cómo el Tratado de Versalles y el Protocolo de Ginebra criminalizaron al adversario y menoscabaron la soberanía de ciertos estados. Schmitt se posiciona en contra de relacionar la guerra con la sanción o el delito,22 como hace Kelsen, pues para él toda guerra es justa en la medida en que es llevada a cabo por estados soberanos: “De este modo, la imposibilidad de determinar por un tercero con cierta objetividad y con cierta eficacia la causa justa de los conflictos bélicos, si estos son aplicación del derecho o violación del mismo, deriva no en la consideración de cualquier guerra como injusta, sino en la declaración de toda guerra como legítima o justa. Pensar de otro modo sería pretender limitar lo que por definición no puede ser limitado: la soberanía de los Estados, especialmente la de aquellos creadores y portadores del jus publicum europaeum”23. La autora observa que, para Schmitt, aceptar el conflicto limitado entre estados es lo que precisamente garantiza el equilibro interestatal y lo que permite “acotar la guerra pero no eliminarla, regular el ejercicio de la guerra pero no el derecho a la misma”24. Esto escondería una contradicción, la contradicción de una paz basada en la soberanía absoluta: “Lo que no parece claro en la exposición de Schmitt es la razón por la cual un Estado soberano que ejerce la guerra como un derecho propio, debería limitar las consecuencias de esta y no caer en la llamada guerra de aniquilamiento simplemente por el hecho de que también los demás Estados son soberanos y tienen un jus belli ilimitado. Una soberanía sin límites, parece sostener el jurista alemán, genera de facto una situación de soberanías limitadas, por su propio coexis19 20 21 22 23 24 Ibidem, p. 123. En la época en la que escribe Schmitt el Ius publicum europaeum ya estaba superado. Sin embargo, Schmitt, aunque consciente de que el estado soberano ya no puede tener la preeminencia política que tuvo en la Modernidad, intenta recuperar en cierta medida la capacidad de decisión de los entes políticos a través de su teoría de los grandes espacios. Esta teoría schmittiana, pensada para el contexto contemporáneo y que algunos consideran de tintes imperiales, está basada en la creación de grandes bloques regionales que ejercerían su poder de decisión sin la intromisión de terceros. También es crítico con Francisco de Vitoria, por no haber superado el paradigma teológico y por desarrollar una teoría de la guerra en base a causas justas. Schmitt tampoco apoya un normativismo abstracto, sino un decisionismo basado en el orden concreto: “Así, refutando la tesis kelseniana de la norma fundamental como base del orden dinámico que es el derecho, Schmitt considera que las decisiones de los órganos estatales o las decisiones políticas tienen un valor jurídico autónomo que no procede de su remisión a normas generales y abstractas”, C. García Pascual, Norma mundi. La lucha por el Derecho Internacional, op.cit., p. 135. Ibidem, p. 125. Ibidem, p. 126. 170 Yousef Sandoval, L. Res publica (Madrid), 20(1) 2017: 165-172 tir, en la perversidad y deshumanización de la guerra. Al argumento schmittiano le falta el hilo argumental que pueda sostener ese sofisma que constituye el paso del ilimitado poder a la limitada crueldad”25. El cuarto bloque del libro, titulado Del derecho internacional al derecho cosmopolita, estudia la posibilidad del derecho cosmopolita desde la perspectiva de Rawls y Habermas, entre otros autores. The law of peoples es un intento del filósofo John Rawls por mostrar un “kantismo escéptico”: “el autor americano opta […] por una de las posibles interpretaciones del Kant iusinternacionalista, aquella que no toma en consideración las afirmaciones sobre la idea de la república universal como ideal de la razón, sino que, antes bien, ignorando estos argumentos, sostiene aquellos pasajes de la obra del filósofo alemán que muestran su escepticismo hacia el Estado mundial y defienden una federación pacífica como único fin posible”26. La única salida que atisba Rawls no es el derecho cosmopolita, sino el derecho de gentes, ese derecho interestatal que resultaba tan imperfecto para Kant, en comparación con el ideal de la paz mundial. Se podría criticar que este pensamiento liberal presenta ciertas carencias al hacer abstracción de cualquier consideración económica o social. Además, la autora critica su división en “sociedades aceptables” y “sociedades no aceptables”, así como el recurso a la guerra justa, que sólo sirve para mantener el statu quo y no para generar mejoras o innovaciones a nivel internacional. Por su parte Habermas, partiendo de un estudio sobre el ideal cosmopolita kantiano, llega a la conclusión de que no se puede prescindir de la soberanía en el proyecto mundial. Especialmente interesante resulta la crítica que García Pascual realiza al vínculo entre democracia y paz, tan alabado por Habermas en sus obras: “Si echamos la vista atrás y observamos la historia, tal vez concluyamos que el Estado democrático nacional de facto no se ha revelado más pacífico que su predecesor, el Estado autoritario dinástico. La historia de los últimos siglos podría verse como un periodo de consolidación del as democracias europeas que, no obstante, simultáneamente, no han cesado de exportar violencia fuera de sus fronteras. Un periodo de pacificación en el interior que se desarrolla de manera paralela a un periodo de creciente violencia exterior”27. Según Habermas, un elemento que puede fomentar la unión de los estados a nivel internacional, junto con Internet como voz de la opinión pública mundial, es el comercio. Esto recibe de nuevo una crítica contundente por parte de la autora del libro: por un lado, pone de manifiesto que las decisiones que más nos afectan se toman en espacios no democráticos y carentes de participación y transparencia; por otro, subraya que la historia ya nos ha mostrado cómo la expansión del capitalismo no ha ido ligada necesariamente al fomento de la paz internacional28. La parte final del libro se pregunta, bajo el epígrafe Derecho internacional, escepticismo e impotencia. Perspectivas para un nuevo siglo, por la fase del derecho internacional en la que nos encontramos hoy en día, qué efectos tiene el fin de la 25 26 27 28 Ibidem, pp. 126-127. Ibidem, p. 157. Ibidem, p. 177. “Si observamos nuestras circunstancias y analizamos la expansión desbocada del capitalismo en la que vivimos inmersos, nos parece imposible extraer consecuencias positivas del desarrollo de las transacciones comerciales […] Basta pensar que las grandes empresas transnacionales han sido consideradas por muchos analistas como semi-Estados…”. Ibidem, p. 178. Aquí García Pascual hace referencia a Joseph Stiglitz (“La corporación multinacional”, Claves de razón práctica, 167, 2006) y a Ulrich Beck, Un nuevo mundo feliz. La precariedad del trabajo en la era de la globalización, Barcelona, Paidós, 2000. Yousef Sandoval, L. Res publica (Madrid), 20(1) 2017: 165-172 171 soberanía tradicional y qué papel tienen las entidades no estatales que determinan el panorama internacional. El título del último capítulo resume el estado de ánimo actual respecto a lo internacional: “escepticismo e impotencia” y en él se repasan, entre otras, teorías como las de Posner y Koskenniemi. La actitud escéptica del jurista estadounidense Richard Posner parte de la inutilidad del derecho internacional ante su incapacidad para resolver conflictos. Tomando como herencia las ideas de Hans Morgenthau, establece que la clave para entender las relaciones internacionales es el interés estatal, lo que permite a los estados incumplir las normas internacionales. El derecho internacional tiene poco margen de acción para constreñirles, y si así sucediera significaría que un derecho supranacional se ha impuesto, sin procedimiento democrático mediante, sobre cada uno de los estados soberanos29. El escepticismo de Martti Koskenniemi, dista bastante del de Posner. El diplomático y académico finlandés bebe de las fuentes de la corriente Critical Legal Studies y pretende demostrar que el derecho internacional carece de objetividad porque es contradictorio, sin menoscabo de reconocer al mismo tiempo que es necesario para guiar la acción política internacional30. Según Koskenniemi, el problema radica en que los juristas creen que están situados en un espacio neutral cuando, en realidad, el derecho está atravesado por la parcialidad y además es indeterminado, subjetivo, confuso y crea situaciones de hegemonía: “Koskenniemi parte, pues, de un claro y radical escepticismo o, si se quiere, de un anticognoscitivismo radical. El derecho internacional sería un discurso que puede justificar cualquier pretensión, una cosa y la contraria”31. El jurista internacional se ve atrapado entonces entre dos situaciones sin salida, entre lo que él denomina, el utopismo y el apologismo, esto es, “entre mantener el discurso descriptivo de la política o de las relaciones de poder o mantener un discurso prescriptivo, falto de normatividad y carente de relación con la realidad”32. La tesis de Koskenniemi es que el derecho internacional es tanto política como derecho y quizás más lo primero que lo segundo. Para ilustrarlo se sirve de una ilustración wittgensteiniana: “A Koskenniemi le gusta utilizar la imagen duck-rabbit illusion de Wittgenstein para hacer frente a la pregunta acerca de qué es el derecho internacional: ¿objetivamente derecho u objetivamente política? Como en el duck-rabit illusion, la realidad es una, aunque contenga dos rostros o la podamos observar desde dos perspectivas: la de la política y la del derecho”33. Como se puede observar, ese dilema entre política y derecho es el que recorre la historia del derecho internacional, la cual, según García Pascual “podría contarse como la historia de los fracasos de la comunidad internacional”34 y es así mismo la lucha conceptual que atraviesa el libro. Una de las muchas conclusiones que se extraen de su lectura es que, o insistimos en entender el derecho internacional como un mero conjunto de normas jurídicas, o reconocemos la presencia de la política junto con esas normas y en esas normas. Afirmar la última tesis, esto es, reconocer el carácter político del derecho no menoscaba la necesidad de un principio de juridicidad, 29 30 31 32 33 34 “La conversión del derecho internacional en derecho interno a través de procesos de reconocimiento normativo sería una concesión a un derecho que, en principio, no tiene la legitimidad que dan las instituciones democráticas”. C. García Pascual, Norma mundi. La lucha por el Derecho Internacional, op.cit., p. 205. Cfr. Ibidem, p. 220. Ibidem, p. 222. Ibidem, p. 225. Ibidem, p. 227. Ibidem, p. 243. Yousef Sandoval, L. Res publica (Madrid), 20(1) 2017: 165-172 172 más bien hace ineludible postular escenarios ideales donde la praxis esté informada por las normas. Y esta indefectible unión entre el reino del derecho y el reino de la política queda magníficamente expresada en la frase que cierra el libro: “La tarea de someter a reglas preexistentes los conflictos y no abandonarlos a la lógica del poder, de hacer hablar al derecho tras una guerra o tras masivas violaciones de derechos humanos, la tarea de trabajar con el lenguaje de la legalidad o ilegalidad, con el argumento de las decisiones judiciales que deben ser cumplidas o de las infracciones de normas que deben ser sancionadas; la consolidación en definitiva, de una norma mundi que nos permita vivir en paz es algo que está dentro del discurso del derecho y que, al mismo tiempo, señala que el derecho no lo puede todo, y menos aún sin contar con el soporte de la política y de la moral”35. 35 Ibidem, p. 254.
https://openalex.org/W1986946432	https://univoak.eu/islandora/object/islandora%3A54922/datastream/PDF/view	English	null	RNA structures, genomic organization and selection of recombinant HIV	RNA biology	2,011	cc-by	7,960	RNA Biology ISSN: 1547-6286 (Print) 1555-8584 (Online) Journal homepage: http://www.tandfonline.com/loi/krnb20 Date: 27 June 2017, At: 02:51 Architecture et Réactivité de l’ARN; Université de Strasbourg; CNRS; IBMC; Strasbourg, France †Current address: Center for Infectious Disease Dynamics; Department of Biology; The Pennsylvania State University; University Park, PA USA ddress: Center for Infectious Disease Dynamics; Department of Biology; The Pennsylvania State University; University Park, PA USA Key words: RNA structures, recombination, HIV, genomic organization, selection, evolution ©2011 Lande Do not d Recombination is an evolutionary mechanism intrinsic to the evolution of many RNA viruses. In retroviruses and notably in the case of HIV, recombination is so frequent that it can be considered as part of its mode of replication. This process not only plays a central role in shaping HIV genetic diversity worldwide, but has also been involved in immune escape and development of resistance to antiviral treatments. Recombination does not create new mutations in the existing genetic repertoire of the virus, but creates new combinations of pre-existing polymorphisms. The simultaneous insertion of multiple substitutions in a single replication cycle leaves little room for the progressive coevolution of regions of proteins, RNA or, more in general, genomes, to accommodate these drastic sequence changes. Therefore, recombination, while allowing the virus to rapidly explore larger sequence space than the slow accumulation of point mutations, also runs the risk of generating non functional viruses. Recombination is the consequence of a switch in the template used during reverse transcription and is promoted by the presence of structured regions in the genomic RNA template. In this review, we discuss new observations suggesting that the distribution of RNA structures along the HIV genome may enhance recombination rates in regions where the resultant progeny is less likely to be impaired, and could therefore maximize the evolutionary value of this source of genetic diversity. gp41, which mediate viral entry into the cell. During infection, upon the release of the viral core in the cytoplasm, the viral polymerase, the reverse transcriptase (RT), converts the single stranded RNA genome into double stranded DNA, the provirus, which is then translocated to the nucleus and integrated in the host genome by the viral integrase. s Bioscience. istribute For the conversion of the genomic RNA into a double stranded DNA, first, the RT synthesizes a single stranded DNA (minus strand) using the genomic RNA as a template. This latter is progressively degraded, once copied, by the RNAse H activity encoded by the RT. Etienne Simon-Loriere, Paola Rossolillo & Matteo Negroni To cite this article: Etienne Simon-Loriere, Paola Rossolillo & Matteo Negroni (2011) RNA structures, genomic organization and selection of recombinant HIV, RNA Biology, 8:2, 280-286, DOI: 10.4161/rna.8.2.15193 To link to this article: http://dx.doi.org/10.4161/rna.8.2.15193 Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=krnb20 Download by: [Université de Strasbourg, SCD ] Download by: [Université de Strasbourg, SCD ] Download by: [Université de Strasbourg, SCD ] RNA Biology 8:2, 280-286; March/April 2011; © 2011 Landes Bioscience Architecture et Réactivité de l’ARN; Université de Strasbourg; CNRS; IBMC; Strasbourg, France †Current address: Center for Infectious Disease Dynamics; Department of Biology; The Pennsylvania State University; University Park, PA USA This feature is essential to ensure that during first DNA strand synthesis, the nascent DNA is transferred from the repeated sequence R, located at 5' end of the genome, to the homologous region located at the 3' end, to complete synthesis. The minus strand DNA then serves as template for the synthesis of the complementary positive strand. stribute. Evidence has been provided that a single genomic RNA copy can be sufficient to ensure the entire process of reverse transcrip- tion in the spleen necrosis virus.1 However, reverse transcription shifts from one RNA strand to the other with a high frequency during minus strand DNA synthesis and one could consider that, at least in HIV, both genomic RNA copies are used, alter- natively, as templates. Indeed, the frequency of copy choice in HIV-1, between highly similar template RNAs, is estimated at 3 x 10-4 to 1.4 x 10-3 events per nucleotide,2-5 which extrapolates to approximately 3 to 12 template switch per genome replication. As discussed below, if the two RNA strands do not have identi- cal sequences, the process results in the generation of a recombi- nant provirus.6,7 Importantly, the formation of virions containing two different genomic RNAs (i.e., heterozygous virions) requires the fulfilment of certain prerequisites: first, two or more viruses with different genotypes must infect the same cell and, second, genomic RNAs of different origin must co-package. Correspondence to: Matteo Negroni; Email: m.negroni@ibmc-cnrs.unistra.fr Submitted: 12/06/10; Revised: 02/07/11; Accepted: 02/08/11 DOI: 10.4161/rna.8.2.15193 Etienne Simon-Loriere,† Paola Rossolillo and Matteo Negroni Architecture et Réactivité de l’ARN; Université de Strasbourg; CNRS; IBMC; Strasbourg, France HIV, Retroviruses and Viral Diploidy As with all retroviruses, the human immunodeficiency virus (HIV) carries two copies of genomic RNA in each viral particle. As such, it can be considered as diploid, a feature that strongly potentiates the mechanism of recombination. The HIV genome is constituted by two positive sense, single stranded RNA mol- ecules non-covalently linked near their 5' end. Associated to the nucleocapsid protein (NC) and to viral enzymes, they are “protected” within the viral capsid, a conical structure formed by the p24 protein. The capsid is itself surrounded by a protein layer (the matrix, p17) and finally by the viral membrane, a lipid bilayer decorated with the products of the env gene, gp120 an Coinfection of a cell can be due to either the transmission of more than one virus before the immune response has settled or to superinfection.8-10 However, the fact that the Nef and Vpu pro- teins downmodulate the expression of CD4 and co-receptors dur- ing HIV-1 infection has long suggested that superinfections are extremely rare events. Nevertheless, in situ-hybridization of cells from patients revealed that single cells could contain as many as 4 proviruses or more11,12 and, even if recent data provide a lower evaluation for circulating blood cells,13 the high recombination rates estimates for HIV-1 further suggest that coinfection occurs RNA Biology Volume 8 Issue 2 Volume 8 Issue 2 280 REVIEW REVIEW at some substantial frequency in vivo. The cell-to-cell mode of transmission is also emerging as an important mechanism that may increase the frequency of single-cell coinfection and, conse- quently, recombination.14-17 The error rate of the RT is approximately 3.4 x 10-5 mutations per nucleotide per replication cycle;26 considering that roughly 1010 viral particles are produced in an individual each day,27 millions of HIV-1 variants are generated everyday in infected patients. However, if HIV displayed a limited tolerance to genetic change, the majority of the variants generated by the error prone RT and fast replication cycle would be transient and unobserved. For these reasons, an intrinsic genetic flexibility is likely to contribute to the genetic variability observed in HIV. ©2011 Lande Do not di The production of heterozygous virions via the copackag- ing of different genomic RNAs requires the packaging RNA sequences to be compatible. The integrated provirus (produced from reverse transcription of the genomic RNA) is transcribed by the host DNA-dependent RNA polymerase into messenger and genomic RNAs. i i HIV-1 Distribution Worldwide s Bioscience. stribute. Two types of HIV have been identified to date: HIV-1 and HIV-2, both of which derive from cross-species infections. Specifically, the primate reservoir of HIV-2 has been identified as Simian Immunodeficiency Virus of sooty mangabey (SIVsm), while HIV-1 originated from chimpanzee SIV (SIVcpz; Pan troglodytes troglodytes).32 Three independent transmission events have generated the three major HIV-1 groups: M (main), O (Outlier) and N (Non-M Non-O); in 2009 a new HIV-1 group, closely related to SIV of Gorillas (SIVgor), was identified in Cameroonian women and is now called group P.33 Group M is responsible for the majority of infections worldwide and can be divided into 9 subtypes (A–D to F–K), within which it is possible to identify phylogenetic subtypes, that comprise viral isolates more closely related to each other than to isolates from other subtypes.34 For example, subtypes A and F are divided into subsubtypes A1-A2-A3-A4 and F1-F2 respectively.35,36 The degree of genetic variation within a subtype is 15–20% and among subtypes is 25–35%. There is a specific geographic dis- tribution pattern for HIV-1 subtypes worldwide,37 which may reflect population migrations combined with founder effects or more prevalent routes of transmission.i HIV, Retroviruses and Viral Diploidy While the spliced mRNAs will be translated into viral proteins, the genomic RNAs will dimerize and be pack- aged into the budding virion to constitute its genome. The linked processes of dimerization and packaging result from interactions between the NC portion of the Gag precursor and the Dimer Linkage Structure (DLS/PSI), a sequence of 300 base-pairs (bp) located at the 5'UTR of the HIV-1 genome. This region folds into a highly ordered secondary structure consisting of 6 hairpins (or stem-loops); the SL1 hairpin, which contains two internal bulges and an apical loop of 9 bps, with a 6 bp palindrome, is widely accepted as the Dimerization Initiation Site (DIS). The palindromic sequences contained in the DIS allow one RNA to base pair via “kissing” interactions with a second RNA; this intermolecular base pairing is then extended through structural transition to form the encapsidated dimer.18-20 As packaging and dimerization are coupled, the formation of heterozygous virions depends on the ability of genomic RNAs to form heterodimers. The sequence responsible for beginning the dimerization process has therefore been regarded as a crucial determinant for recombi- nation. In support of this view, it has been reported that genomic RNAs from HIV-1 strains with the same DIS are co-packaged more efficiently than strains with discordant DIS sequences.21,22 However, data from epidemic and cell culture studies indicate that the presence of compatible DIS sequences is not manda- tory for recombination to occur, and that heterodimers do form between genomic RNAs containing incompatible DIS, albeit most likely at a lower efficiency.23-25 This requisite seems there- fore important for allowing the frequent occurrence of recombi- nation, but does not constitute an absolute condition for genetic exchange between two HIV variants to occur. Additionally, frequent recombination can further increase genetic variability by reshuffling, in a single infectious cycle, genetic polymorphisms that previously appeared in distinct viruses. Therefore, genetic predisposition to tolerate recombina- tion is also an extremely important feature that allows a virus so prone to recombine, to limit the potential negative consequences of such frequent recombination. Recombination is equally thought to facilitate the combination of advantageous mutations and the removal of deleterious ones from the viral populations. In this manner, recombination contributes to HIV’s dynamic evasion of the immune response and its continual evolution of drug resistance.28-31 www.landesbioscience.com Recombination in Retroviruses This process is crucial for guiding the transfer of DNA synthesis from the donor RNA onto the acceptor template.46 The importance of the deg- radation of the template RNA for template switching is also sup- ported by the observation that RTs depleted of RNase H activity are almost unable to carry out strand transfer in both in vitro reconstituted reactions and in cell culture.46-48 Reverse transcrip- tases posses two types of RNase H activity, one coupled to DNA synthesis and one independent from it and both can contribute to increase strand transfer by degradation of the template RNA once copied. In particular, the molar excess of RT present in the viral particle with respect to the genomic RNA, provides a reservoir of enzyme that can contribute to donor RNA degradation through the polymerase-independent RNaseH activity. Nevertheless, in vivo and in vitro studies showed that the polymerase-indepen- dent cleavage of the RNA strand only mildly stimulates strand transfer, as the propagation and terminus transfer steps of the process only depend from the polymerase-dependent RNaseH activity.49,50 Subsequent studies have identified RNA structures of template strands of being of particular importance. For instance, the role of RNA structures in recombination has been suggested from the modulation of template switching by RNA chaperons, like the viral NC protein.62 Indeed, in reconstituted in vitro reactions, template switching did not occur in the same positions in the absence or in the presence of the NC protein, suggesting that the NC modulates the secondary structure of the template RNA.63 Later, RNA structures have been shown directly to be involved in favouring template switching in different ways. For example, they have been shown to induce stalling of DNA synthesis.64 However, template switching was not found to be associated with the position at which DNA synthesis paused. According to the proposed model, the role of this RNA structure-induced stalling is to favour strand invasion by the acceptor RNA, by providing more time for the acceptor RNA to invade the nascent DNA- donor RNA heteroduplex.64-66 Sequence matching between the nascent DNA and the accep- tor RNA is therefore extremely important for recombination to occur efficiently. Recombination in Retroviruses s Bioscience. stribute. In addition to the crucial role of sequence identity, the mecha- nism of recombination has been shown to be influenced by sev- eral parameters such as the kinetics of reverse transcription or the secondary structure of the RNA templates. Early studies identi- fied the stalling of reverse transcription at specific positions during synthesis of the first DNA strand as particularly relevant for the occurrence of recombination.47 Several factors can induce pausing during reverse transcription, as the secondary and, possibly, the ternary structure of the template RNA. Specific factors, such as the presence of a guanosine quartet in Gag, can also enhance the local rate of recombination through stalling of the RT.58 A correlation between a reduction of the kinetic of DNA synthesis and increased template switching has also been deduced from the observation that nucleoside analogues-resistant HIV RT that display a decrease in binding affinity for dNTPs, and thereby are expected to have a slower kinetic of reverse transcription, display an increased fre- quency of template switching in vivo.47,59 Other mutants affecting the kinetic of reverse transcription could therefore affect in differ- ent ways the recombination rates.60 Mechanistically, stalling was deemed important to induce a more extensive degradation of the donor RNA by the RNase H.61 ©2011 Lande Do not di It is now widely accepted that, in the vast majority of the cases, recombination occurs during synthesis of the first DNA strand, when the genomic RNA serves as template.3,43-45 The molecu- lar process leading to recombination is a transfer of the nascent DNA strand from one genomic RNA (defined as the donor) onto the other strand (therefore called the acceptor). In retroviruses, a peculiar feature of first strand DNA synthesis is required for recombination to be frequent: the degradation by the RNAse H of the template once it has been copied. This creates an extended region of complementarity between the nascent DNA and the second copy of genomic RNA packaged in the capsid, allowing the annealing of these two nucleic acid moieties. Genetic Flexibility If perpetual change in genomic sequence is synonymous with adaptability to environmental changes, HIV certainly provides a remarkable example of this phenomenon. HIV genetic flexibility was first observed by sequencing viruses isolated from patients and, later, studied through the biochemical characterization of the mechanisms of viral replication by the RT. More recently, the impact of this variability on the generation and selection of viral variants has been addressed more extensively, both in cell culture studies and through the characterization of intra-patient fluctua- tions of the viral sub-populations. While classification of HIV-1 was originally based on sequences of subgenomic regions or individual genes, recent improvements in sequencing methods have made it possible to classify HIV-1 based on full-length genomes or on sequences from multiple subgenomic regions. This has allowed identifying isolates with distinctive parts of their genomes corresponding to different subtypes: these isolates are clearly the products of recombination between parental strains belonging to different subtypes. When a given recombinant form is identified in 3 or more individuals with no direct epidemiological linkage, it is classified as Circulating Recombinant Form (CRF). At present, 48 different CRF are The high genetic variability of HIV is due to several param- eters. The error-prone nature of the viral replication machinery coupled with the short viral replication times are the two factors most accountable for the rapid generation of new genetic variants. 281 RNA Biology referenced in the Los Alamos Sequence Database (http://www. hiv.lanl.gov/content/sequence/HIV/CRFs/CRFs.html) and, when combined, they are responsible for at least 20% of global epidemics HIV, being the locally predominant form in several regions, such as Southeast Asia (CRF01-AE),38-40 and West and West Central Africa (CRF02-AG).41,42 recombination.54 The role of sequence identity has been further underscored when recombination studies where shifted from non- viral model sequences to natural sequences. Genetic Flexibility By crossing increas- ingly divergent HIV isolates in cell culture, the local degree of identity between the templates was shown to be crucial in the region located immediately upstream, in the sense of reverse tran- scription, with respect to the breakpoint position.55-57 Statistical analysis of 162 individual breakpoints generated in the env gene in the absence of selection underlined the presence of an identity threshold below which the probability of template switching dra- matically drops.55 Notably, the distribution of subtypes and recombinant forms in HIV-1 epidemic is highly dynamic: contemporary HIV-1 epidemic are mixtures of recombinants that originated early in the global epidemic and others that are of more recent origin, all of which contribute to the creation of more complex recombi- nant forms that will contribute to the future dynamics of global HIV-1 populations. Recombination in Retroviruses Supporting this view, when the frequency of recombination between templates with varying degrees of sequence identity was investigated using retroviral vectors, a correlation between the level of similarity between the tem- plates and the frequency of recombination was observed.51-53 The extreme case where recombination was studied using templates with no relevant identity indicated that non-homologous recom- bination is hundreds of times less frequent than homologous Finally, recombination hot sites have been observed to co- localize frequently with the presence of structured regions on the genomic RNA, independently from the pausing pattern of RNA Biology 282 Volume 8 Issue 2 reverse transcription.63 In tissue culture studies of a well charac- terized recombination hot spot, it was observed that copy choice occurred mostly in the descending portion of the stem of the hairpin that characterized that hot spot.2,57,67 Template switching was proposed to be favoured by the presence of a double stranded portion at the corresponding position in the acceptor RNA, which would allow strand exchange between donor and accep- tor RNA following a mechanism of branch migration, similar to recombination involving double stranded DNA. Interestingly, destabilizing the RNA structure of the acceptor template led to a three- to four-fold drop in the frequency of recombination, while no effect was observed upon destabilization of the donor RNA, thus demonstrating that the kinetics of DNA synthesis, ongoing on the donor RNA and template switching may be uncoupled.67 mechanistic enhancement of the strand transfer process, RNA secondary structure may also form hot spots of recombination for crossing strains with low global identity: highly structured regions may act as ‘hinges’ for the modular assembly of different parts of the genome. A clear case in favour of this view comes from the crossing of divergent isolates from different HIV-1 groups (group M and O) in cell culture experiments, where recombina- tion was strongly oriented towards more conserved regions of the Rev Responsive Element (RRE).77 s Bioscience. stribute. What is true when comparing individual crosses between iso- lates with varying overall levels of sequence identity, is also true when considering crosses between two isolates and comparing regions of the genome where the level of sequence identity var- ies locally. In this regard, the peculiar organization of the HIV env gene made it an ideal model to study. Using a tissue culture system in the absence of selection on recombinants, two types of observations have been made. Recombination in Retroviruses First, regions of high diversity such as the variable loops of gp120 contained far less breakpoints than those encoding the conserved regions of the protein.55,77 Second, strong disparities were observed for comparable degree of sequence identity in conserved regions, indicating that, among these portions of the gene, parameters other than the level of sequence identity modulated recombination.78 The determination of a model of secondary structure for the HIV genome79 allowed to push further this analysis and to directly link enhanced recom- bination rates to higher secondary structure organization. It was indeed shown that regions of the genomic RNA with a high pro- portion of residues involved in the formation of secondary struc- tures contained significantly more breakpoints.78 The extent of RNA structures along HIV genome seems to provide us with a relatively accurate picture of the pattern of recombinant genomes generated by the mechanism of recombination. However, only a fraction of the recombinant HIV genomes produced will be able to expand in the infected organism and have the opportunity to become epidemiologically relevant. There is where selection comes into play. It is important to remember, however, that the findings reviewed above come from studies of specific cases, and that the ability of these models to explain the natural process of recombi- nation that is ongoing in HIV infections in human hosts remains an open question. When RNA Templates for Recombination Become HIV Genomic RNAs ©2011 Lande Do not d In nature, recombination occurs between viruses with differ- ent degrees of genetic divergence. HIV undoubtedly presents a high degree of recombination between viral quasispecies evolv- ing within each infected individual68-75 (also, De Crignis and Graziosi, personal communication). However, the most visible effect of recombination involves the crossing of divergent strains of the AIDS pandemics and highlights the ability of this process for creating phylogenetic shortcuts. Under these different situ- ations (recombination between low diversity vs. high diversity strains), the various parameters driving recombination might have different weights. For the crossing of closely related parental RNA genomes, which is the most frequent situation for viruses evolving in an infected individual, the homogeneous local degree of sequence identity along the genome allows strand transfer to occur with nearly similar chances all along the molecule. Under these condi- tions, the parameters that have been shown to be important for recombination in purely mechanistic studies using highly similar templates, such as pause sites and RNA structures, may be pre- dominant. Although, presently, no characterization of the molec- ular determinants of intrasubtype breakpoint location has been carried out, a recent cell culture study of intrasubtype B recombi- nants, has shown a non-homogeneous distribution of recombina- tion breakpoints in the pol-coding region,76 which suggests that determinants of local fluctuations in recombination rates most likely exist also for intrasubtype recombinants. www.landesbioscience.com RNA Structures as Recombination Signposts: Selection on the RNA Element Furthermore, when the products of recombinant env genes gen- erated in absence of selection were tested in functionality assays, the general trend was of decreased functionality with respect to the parental strains, thus underlining the importance of con- straints imposed on the functionality of the recombinant forms of this gene despite its legendary genetic variability.77 ©2011 Lande Do not d Structured regions of the genome are means for viruses to con- vey functional information. For example, hairpins containing the signals for packaging of the genomic RNA have been well-char- acterized in the 5' end of the genome, as well as two other struc- tures crucial for viral gene expression: the trans-active responsive element (TAR) and RRE. TAR is the sequence recognized for transactivation of transcription by the viral protein Tat while RRE is essential for exporting non-spliced and partially-spliced RNAs from the nucleus to the cytoplasm. These RNA structures are sites of frequent recombination,77,80 with the consequence that these elements may be constituted of two sequences of different phylogenetic origins in the recombinant progeny. Misfolded or destabilized RNA structures might not be able to fulfil their roles, might these be regulation of the translational machinery, interaction with proteins that regulate the viral cycle, or others.81 Furthermore, both TAR and RRE interact with proteins encoded by a different part of the genome from the ones where they are located, and any recombination breakpoint occurring between the regions constituting the RNA element (TAR or RRE) and those encoding the protein that binds this element (Tat or Rev, respectively) might present the risk of perturbing coevolved inter- actions. However, these functional RNA elements appear very conserved, even for distantly related strains. Associated to the structural tolerance of RNA structures, this conservation could be strong enough to significantly limit the likelihood of recombi- nation generating defective structural elements. This hypothesis is supported by the results obtained from experimental assays in which the functionality of not only intersubtype recombinants but also intergroup M/O recombinant RRE was tested. Indeed, all recombinant RRE that were tested displayed levels of func- tionality comparable to the parental sequences.77 The frequent induction of recombination in other portions of the structured genomic RNA have been highlighted with the example of the DIS sequence,82 but no functional characterization of the result- ing recombinants is currently available to address the issue of the functional tolerance of these RNA structure to recombination. s Bioscience. Recombination, Viral Evolution and RNA Structures In HIV, the high rate of single residue substitutions occurring during each replication, coupled with the fast viral turnover, cre- ates very favourable conditions for maximizing the potentials of recombination as a means to spread multiple genetic polymor- phisms across the viral population. Selection of Recombinants Expectedly, recombination occurs where the most favourable mechanistic conditions are encountered along the genome, and this occurs irrespective of the consequences on the functionality of the resulting recombinant product. Proteins and RNA func- tional elements need to fold into specific conformations to be functional; however, concatenated structural components from different genetic origins might not be able to fold into optimally functional structures. Depending on the position in the genome where recombination occurs, different functional elements will be involved, each with its own specific tolerance for preserving its functionality in case of recombination. The localization of RNA structures that, as discussed above, also constitute recom- bination hot regions, is therefore important for selection on the recombinant forms they can generate. Selection may come into play at two major levels: the functionality of the recombinant RNA element itself and the functionality of the recombinant proteins generated. As the genetic distance between the two parental strains increases, the presence of secondary structure elements in spe- cific regions of the genome might play a dual role with regard to recombination. Since these motifs are generally conserved, they form islands of local higher degree of sequence identity, where strand transfer is much more likely to occur than in the rest of the genome, relatively more divergent. Combined with their RNA Biology 283 RNA Structures as Recombination Signposts: Selection on the RNA Element istribute. The fate of the recombinant viral progeny appears also strongly conditioned by the position of the breakpoint. Indeed, studies on non-viral proteins (SCHEMA directed evolution of β-Lactamases),84 showed that the position of the breakpoint in the gene strongly affects the recombinant functionality. Importantly, breakpoints close to the boundaries of autono- mously folding protein domains were shown to present a lower risk of disturbing structural interactions required for the proper folding and function of the recombinant proteins. Recent obser- vations suggest a role of the pattern of RNA structures in HIV genome in regard to the selection on recombinant proteins. The publication of the architecture of the whole genome of the NL4.3 strain of HIV79 has indeed led to the identification of numerous new RNA structures. Intriguingly, the distribution of structured RNA elements appears to be correlated with the arrangement of the genes along the genome, as well as by the structural organiza- tion of the individual proteins.78 Specifically, stable RNA second- ary structures were found at the junctions between most HIV-1 proteins domains and it has been suggested that these structures could modulate ribosome processivity and, consequently, facili- tate the folding of individual protein domains. A striking “side effect” of the specific distribution of RNA secondary structures close to gene or protein domain borders observed by Watts and co-workers would be to enhance strand transfer rates specifically in regions where recombination is less likely to generate genomes encoding dysfunctional proteins.78 RNA Structures as Recombination Signposts: Selection on Proteins The efficiency at which functional diversity is generated by this process, evolved to be highly effective for the peculiar viral replication strategy, is however difficult to evaluate quantita- tively. In that regard, the characterization of recombinant forms generated in single cycle experiment provides a unique insight into the role of recombination in accessing distant regions of the functional sequence space. This evolutionary process is in fact- much more conservative than the introduction of random muta- tions.83 Indeed, as recombination joins patterns of amino acids substitutions already proved to be compatible in their respective parental structures, these mutations are less likely to be incom- patible in the new backbone than randomly introduced muta- tions. Consequently, when sequences encoding divergent but related proteins recombine, large distances may be travelled in sequence space relative to random mutation without disturbing fuction and/or structure. Concerning selection at the protein level, simulations on model proteins suggest that mutation-tolerant proteins are also recom- bination-tolerant.83 While the high diversity observed for HIV could lead one to think that viral proteins could be extremely tolerant to recombination, experimental studies indicate that this is not necessarily the case, in particular for intersubtypes crossings. Notably, directed evolution experiments on non-viral proteins have shown a negative correlation between recombina- tion tolerance and parental sequence divergence.83 When intra and intersubtypes recombinant pol genes were generated in single cycle assay, both populations showed complex patterns of break- points.76 However, only the intersubtype recombinant population showed a strong decrease of diversity in multiple-cycle assays. Volume 8 Issue 2 Volume 8 Issue 2 RNA Biology 284 well adapted to tolerate recombination between both closely and distantly related strains. At the scale of the genome, for distantly related strains of HIV, the particular disposition of RNA structures close to gene and domain borders appears to mechanistically favour the exchange of proteins blocks, further limiting the risk of generating non functional products. It is of particular interest to note that the extreme case of such genetic compartmentalization is observed in segmented viral genomes, where the genetic reassortment proceeds through the redistribution of various portions of the genome encoded by physically distinct molecules. While mech- anistically limiting the potential sequence space explored, this RNA-mediated orientation of recombination is likely to actually enhance the evolutionary value of this process for poorly related viruses. References 29. Labrosse B, Morand-Joubert L, Goubard A, Rochas S, Labernardiere JL, Pacanowski J, et al. Role of the enve- lope genetic context in the development of enfuvirtide resistance in human immunodeficiency virus type 1-infected patients. J Virol 2006; 80:8807-19. 15. Chen P, Hubner W, Spinelli MA, Chen BK. Predominant mode of human immunodeficiency virus transfer between T cells is mediated by sustained Env- dependent neutralization-resistant virological synapses. 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https://openalex.org/W3091866416	https://www.dora.lib4ri.ch/psi/islandora/object/psi%3A34719/datastream/PDF/Henthorn-2020-Mapping_the_future_of_particle-%28published_version%29.pdf	English	null	Mapping the Future of Particle Radiobiology in Europe: The INSPIRE Project	Frontiers in physics	2,020	cc-by	9,465	Mapping the Future of Particle Radiobiology in Europe: The INSPIRE Project Nicholas T. Henthorn 1,2†, Olga Sokol 3†, Marco Durante 3,4, Ludovic De Marzi 5, Frederic Pouzoulet 6, Justyna Miszczyk 7, Pawel Olko 7, Sytze Brandenburg 8,9, Marc Jan van Goethem 8,9, Lara Barazzuol 9,10, Makbule Tambas 9, Johannes A. Langendijk 9, Marie Davídková 11, Vladimír Vondrá ˆcek 12, Elisabeth Bodenstein 13, Joerg Pawelke 13,14, Antony J. Lomax 15,16, Damien C. Weber 15,17,18, Alexandru Dasu 19,20, Bo Stenerlöw 20, Per R. Poulsen 21, Brita S. Sørensen 21, Cai Grau 21, Mateusz K. Sitarz 21, Anne-Catherine Heuskin 22, Stephane Lucas 22, John W. Warmenhoven 1,2, Michael J. Merchant 1,2, Ran I. Mackay 1,23 and Karen J. Kirkby 1,2 REVIEW REVIEW published: 06 October 2020 doi: 10.3389/fphy.2020.565055 Keywords: proton therapy, radiotherapy, radiobiology, beamline, irradiation Edited by: Pablo Cirrone, Laboratori Nazionali del Sud (INFN), Italy Correspondence: Marco Durante m.durante@gsi.de †These authors have contributed equally to this work †These authors have contributed equally to this work Specialty section: This article was submitted to Medical Physics and Imaging, a section of the journal Frontiers in Physics Received: 23 May 2020 Accepted: 02 September 2020 Published: 06 October 2020 Citation: Henthorn NT, Sokol O, Durante M, De Marzi L, Pouzoulet F, Miszczyk J, Olko P, Brandenburg S, van Goethem MJ, Barazzuol L, Tambas M, Langendijk JA, Davídková M, Vondrá ˆcek V, Bodenstein E, Pawelke J, Lomax AJ, Weber DC, Dasu A, Stenerlöw B, Poulsen PR, Sørensen BS, Grau C, Sitarz MK, Heuskin A-C, Lucas S, Warmenhoven JW, Merchant MJ, Mackay RI and Kirkby KJ (2020) Mapping the Future of Particle Radiobiology in Europe: The INSPIRE Project. Front. Phys. 8:565055. doi: 10.3389/fphy.2020.565055 Specialty section: This article was submitted to Medical Physics and Imaging, a section of the journal Frontiers in Physics Received: 23 May 2020 Accepted: 02 September 2020 Published: 06 October 2020 Particle therapy is a growing cancer treatment modality worldwide. However, there still remains a number of unanswered questions considering differences in the biological response between particles and photons. These questions, and probing of biological mechanisms in general, necessitate experimental investigation. The “Infrastructure in Proton International Research” (INSPIRE) project was created to provide an infrastructure for European research, unify research efforts on the topic of proton and ion therapy across Europe, and to facilitate the sharing of information and resources. This work highlights the radiobiological capabilities of the INSPIRE partners, providing details of physics (available particle types and energies), biology (sample preparation and post-irradiation analysis), and researcher access (the process of applying for beam time). The collection of information reported here is designed to provide researchers both in Europe and worldwide with the tools required to select the optimal center for their research needs. We also highlight areas of redundancy in capabilities and suggest areas for future investment. Edited by: Citation: Henthorn NT, Sokol O, Durante M, De Marzi L, Pouzoulet F, Miszczyk J, Olko P, Brandenburg S, van Goethem MJ, Barazzuol L, Tambas M, Langendijk JA, Davídková M, Vondrá ˆcek V, Bodenstein E, Pawelke J, Lomax AJ, Weber DC, Dasu A, Stenerlöw B, Poulsen PR, Sørensen BS, Grau C, Sitarz MK, Heuskin A-C, Lucas S, Warmenhoven JW, Merchant MJ, Mackay RI and Kirkby KJ (2020) Mapping the Future of Particle Radiobiology in Europe: The INSPIRE Project. Front. Phys. 8:565055. doi: 10.3389/fphy.2020.565055 Edited by: Edited by: Federico Giove, Centro Fermi - Museo storico della ﬁsica e Centro studi e ricerche Enrico Fermi, Italy 1 Division of Cancer Sciences, Faculty of Biology, Medicine and Health, School of Medical Sciences, The University of Manchester, Manchester, United Kingdom, 2 Manchester Academic Health Science Centre, The Christie NHS Foundation Trust, Manchester, United Kingdom, 3 Department of Biophysics, GSI Helmholtzzentrum für Schwerionenforschung, Darmstadt, Germany, 4 Department of Condensed Matter Physics, Technische Universität Darmstadt, Darmstadt, Germany, 5 Radiation Oncology Department, Institut Curie, PSL Research University, Paris, France, 6 Translational Research Department, Experimental Radiotherapy Platform, Institut Curie, PSL Research University, Orsay, France, 7 Department of Experimental Physics of Complex Systems, Institute of Nuclear Physics Polish Academy of Sciences, Krakow, Poland, 8 KVI-Center for Advanced Radiation Technology, University of Groningen, Groningen, Netherlands, 9 Department of Radiation Oncology, University Medical Center Groningen, University of Groningen, Groningen, Netherlands, 10 Section of Molecular Cell Biology, Department of Biomedical Sciences of Cell and Systems, University Medical Center Groningen, University of Groningen, Groningen, Netherlands, 11 Department of Radiation Dosimetry, Nuclear Physics Institute of the CAS, Prague, Czechia, 12 Proton Therapy Center Czech, Prague, Czechia, 13 OncoRay – National Center for Radiation Research in Oncology, Faculty of Medicine and University Hospital Carl Gustav Carus, Helmholtz-Zentrum Dresden – Rossendorf, Technische Universität Dresden, Dresden, Germany, 14 Helmholtz-Zentrum Dresden – Rossendorf, Institute of Radiooncology - OncoRay, Dresden, Germany, 15 Centre for Proton Therapy, Paul Scherrer Institute, Villigen, Switzerland, 16 Department of Physics, ETH, Zurich, Switzerland, 17 Department of Radiation Oncology, University of Zurich, Zurich, Switzerland, 18 Department of Radiation Oncology, University of Bern, Bern, Switzerland, 19 The Skandion Clinic, Uppsala, Sweden, 20 Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden, 21 Danish Center for Particle Therapy, Aarhus University Hospital, Aarhus, Denmark, 22 LARN-Laboratory, Namur Research Institute for Life Sciences (NARILIS), University of Namur, Namur, Belgium, 23 Christie Medical Physics and Engineering, The Christie NHS Foundation Trust, Manchester, United Kingdom Reviewed by: Michela Maraﬁni, Centro Fermi - Museo storico della ﬁsica e Centro studi e ricerche Enrico Fermi, Italy Giuseppe A. INTRODUCTION 2) Enabling access to research infrastructure for researchers in both the public and private sector. 2) Enabling access to research infrastructure for researchers in both the public and private sector. There is a growing investment in proton and heavy ion therapy worldwide, with 89 proton centers and 12 carbon centers currently in clinical operation [according to the Particle Therapy Co-Operation Group (PTCOG)] [1]. Of these worldwide facilities, 31 proton centers (∼35%) and four carbon centers (∼33%) are located in Europe [2]. Despite the increasing adoption of particle therapy there remains a number of unanswered questions about this relatively new treatment modality [3]. These questions range widely in scope and include physical (e.g., range uncertainties or organ motion), biological (e.g., uncertainties in relative biological eﬀectiveness and lack of clinically relevant in vivo data), and societal aspects (e.g., cost-eﬀectiveness and radiotherapy demand) [4]. Many clinical centers oﬀer beam time for research activities to address some of these questions [5]. However, access and utilization of this beam time can be diﬃcult due to a lack of supply and/or funding. Rectifying this situation requires targeted eﬀorts from both researchers and funders alike. 3) Providing training for the next generation of researchers in the ﬁeld. 4) Facilitating knowledge exchange to promote best research practices throughout Europe. g 5) Developing joint research activities (JRAs) that will improve the facilities available within the infrastructure. 6) Developing JRAs in ﬁelds where technological challenges exist to improve European competitiveness. 7) Developing an innovation pipeline to translate research into clinical practice and industrial products. 8) To conduct research within the principles of responsible research and innovation. The project is comprised of 17 European partners, 11 of which oﬀer beam time through transnational access (TNA) (Table 1); a complete list of the INSPIRE partners can be found at https:// protonsinspire.eu/. Further to the partners discussed in this work, the University of Namur (Belgium) is also an INSPIRE partner taking part in radiobiological research, but with their nearby partner center under development does not oﬀer TNA through INSPIRE. However, once operational their resources will be available outside of the current INSPIRE project. Most of these partners are either clinical centers or have very close connections to clinical centers (Figure 1), for example the radiobiological capabilities of CHRISTIE and UNIMAN are shared. Citation: October 2020 \| Volume 8 \| Article 565055 Frontiers in Physics \| www.frontiersin.org 1 INSPIRE Radiobiology Centers Henthorn et al. INTRODUCTION A close clinical link is essential to aid the design of the research at inception and to ensure its relevance and future translation to the clinic. The European project “Infrastructure in Proton International Research” (INSPIRE) was created to allow researchers across Europe access to “state-of-the-art” research capabilities in centers for proton therapy. In addition, multi-ion research centers (research facility of UMCG, Groningen, the Netherlands; GSI, Darmstadt, Germany) augment the particle research portfolio. INSPIRE aims to integrate research activities in protons (and heavy ions) across Europe through eight objectives: 1) Developing new infrastructure by bringing together clinical, academic, and industrial research activities. 1) Developing new infrastructure by bringing together clinical, academic, and industrial research activities. TABLE 1 \| The INSPIRE partners offering equipment and support for radiobiological experiments through transnational access. Center Abbreviation Location Website Aarhus University AU Aarhus, Denmark https://www.en.auh.dk/departments/the-danish-centre-for- particle-therapy/ The Christie NHS foundation trust CHRISTIE Manchester, UK https://www.christie.nhs.uk GSI Helmholtz center for heavy Ion research GSI Darmstadt, Germany https://www.gsi.de/work/forschung/biophysik.htm The Henryk Niewodnicza ´nski institute of nuclear physics polish academy of sciences IFJ PAN Kraków, Poland https://inspire.ifj.edu.pl/en/index.php/dostep-do- infrastruktury-badawczej/ Curie institute Institut curie Paris, France https://institut-curie.org/page/research-and-development- proton-therapy-center Nuclear physics institute of the Czech academy of sciences NPI-CAS Prague, Czech Republic http://www.ujf.cas.cz/en/ Paul Scherrer institute PSI Zurich, Switzerland https://www.psi.ch/en Skandion clinic Skandion Uppsala, Sweden https://skandionkliniken.se/ Technical University of Dresden TUD Dresden, Germany https://www.oncoray.de/research/offer-for-users/ University medical center Groningen UMCG Groningen, Netherlands Clinical facility: https://www.umcgradiotherapie.nl/en/umc-groningen- department-of-radiation-oncology Research facility: https://www.rug.nl/kvi-cart/research/facilities/agor/ University of Manchester UNIMAN Manchester, UK https://www.bmh.manchester.ac.uk/research/domains/ cancer/proton/ TABLE 1 \| The INSPIRE partners offering equipment and support for radiobiological experiments through transnational access. Frontiers in Physics \| www.frontiersin.org October 2020 \| Volume 8 \| Article 565055 2 Henthorn et al. INSPIRE Radiobiology Centers FIGURE 1 \| European clinical proton therapy centers (closed triangle, 26 centers), carbon therapy centers (closed circle, 4 centers), and INSPIRE partners offering radiobiological TNA (closed squares, 11 centers—there is some overlap between centers). Open symbols show centers currently in the planning stage or under construction. Information is from the PTCOG website [1]. FIGURE 1 \| European clinical proton therapy centers (closed triangle, 26 centers), carbon therapy centers (closed circle, 4 centers), and INSPIRE partners offering radiobiological TNA (closed squares, 11 centers—there is some overlap between centers). Open symbols show centers currently in the planning stage or under construction. Information is from the PTCOG website [1]. Frontiers in Physics \| www.frontiersin.org INTRODUCTION Further to the information hosted by each institute’s website, and the information presented in this work, the following references give more information and available setups for Institut Curie [6–8], TUD [9–16], IFJ PAN [17], UMCG [18–24], and GSI [25–31]. best practices for proton radiobiology research and build upon previous work on the topic [36]. Alongside coordinated research INSPIRE also seeks to improve the infrastructure available to European researchers through its TNA. Many research centers have invested signiﬁcantly to develop their research, constructing accelerators, beamlines, and purchasing experimental equipment. INSPIRE also continually upgrades its research capabilities by taking research developed through JRA and making it available to the wider research community via TNA. This means that INSPIRE is able to oﬀer the very latest technology and capabilities. Through INSPIRE we are able to investigate important research questions together and beneﬁt from cross-validation. An immediate example is the variability in data for proton relative biological eﬀectiveness (RBE) that has been seen in the literature over the years [32–35]. A coordinated eﬀort amongst the INSPIRE partners is allowing this variability to be investigated both computationally and experimentally, and the results made available to researchers across Europe through INSPIRE’s experimental and modeling JRA. This systematic and coordinated approach will highlight factors leading to variation and propose mitigation strategies for future studies. These mitigation strategies will help to develop TNA provides researchers an opportunity to access beam time and funding for experiments at INSPIRE partners. The beam time is oﬀered to all researchers and is not limited to INSPIRE partners. Furthermore, whilst the beam time is largely accessible for European researchers, up to 30% of the hours are available to researchers outside the EU. The application process October 2020 \| Volume 8 \| Article 565055 3 INSPIRE Radiobiology Centers Henthorn et al. is managed through the INSPIRE website (https://protonsinspire. eu). Prior to submitting the application through the online form, the researchers are advised to contact the representative of the relevant partner site to discuss the technical details of their proposed experiment. Before being transferred to an independent international user selection panel (USP), the reﬁned application, submitted via the online form, is ﬁrst assessed to ensure that the requested TNA site has the capacity and infrastructure to perform the experiment. Afterwards, the application is evaluated by at least two members of the USP for its technical and scientiﬁc excellence, as well as future potential and impact. INTRODUCTION Priority is given to users who have not had access to the TNA before. The INSPIRE website contains details about each center, links to websites, and contact information for general enquiries aimed to aid the potential researcher. only the beamline for the sample irradiation, but also of the available equipment and capabilities of the biological laboratories on site. The latter are essential for the sample preparation and post-processing. In this work, we aim to provide comprehensive information on the facilities available across INSPIRE. We specify details of the “physics,” including location, beamlines, particle types, energies, and ﬁeld sizes. We specify details of the “logistics,” including details of sample types, positioning, and automation. We specify details of the “biology,” including the available equipment for sample preparation and post-irradiation processing. Finally, we discuss future perspectives for ongoing development and further investment. The details provided here act as a resource for the potential researcher to select the optimal center for their experimental needs. However, it should be noted that there is often ﬂexibility in many of the aspects we report. As such the information we provide should be used as a guide and more speciﬁc details can be obtained through communication with a speciﬁc partner or through INSPIRE’s help desk. It is apparent that the capabilities, at least in terms of “physics,” between many partners are similar. This level of redundancy is desirable, enabling repetition to ensure scientiﬁc rigor, however, establishing these centers requires a large investment and The information provided in this paper acts as a corollary to the INSPIRE website, where up-to-date information is maintained. Here, we provide details of the TNA radiobiology capabilities of each INSPIRE partner. Similar information, at least in terms of the physics capabilities, has previously been presented by the European Particle Therapy Network [37] and can be used alongside this work. Planning of a radiobiological experiment requires the knowledge of not FIGURE 2 \| INSPIRE partners offering radiobiological investigation with particles. The quoted energies are as extracted from the beamlines, lower energies are available with beam degraders. Centers offering both in vitro and in vivo experiments are marked with orange circles, while those offering only in vitro experiments are shown as blue squares. Protons, Helium, Carbon, and Oxygen ions are available at the research facility of UMCG. Protons and ions up to Uranium ions are available at GSI. INTRODUCTION FIGURE 2 \| INSPIRE partners offering radiobiological investigation with particles. The quoted energies are as extracted from the beamlines, lower energies are available with beam degraders. Centers offering both in vitro and in vivo experiments are marked with orange circles, while those offering only in vitro experiments are shown as blue squares. Protons, Helium, Carbon, and Oxygen ions are available at the research facility of UMCG. Protons and ions up to Uranium ions are available at GSI October 2020 \| Volume 8 \| Article 565055 Frontiers in Physics \| www.frontiersin.org 4 Henthorn et al. INSPIRE Radiobiology Centers FIGURE 3 \| Proton energies available at INSPIRE partners as extracted from the accelerator. The highest proton energy is available at GSI (up to 4.5 GeV). The lowest energies are available at the UMCG research facility (15 MeV) and Institut Curie (20 MeV). The overlapping region (shaded area) is between 120 and 190 MeV. Energies can be further degraded in front of the sample. FIGURE 3 \| Proton energies available at INSPIRE partners as extracted from the accelerator. The highest proton energy is available at GSI (up to 4.5 GeV). The lowest energies are available at the UMCG research facility (15 MeV) and Institut Curie (20 MeV). The overlapping region (shaded area) is between 120 and 190 MeV. Energies can be further degraded in front of the sample. through INSPIRE they are able to work eﬀectively together to ensure optimum utilization. reaching up to 1 GeV/u for heavy ions and 4.5 GeV/u for protons, with relevance to proton radiography [40] experiments, while most of the other institutes are limited to a maximum of 230–240 MeV/u. The lowest possible proton energies are oﬀered at the research facility of UMCG (15 MeV) and Institut Curie (20 MeV). Energies can be further degraded before the sample to investigate increased proton linear energy transfer, with a relevance for end of range eﬀects. Access to even lower energies can be obtained through the EU project RADIATE [41]. Frontiers in Physics \| www.frontiersin.org PHYSICS – LOCATION, BEAMLINES, PARTICLES, ENERGIES, AND FIELDS A researcher often faces large heterogeneity when performing experiments between centers, with diﬀerences in protocol, setup, irradiation, and sample processing. Despite this there are a number of overlaps in beam properties and possible experiments between centers. Figure 2 shows a summary of capabilities for the INSPIRE TNA partners. Eight TNA providers have a dedicated research room. This can be useful for studies that require longer irradiations and/or longer follow-up, it also gives more freedom to experiments that require a complex or non-standard sample setup. However, the cost of such studies should always be considered. Whilst the sample may be able to remain in the room post-irradiation this will often inactivate the room using valuable resources. A shared room has the downside of limited usage, due to clinical commitments, although it has the added beneﬁt of rigorous quality assurance to a clinical standard. However, it should be noted that all partners undertake measures to ensure dosimetry and quality of beam delivery in their research rooms. TNA providers mainly cover central and northern Europe, with a similar distribution to clinical centers (Figure 1). Geographic positioning of centers is an important factor to minimize both travel expenses and logistics. A new initiative with the South East European International Institute for Sustainable Technologies (SEEIST) [38, 39] aims to enable researchers from the south east of Europe to access INSPIRE’s capabilities while they are developing their own facilities. Figure 4 shows examples of beamlines for the CHRISTIE + UNIMAN, Skandion, the research facility of UMCG, TUD, GSI, and Institut Curie partners. All of the TNA providers can supply protons, with two centers, GSI and the research facility of UMCG, additionally oﬀering other ion types of clinical interest, such as carbon, helium, or oxygen. As can be seen from Figure 3, in general, the energies available from the accelerator are similar between providers. The most overlapping energy region is between 120 and 190 MeV— experiments at this energy can be done at all of the partner centers. The highest possible energies can be achieved at GSI, There is a range of maximum available scanned ﬁeld sizes across the INSPIRE partners, shown in Figure 5. Six partners, PSI, Skandion, NPI-CAS, IFJ PAN, AU, and Institut Curie, oﬀer the same ﬁeld size (30 × 40 cm2). PHYSICS – LOCATION, BEAMLINES, PARTICLES, ENERGIES, AND FIELDS Institut Curie shows three irradiation rooms; “Room Y1” —horizontal beam up to 201 MeV (left), “Room Y2” —horizontal beam up to 76 MeV (right), and “IBA Room” —gantry up to 230 MeV (bottom). FIGURE 4 \| Beamline setup for (A) UNIMAN + CHRISTIE, (B) Skandion, (C) the research facility of UMCG, (D) TUD, (E) GSI, and (F) Institut Curie. UNIMAN has a beamline leading to a Varian scanning nozzle, samples are placed in front of the nozzle (pictured is a hypoxia cabinet). Skandion has a beamline leading to an IBA scanning nozzle, samples are placed on an adjustable table in front of the nozzle. The research facility of UMCG has a ﬂexible beamline setup using optical benches; picture shows a study on the effect of magnetic ﬁelds in combination to proton irradiation [23]. TUD has two beamlines in the dedicated experimental room, one with a pencil beam scanning nozzle (left) and one static beamline (right). In the picture, setups with water tank and beam dump at the scanning beamline and passive double scattering setup for radiobiological experiments at the static beamline are shown. GSI shows the beamline setup for “Cave A,” equipped with the robotic system for sample exchange. Institut Curie shows three irradiation rooms; “Room Y1” —horizontal beam up to 201 MeV (left), “Room Y2” —horizontal beam up to 76 MeV (right), and “IBA Room” —gantry up to 230 MeV (bottom). orientation (40 × 30 cm2). All partners oﬀer a ﬁeld size large enough to irradiate most in vitro sample types, such as tissue culture ﬂasks or microplates. The ﬁeld size may become a limitation for larger non-standard samples, or simultaneous irradiation of multiple samples. Though in some cases the ﬁeld size may be increased by introducing scatterers. reference radiation may lead to slight diﬀerences in relative eﬀect measurements, making inter-center comparisons more complicated. However, it should be noted that this is a problem for radiobiology in general and is not limited to INSPIRE partners [42]. Choice of reference radiation is an important aspect in general for radiobiology. The biological eﬀects of protons are often quoted relative to the more familiar photon case, most notably the relative biological eﬀectiveness for cell kill. A variety of reference photon qualities are used between the INSPIRE partners. PHYSICS – LOCATION, BEAMLINES, PARTICLES, ENERGIES, AND FIELDS Several partners have the possibility to choose between clinical LINACs and kilovoltage X-ray machines (CHRISTIE + UNIMAN, TUD, NPI-CAS, Institut Curie, UMCG), whilst the capabilities of others are more limited. The diﬀerence in Frontiers in Physics \| www.frontiersin.org PHYSICS – LOCATION, BEAMLINES, PARTICLES, ENERGIES, AND FIELDS TUD and CHRISTIE + UNIMAN oﬀer the same ﬁeld size but in the landscape October 2020 \| Volume 8 \| Article 565055 5 Henthorn et al. INSPIRE Radiobiology Centers FIGURE 4 \| Beamline setup for (A) UNIMAN + CHRISTIE, (B) Skandion, (C) the research facility of UMCG, (D) TUD, (E) GSI, and (F) Institut Curie. UNIMAN has a beamline leading to a Varian scanning nozzle, samples are placed in front of the nozzle (pictured is a hypoxia cabinet). Skandion has a beamline leading to an IBA scanning nozzle, samples are placed on an adjustable table in front of the nozzle. The research facility of UMCG has a ﬂexible beamline setup using optical benches; picture shows a study on the effect of magnetic ﬁelds in combination to proton irradiation [23]. TUD has two beamlines in the dedicated experimental room, one with a pencil beam scanning nozzle (left) and one static beamline (right). In the picture, setups with water tank and beam dump at the scanning beamline and passive double scattering setup for radiobiological experiments at the static beamline are shown. GSI shows the beamline setup for “Cave A,” equipped with the robotic system for sample exchange. Institut Curie shows three irradiation rooms; “Room Y1” —horizontal beam up to 201 MeV (left), “Room Y2” —horizontal beam up to 76 MeV (right), and “IBA Room” —gantry up to 230 MeV (bottom). FIGURE 4 \| Beamline setup for (A) UNIMAN + CHRISTIE, (B) Skandion, (C) the research facility of UMCG, (D) TUD, (E) GSI, and (F) Institut Curie. UNIMAN has a beamline leading to a Varian scanning nozzle, samples are placed in front of the nozzle (pictured is a hypoxia cabinet). Skandion has a beamline leading to an IBA scanning nozzle, samples are placed on an adjustable table in front of the nozzle. The research facility of UMCG has a ﬂexible beamline setup using optical benches; picture shows a study on the effect of magnetic ﬁelds in combination to proton irradiation [23]. TUD has two beamlines in the dedicated experimental room, one with a pencil beam scanning nozzle (left) and one static beamline (right). In the picture, setups with water tank and beam dump at the scanning beamline and passive double scattering setup for radiobiological experiments at the static beamline are shown. GSI shows the beamline setup for “Cave A,” equipped with the robotic system for sample exchange. LOGISTICS – SAMPLES, POSITIONING, AND AUTOMATION The mode of sample irradiation is an important consideration, including sample orientation and possibility of automated handling. Monolayers of cells, grown in a ﬂask or microplate, should not be free from media for a long duration of time to avoid drying. As such, several centers, particularly with horizontal October 2020 \| Volume 8 \| Article 565055 Frontiers in Physics \| www.frontiersin.org 6 INSPIRE Radiobiology Centers Henthorn et al. FIGURE 5 \| Maximum available scanned ﬁeld sizes of the INSPIRE partners, ranging from 10 × 10 cm2 to 30 × 40 cm2. Larger ﬁeld sizes may be available by introducing scatterers. FIGURE 5 \| Maximum available scanned ﬁeld sizes of the INSPIRE partners, ranging from 10 × 10 cm2 to 30 × 40 cm2. Larger ﬁeld sizes may be available by introducing scatterers. FIGURE 5 \| Maximum available scanned ﬁeld sizes of the INSPIRE partners, ranging from 10 × 10 cm2 to 30 × 40 cm2. Larger ﬁeld sizes may be available by introducing scatterers. able scanned ﬁeld sizes of the INSPIRE partners, ranging from 10 × 10 cm2 to 30 × 40 cm2. Larger ﬁeld sizes may be available by Figure 6 shows examples of sample presentation to the beam at Christie + UNIMAN, the research facility of UMCG, Institut Curie, GSI, and AU. The system at CHRISTIE + UNIMAN (Figure 6A) employs a 6-axis robot mounted inside a hypoxia end station. The space limitations of the hypoxia cabinet mean that at most a mix of up to 36 samples can be housed at a time. The ﬁngers of the robot are designed for T75 ﬂasks or 96-well-plates, limiting the sample type. However, other samples can be used so long as they have the same footprint as a 96-well-plate or through use of customized sample holders, alternatively a large range of samples can be used without the robot. Similar to the CHRISTIE + UNIMAN system, the GSI system (Figure 6D) holds samples in the horizontal position lifting them to the beam for irradiation. This change in orientation minimizes the time that cells are free from media, ensuring a good cellular environment and avoiding beamlines, employ automated sample handling. Here, the sample can remain in a horizontal orientation and is lifted up only when presented to the beam for irradiation. Automated sample handling also has the added beneﬁts of improving repeatability and minimizing access to the irradiation room, increasing sample throughput. Frontiers in Physics \| www.frontiersin.org BIOLOGY – SAMPLE PREPARATION AND PROCESSING Alongside the physics capabilities, the biological equipment available at a center will often deﬁne the type and complexity of experiments that are possible. This impacts both the pre- irradiation sample preparation and post-irradiation analysis. For some experiments it is not possible to prepare samples prior to transport to the irradiating center. Similarly, it is not always possible to ﬁx samples following irradiation ready for transport to the home institute. Table 3 gives details of the in vitro biological equipment available at INSPIRE partners. In most cases the equipment detailed in Table 3 is shared between the INSPIRE partner and other groups at the same institute. Therefore, these details should be used as a guide for maximum available equipment. Similarly, extra resources may be available at a partner’s sister institute. Researchers requiring the use of any of this equipment should discuss their needs with the relevant partner. All centers can offer a horizontal beamline, with the four able to irradiate samples from above or at user-deﬁned angles using a gantry. There is ﬂexibility in sample types, but the majority of centers have the ability to irradiate ﬂasks and well-plates. All centers can offer a horizontal beamline, with the four able to irradiate samples from above or at user-deﬁned angles using a gantry. There is ﬂexibility in sample types, but the majority of centers have the ability to irradiate ﬂasks and well-plates. Common amongst all centers is the availability of ﬂow hoods and incubators, with TUD oﬀering the largest capacity for sample preparation and storage. At the moment, only one center, UNIMAN, has a hypoxia station for irradiation of samples under variable oxygen tension. This oﬀers the capability for studying the oxygen enhancement ratio and probing new ﬁelds such as the FLASH eﬀect under strictly controlled conditions. The hypoxia station at UNIMAN is positioned directly at the beam nozzle, which prevents O2 ﬂuctuations in the sample while it is being transported from the laboratory to the irradiation facility. Additionally, the irradiation in hypoxic conditions is possible at AU and GSI, where the samples can be gassed inside specially designed containers prior the transportation to the experimental room. The availability of more sophisticated post-irradiation analysis, such as ﬂow cytometry, FACS, mass spectrometry, PCR, and sequencing is varied amongst the partners. Similarly, the advanced microscopy available amongst the partners is varied, though the majority have ﬂuorescent and confocal microscopes available. LOGISTICS – SAMPLES, POSITIONING, AND AUTOMATION Four centers employ automated sample handling. All the centers have the capability of a horizontal beamline, though four can additionally oﬀer a vertical beam direction, and six oﬀer more irradiating angles by using gantries. The sample type that can be irradiated is a limitation deﬁned by the system. Most centers have ﬂexibility here, with all capable of irradiating at least ﬂasks and well-plates. The sample type capability may go beyond this (as long as it can be ﬁxed in front of the beam and meet the safety regulations of the experimental room) and should be further discussed with the partner institute. Table 2 shows a summary of these details. beamlines, employ automated sample handling. Here, the sample can remain in a horizontal orientation and is lifted up only when presented to the beam for irradiation. Automated sample handling also has the added beneﬁts of improving repeatability and minimizing access to the irradiation room, increasing sample throughput. Four centers employ automated sample handling. All the centers have the capability of a horizontal beamline, though four can additionally oﬀer a vertical beam direction, and six oﬀer more irradiating angles by using gantries. The sample type that can be irradiated is a limitation deﬁned by the system. Most centers have ﬂexibility here, with all capable of irradiating at least ﬂasks and well-plates. The sample type capability may go beyond this (as long as it can be ﬁxed in front of the beam and meet the safety regulations of the experimental room) and should be further discussed with the partner institute. Table 2 shows a summary of these details. October 2020 \| Volume 8 \| Article 565055 Frontiers in Physics \| www.frontiersin.org 7 INSPIRE Radiobiology Centers Henthorn et al. TABLE 2 \| Beamline and radiobiological sample details of the INSPIRE partners. LOGISTICS – SAMPLES, POSITIONING, AND AUTOMATION Center Beam direction (H/V) Gantry Automated sample exchange Sample type Flask Petri dish Well-plate Other vessels AU H x x x GSI H x x IFJ PAN H x Institut Curie H + V x NPI-CAS H + V x x PSI H + V x UMCG H x Skandion H + V x TUD H x x CHRISTIE + UNIMAN H x All centers can offer a horizontal beamline, with the four able to irradiate samples from above or at user-deﬁned angles using a gantry. There is ﬂexibility in sample types, but the majority of centers have the ability to irradiate ﬂasks and well-plates. TABLE 2 \| Beamline and radiobiological sample details of the INSPIRE partners. sample drying. Alternatively, samples can be prepared so that the culture vessel is full of cell media, which is the case for the research facility of UMCG (Figure 6B) and Institut Curie (Figure 6C). BIOLOGY – SAMPLE PREPARATION AND PROCESSING both the oﬃcial regulations of their country/state as well as those of the TNA provider. Moreover, these regulations might vary from one state to another within the same country (for example, in Germany). Ethics applications in EU generally require a FELASA (Federation of European Laboratory Animal Science Associations) certiﬁcation for participating scientists that cover the duration of the relevant research. In addition to that, country- speciﬁc licenses might be required. In the latter case, exceptions can be made when the guest scientists are only irradiating the animals without leaving them at the TNA facility. The application for the ethical approval is normally done well in advance, as the review procedure can last up to several months. All of the paperwork relating to ethical approval is retained by the partner and made available to the EU upon request. In addition, for some experiments the EU requires copies of the ethical permissions prior to any experiment taking place. While all the INSPIRE TNA partners mentioned in this work oﬀer the environment for in vitro studies, the in vivo capabilities are slightly more limited, as seen in Figure 2. Despite the data from cell experiments being a valuable preliminary tool for studying the eﬀects of proton beams, all of the physiological processes and their complex interplay cannot be reproduced in vitro, and thus the clinical treatments must ﬁrst be simulated using animal models before moving onto human trials. Table 4 shows the in vivo capabilities of the INSPIRE TNA providers. Frontiers in Physics \| www.frontiersin.org FUTURE PERSPECTIVES As has been shown, the resources available within the INSPIRE network are state-of-the-art. Further to this a number of new centers are under development and will soon be accessible to the research community. For example, the Proteus ONE IBA center at Charleroi (Belgium) will oﬀer both in vitro and in vivo capabilities complete with a basic in vitro lab and animal facility on site, with researcher access oﬀered through partnership with Namur. Belgium is also developing a center at Leuven, which will also oﬀer in vitro and in vivo research capabilities. Furthermore, the European In vivo experiments bring the added complexity of ethical review. INSPIRE has a well-established ethics platform for both its TNA and JRA, which is overseen by an ethics panel comprised of international experts in the ﬁeld. The partners must also follow October 2020 \| Volume 8 \| Article 565055 Frontiers in Physics \| www.frontiersin.org 8 INSPIRE Radiobiology Centers Henthorn et al. sample irradiation at (A) CHRISTIE + UNIMAN, (B) UMCG research facility, (C) Institut Curie, (D) GSI, and (E) AU. The CHRISTIE + UNIMAN otic arm mounted in a hypoxia cabinet, allowing irradiation at different oxygen tensions from 0.1 to 20%. The robot picks samples from a “hotel” t of a beam window within the cabinet, before either replacing the sample to the hotel or moving to an automated ﬁxation system (left). The hotel (Continued) ww.frontiersin.org 9 October 2020 \| Volume 8 \| Article 565055 FIGURE 6 \| Setup for sample irradiation at (A) CHRISTIE + UNIMAN, (B) UMCG research facility, (C) Institut Curie, (D) GSI, and (E) AU. The CH system is a 6-axis robotic arm mounted in a hypoxia cabinet, allowing irradiation at different oxygen tensions from 0.1 to 20%. The robot picks s and holds them in front of a beam window within the cabinet, before either replacing the sample to the hotel or moving to an automated ﬁxation s FIGURE 6 \| Setup for sample irradiation at (A) CHRISTIE + UNIMAN, (B) UMCG research facility, (C) Institut Curie, (D) GSI, and (E) AU. The CHRISTIE + UNIMAN system is a 6-axis robotic arm mounted in a hypoxia cabinet, allowing irradiation at different oxygen tensions from 0.1 to 20%. FUTURE PERSPECTIVES The robot picks samples from a “hotel” and holds them in front of a beam window within the cabinet, before either replacing the sample to the hotel or moving to an automated ﬁxation system (left). The hotel (Continued) October 2020 \| Volume 8 \| Article 565055 Frontiers in Physics \| www.frontiersin.org 9 Henthorn et al. INSPIRE Radiobiology Centers FIGURE 6 \| can house up to 36 samples, a mix of T75 ﬂasks or 96-well-plates (right). The system of the UMCG research facility shows the sequential irradiation of three 12-well-plates. Wells are ﬁlled with cell media and sealed with paraﬁlm. The Institut Curie system shows sequential irradiation of six in vitro samples (left), and immobilized in vivo irradiation (right). The GSI system allows for sequential irradiation of 16 tissue culture ﬂasks. The ﬂasks remain in the horizontal position whilst not being irradiated (left), preventing the cell layer inside from drying. The robotic system lifts the sample and presents it to the beam (right), replacing it when irradiation is complete. The AU system shows an in vivo setup for mouse leg irradiation. TABLE 3 \| In vitro biological analysis equipment available at the INSPIRE partners. Center # of laminar ﬂow cabinets # of incubators Hypoxia irradiation station Chemical hood Flow cytometry FACS Biological mass spectrometry PCR Sequencing Fluorescent microscope Confocal microscope Super resolution microscope AU 1 1 x x x x x x x x GSI 2 4 x x x x IFJ PAN 2 1 x x x x x x Institut curie 1 1 x NPI-CAS 2 3 x x x x x x PSI* 0 1 x x x x x x x x x x UMCG 2 2 x x Skandion 4 4 x TUD 6 12 x x x x x CHRISTIE + UNIMAN 5 5 Biological equipment at PSI is available at a partner institute and will need to be discussed. TABLE 4 \| In vivo capabilities available at the INSPIRE partners. Center Animals Capacity (max. No. of animals) Max. FUTURE PERSPECTIVES days before irradiation Onsite immobilization Onsite anesthesia Models used Imaging Histology AU Rats, mice 80 rats, 200 mice 7 x x Normal tissue and a range of tumor models (syngenic and xenografts) x x GSI Rats, mice 80 7 x x x x IFJ PAN Rats, mice, hamsters 100 7 x MRI x Institut Curie Rats, mice 100 rats, 40 mice A few months Normal tissue and a range of tumor models (syngenic and xenografts), orthotopic grafts, speciﬁc tissue toxicity assays CT, X-ray, OCT, Bioluminescence PSI Mice, Zebraﬁsh – – – – – UMCG Rats, mice, zebraﬁsh 132 rats, 264 mice 7 Normal tissue and a range of tumor models TUD Rats, mice, zebraﬁsh 100 7 Zebraﬁsh embryo strain wild type AB; NMRI nu/nu Nude, C57Bl/6JRj and C3H/HeNRj CT, X-ray, MRI, Proton radiography, Bioluminescence, PET, Ultrasound In vivo irradiation at PSI has previously been done, but capacities and equipment need to be discussed. project SEEIST [38, 43] will develop capabilities in South- There is a growing European interest into studying the TABLE 3 \| In vitro biological analysis equipment available at the INSPIRE partners. Biological equipment at PSI is available at a partner institute and will need to be discussed. TABLE 4 \| In vivo capabilities available at the INSPIRE partners. In vivo irradiation at PSI has previously been done, but capacities and equipment need to be discussed. In vivo irradiation at PSI has previously been done, but capacities and equipment need to be discussed. project SEEIST [38, 43] will develop capabilities in South- eastern Europe, ﬁlling in some geographical gaps shown in Figure 1. As well as developing a new heavy ion center the SEEIST project will have access to resources provided by INSPIRE. There is a growing European interest into studying the eﬀectiveness of heavy ions, with four operational carbon centers and two new centers under construction. A 2019 meeting of UK clinicians, scientists, engineers, and stakeholders began the process of considering future UK development of heavy ion Frontiers in Physics \| www.frontiersin.org October 2020 \| Volume 8 \| Article 565055 10 INSPIRE Radiobiology Centers Henthorn et al. including how the resources can be accessed. It is clear that whilst there are a number of diﬀerences between the partners there are also a number of similarities. CONCLUSION This work was funded by the European Union’s Horizon 2020 research and innovation programme under grant agreement no 730983 (INSPIRE). In this work we have given details about the radiobiological capabilities of partners involved in the INSPIRE project, AUTHOR CONTRIBUTIONS In vivo radiobiological research is a crucial step along the path to clinical implementation. Seven of the 11 partners discussed in this work are currently performing in vivo research (AU, GSI, IFJ PAN, Institut Curie, PSI, TUD, UMCG). Further to this, CHRISTIE + UNIMAN are beginning development of a second beamline for in vivo work. Skandion are also in the early stages of planning future in vivo work. This added capacity, and the currently available capacity, is sure to aid in the clinical eﬃcacy of proton therapy. MDu designed the structure of the manuscript. NH and OS wrote the manuscript with input from the other authors. OS and MDu provided information for GSI. LD and FP provided information for Institut Curie. JM and PO provided information for IFJ PAN. SB, MG, LB, MT, and JL provided information for UMCG. MDa and VV provided information for NPI-CAS. EB and JP provided information for TUD. AL and DCW provided information for PSI. AD and BS provided information for Skandion. PP, BSS, CG, and MS provided information for AU. A-CH and SL provided information for Namur. NH, JW, MM, RM, and KK provided information for UNIMAN and CHRISTIE. MDu leads the radiobiology work package of the INSPIRE project. KK leads the INSPIRE project. All authors reviewed and agreed the manuscript. The connection between research activities and clinically relevant questions must be made stronger. There are close links between many INSPIRE partners and clinical centers, which aids in this connection. However, it is important that the clinical community become more involved with research at inception. With a limited amount of ﬁnances this will ensure prioritization of the most pertinent research and advance clinical translation, all for the beneﬁt of the patient. FUTURE PERSPECTIVES This allows for investigations into the cause of variance in published radiobiological data, such as the planned joint experiment of the INSPIRE partners. However, establishing these research centers requires signiﬁcant investment and, as can be seen, many of the capabilities are already in place. More eﬀort must be made to develop and utilize the resources currently available to us. Eﬀorts are being made to further increase in vivo capabilities, whilst in vitro research is invaluable for identifying and probing mechanisms, in vivo research is crucial for clinical adoption. Also required here is a closer relationship with clinical partners, ensuring a good direction for future research. With a renewed interest in radiotherapy delivery techniques, and the unknown biological mechanisms, now is certainly and exciting time for particle radiobiology. Mechanisms that the INSPIRE network is well-placed to address. therapy. There are also ongoing investigations into the clinical utilization of other particle types. For example, Helium has been seen as an intermediate between protons and carbon [44–46]. Other studies investigate the possibilities of combining multiple beams within one treatment plan to ensure a more uniform RBE distribution [47], or better treatment of hypoxic tumors [48]. The INSPIRE network is well-placed for the associated radiobiological investigations here, in particular with the partner institutes GSI and UMCG. There has been a worldwide renewed interest in radiotherapy delivery techniques and improved normal tissue sparing. For example, spatially fractionated proton therapy [49–52] and ultra-high dose rate (FLASH) [53–56]. In these cases, the radiobiological mechanism driving the eﬀect remains elusive. In particular, the diﬀerences between photon and particle therapy requires further investigation. Alongside this, the combination of particle therapy with immunotherapy [57, 58] is an exciting treatment that requires mechanistic understanding. Again, the INSPIRE network provides resources for investigation here, particularly through in vivo work, with results being directly useful for clinical adoption. REFERENCES Design and implementation of a robust and cost-eﬀective double- scattering system at a horizontal proton beamline. J Instrum. 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(2018) 56:81– 9. doi: 10.1016/j.ejmp.2018.10.025 Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. 51. González W, Prezado Y. Spatial fractionation of the dose in heavy ions therapy: An optimization study. Med Phys. (2018) 45:2620–27. doi: 10.1002/mp.12902 52. Prezado Y, Jouvion G, Hardy D, Patriarca A, Nauraye C, Bergs J, et al. Proton minibeam radiation therapy spares normal rat brain: long-term clinical, radiological and histopathological analysis. Sci Rep. (2017) 7:1– 7. doi: 10.1038/s41598-017-14786-y Copyright © 2020 Henthorn, Sokol, Durante, De Marzi, Pouzoulet, Miszczyk, Olko, Brandenburg, van Goethem, Barazzuol, Tambas, Langendijk, Davídková, Vondráˆcek, Bodenstein, Pawelke, Lomax, Weber, Dasu, Stenerlöw, Poulsen, Sørensen, Grau, Sitarz, Heuskin, Lucas, Warmenhoven, Merchant, Mackay and Kirkby. 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. 53. REFERENCES van Luijk P, Novakova-Jiresova A, Faber H, Schippers JM, Kampinga HH, Meertens H, et al. Radiation damage to the heart enhances early radiation-induced lung function loss. Cancer Res. (2005) 65:6509– 11. doi: 10.1158/0008-5472.CAN-05-0786 41. RADIATE. Research And Development with Ion Beams – Advancing Technology in Europe. 42. Paganetti H, Blakely E, Carabe-Fernandez A, Carlson DJ, Das IJ, Dong L, et al. Report of the AAPM TG-256 on the relative biological eﬀectiveness of proton beams in radiation therapy. Med Phys. 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Merk B, Voss KO, Müller I, Fischer BE, Jakob B, Taucher-Scholz G, et al. Photobleaching setup for the biological end-station of the darmstadt heavy- ion microprobe. Nucl Instruments Methods Phys Res Sect B Beam Interact Mater Atoms. (2013) 306:81–4. doi: 10.1016/j.nimb.2012.11.043 45. Knäusl B, Fuchs H, Dieckmann K, Georg D. Can particle beam therapy be improved using helium ions? – A planning study focusing on pediatric patients. Acta Oncol. (2016) 55:751–9. doi: 10.3109/0284186X.2015.1125016 26. Abdollahi E, Taucher-Scholz G, Durante M, Jakob B. Upgrading the GSI beamline microscope with a confocal ﬂuorescence lifetime scanner to monitor charged particle induced chromatin decondensation in living cells. Nucl 46. Krämer M, Scifoni E, Schuy C, Rovituso M, Tinganelli W, Maier A, et al. Helium ions for radiotherapy? Physical and biological veriﬁcations of a novel treatment modality. Med Phys. 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https://openalex.org/W4367680426	https://journals.library.columbia.edu/index.php/cja/article/download/11112/5541	English	null	Ecofeminism in the Early Twentieth Century Bengali Literature: Rokeya Sakhawat Hossain’s Sultana’s Dream	Columbia journal of Asia	2,023	cc-by	2,782	The Columbia Journal of Asia The Columbia Journal of Asia ECOFEMINISM IN THE EARLY TWENTIETH CENTURY BENGALI LITERATURE: ROKEYA SAKHAWAT HOSSAIN’S SULTANA’S DREAM LABIBA RIFAH NANJEEBA B.A. Candidate in English Literature BRAC University, Class of 2023 1 Plumwood, “Ecofeminism: An overview,” 120. 2 Mookerjea-Leonard, "Futuristic technologies and purdah in the feminist utopia,” 144. 3 Rajan, “Feminism’s Futures,” 39. Introduction E cofeminism is a field of feminist inquiry and activism that has been used in literary criticism to illustrate the connection between ecology and feminism. To end women’s oppression, ecofeminism believes that it is necessary to end all forms of oppression, particularly environmental oppression. In the last few decades, a wide range of literary works delve into the “link between the domination of women and the domination of nature.”1 Ecofeminism in literature explores the intersection between gender, nature, and patriarchal domination. This essay seeks to argue for the presence of ecofeminist thinking in early twentieth-century Bengali literature by analyzing Rokeya Sakhawat Hossain’s short story, Sultana’s Dream. E Hossain was a pioneering reformer of Bengali Muslim society and an early twentieth-century utopian feminist writer who explored the close connection between the oppression of women and the environment through her works. She was born in colonial India in 1880, during a time when women were subjected to the conservative upbringing of purdah which included “veiling, dressing modestly, gender segregation, and the seclusion of women in the zenana, or women’s quarters.”2 Despite the social restrictions women experienced at that time, Hossain managed to learn English and Bangla with the help of her brother and sister. However, her strict, traditional father did not encourage women’s education and prevented Hossain from beginning her literary career at home. Her literary career began after her marriage to Syed Sakhawat Hossain, a highly educated man and, as Roushan Jahan puts it, “a man of liberal attitude,” who encouraged her to pursue writing. 3 Rokeya Sakhawat Hossain produced many prominent works that still shape Bengali literature, and she also greatly contributed to the education of Muslim women in Bengal by establishing the Sakhawat Memorial Girls School in Calcutta. Hossain published Sultana’s Dream in the journal The Indian Ladies’ Magazine, a Madras-based English-language journal, in 1905 and it was the only short story she wrote in English. Sultana’s Dream is critical of the patriarchal system that existed in the Indian subcontinent, particularly the enforcement of purdah. In Sultana's Dream, she represents a feminist utopian vision of a fictional world in which women and nature Volume II, Issue 1 \| 8 Volume II, Issue 1 \| 8 © 2023 Labiba Rifah Nanjeeba The Columbia Journal of Asia are no longer subjugated but rather dominate the social sphere. © 2023 Labiba Rifah Nanjeeba 4 Gaard, “Ecofeminism,” 5. 5 Rahman and Sarker, “Eco-Feminist Vision,” 39. 6 Chatterjee, “The Nationalist Resolution,” 122-127. Introduction Hossain explores the close relationship between ecology and feminism by tying the slavery of women and nature and demonstrating that if women attain emancipation, nature would thrive as well. The primary ecofeminist goal is to define the interconnectedness of oppression by simultaneously providing a voice to women and nature. Hence, for ecofeminist writers, it is important to break the justification of nature and women’s domination by “feminizing nature and naturalizing women.”4 According to Rahman, Sultana’s Dream is an “ecofeminist celebration” 5 because of Hossain’s creation of the female- dominated “Ladyland” which subverts the reality of male dominance in colonial India. Therefore, in this article, I examine the ecofeminist potential of this short story, as well as how Hossain attempts to combine the concept of freedom for both the environment and women in orthodox Muslim society during British colonial rule of the Indian subcontinent. In addition, I will dissect the reversed gender roles portrayed in the story to argue that the inextricable link Hossain makes between women’s freedom and environmental freedom marks a deep ecofeminist consciousness in Hossain’s writing. Finally, I will explore the ideas of sustainability presented in the story to reveal Hossain’s subtle claim-making surrounding the radical potential of women’s leadership and its possible positive ecological impacts. [Sister Sara:] I have seen some of them [men in colonial offices] doing their work. Do you think they work all the seven hours? [Sultana:] Certainly they do! 4 Gaard, “Ecofeminism,” 5. 4 Gaard, “Ecofeminism,” 5. 5 Rahman and Sarker, “Eco-Feminist Vision,” 39. 6 Chatterjee, “The Nationalist Resolution,” 122-127. The Columbia Journal of Asia No, dear Sultana, they do not. They dawdle away their time in smoking. Some smoke two or three choroots [sic] during the office time. They talk much about their work, but do little. Suppose one choroot [sic] takes half an hour to burn off, and a man smokes twelve choroots [sic] daily; then, you see, he wastes six hours every day in sheer smoking.7 Hossain is thereby asserting that the colonial office culture has removed men from contact with nature and agriculture and repositioned them in an artificial world bereft of meaningful work. According to Rangarajan, India's environment underwent devastating changes during the colonial period, such as large-scale ecological interventions (including marsh- and forest-clearing) that attempted to increase the land available for commercial cropping and housing.8 The colonial environmental history of India is marked by the commodification of nature through railway and dam construction projects that severely disrupted local ecological cycles. In addition, colonial land- mapping rhetoric developed specific forms of ecological antagonisms. The colonial concept of “wasteland,” for instance referred to uncultivated marshes, forests, and chars (a newly emerged land in the middle of an ocean, sea, lake, or stream surrounded by water), which were cleared by human labor for resettlement and farming purposes to generate revenue under British control.9 Evidently, such terminology creates a normative axis of ecological “utility,” wherein the “uncultivated” is undesired and “cultivated” land brought under human control is of value. Consequently, the ecological equilibrium of Bengal suffered. Based on the new heuristic of materialistic ecological priorities, the local peasantry began to clear vast lands containing numerous now-extinct species' habitats. © 2023 Labiba Rifah Nanjeeba 7 Hossain, Sultana’s Dream. 8 Peers and Gooptu, “India and the British Empire,” 212. 9 Iqbal, “The Bengal Delta,” 19-22. 10 Pouchepadass, “Colonialism and Environment in India,” 2064. 11 Iqbal, “The Bengal Delta,” 117-119. 12 Satya, “British Imperial Railways,” 71. British Colonial Influence on the Environment in the Indian Subcontinent In colonial Bengal, according to Chatterjee, men and women were respectively categorized along gender dichotomies such as public and private, world and home, material and spiritual, outward and inner, and so on. Nationalists, as part of framing an anti-colonial rhetoric of resistance, instrumentalized these dichotomies and compelled women to take on roles that aligned with the narrative of the private, inner spirituality of women as bearers of religious traditions and ancestral values, thereby contrasting women with “modern Western society.”6 Consequently, it was essential to separate the realm of women from the colonized, materialistic world. However, Hossain shows in her short story that women can lead by simultaneously grappling with culture, nature, and modernity. She also subtly refers to the colonial office culture’s gender divisions. In the story, the titular character and protagonist, Sultana (who is contending with the possibilities of the utopian feminist world she sees and the realities of gender divisions in her own world) converses with her friend Sister Sara who shows her this utopia: Volume II, Issue 1 \| 9 Volume II, Issue 1 \| 9 © 2023 Labiba Rifah Nanjeeba Volume II, Issue 1 7 Hossain, Sultana’s Dream. 8 Peers and Gooptu, “India and the British Empire,” 212. 9 Iqbal, “The Bengal Delta,” 19-22. 10 Pouchepadass, “Colonialism and Environment in India,” 2064. 11 Iqbal, “The Bengal Delta,” 117-119. 12 Satya, “British Imperial Railways,” 71. q g 12 Satya, “British Imperial Railways,” 71. The Columbia Journal of Asia This extractive colonial logic concerning ecology resulted in mass deforestation, drastic changes in vegetation patterns due to commercial cropping, and excessive pressure on farming land in former colonies (including the Indian subcontinent).10 In the mid-nineteenth century, the British Empire infrastructurally penetrated India through railway lines that cut across not just cultivable land but also forested areas and brought with it a series of famines in Bengal.11 Even though the British Empire projected that the Indian railway system would reduce famines and increase employment for Indian people, it had in fact created an extractive infrastructural complex and “employment system [conducive to the economic interests of] Britain rather than India.”12 As a vigilant observer and writer, Hossain challenges, through her work, the colonial, materialistic worldview which was visibly influencing Indian men who served as low-level colonial administrators and workers in her critique of the dysfunctionality, Volume II, Issue 1 \| 10 © 2023 Labiba Rifah Nanjeeba The Columbia Journal of Asia futility, and inefficiency of the latter. Furthermore, the utopic country in Sultana’s Dream resembles a “garden,” which can be considered to be Hossain’s attempt to “escape the battered [colonial] cities” that bear the marks of a dysfunctional colonial ecological logic and divisive gender relations.13 , , 14 Glazebrook, “Karen Warren’s Ecofeminism,” 13. 13 Rahman and Sarker, “Eco-Feminist Vision,” 39. Futuristic Sustainability Ideas of Nature Furthermore, Hossain attempts to oppose colonial and patriarchal rule by incorporating futuristic ideas such as sustainability. In Sultana’s Dream, Hossain shows that all the men are kept at “mardana,” (men’s area in a house) and leadership is given— almost performatively and emphatically—to women who then bring to effect various revised social policies. As Sister Sara tells the narrator, women in Ladyland convinced all men to confine themselves to the mardana because the latter was defeated in war and needed the help of women in securing the nation’s sovereignty, so the women demanded they be sanctioned in the domestic sphere. The men, being tired and broken, relented: [Sister Sara:] On the following day, the Queen called upon all men to retire into zenanas [women’s area in a house] […] they [the men] took the order for a boon [… and] entered the zenanas without uttering a single word of protest. […] we call the system “mardana” instead of “zenana.”15 As soon as men quit the public sphere and enter the domestic sphere, women take charge, and Hossain explains that the country transitioned to one where “gender hierarchy [exists] not by physical power, but by intellectual power.”16 Through this contentious claim-making, Hossain depicts the way women, using their “intellectual power” and leadership, introduce reforms that bridge science and ecological stability through a series of innovations. For instance, there are “wonderful balloon[s] […] that could draw as much water from the atmosphere as they pleased,” instruments that “could collect as much sun-heat as they wanted,” eco-friendly “air cars,” and even mechanisms wherein fields could be “tilled by means of electricity,” among others – all of which men in Ladyland disregard as “sentimental nightmares.”17 Not only does this present a radical vision of women as individuals with a then-paradoxical, simultaneous affinity to scientific innovation and nature, but it also heightens the antagonistic role men play in this women-science-nature nexus in Ladyland. The Reversed Role of Nature Ecofeminism attempts to highlight the dual marginalization of women and nature as social “others” because of their definition in relation to men. It also highlights that feminist “emancipation” rests on ecological “emancipation,” since both rest on the same or interrelated logics of oppression. As Rosemary Radford Ruether asserted, “women must see that there can be no liberation for them and no solution to the ecological crisis within a society whose fundamental model of relationships continues to be one of domination.”14 This demonstrates the connection between women and nature as being systematically subjugated by men. Therefore, in Sultana’s Dream, Hossain posits a radically different logic of organizing power and interpersonal relations as a way to challenge the contemporary ways of thinking that constituted systems of oppression. She presents a defiant, new vision of society that is inherently different and starts from fundamentally different foundational values: empathy, self-reflection, and compassion constitute the cornerstones of social relations in Ladyland. In the story, the women of Ladyland design a new (in relation to the system in place at the time) punishment system without the death penalty, wherein people who are otherwise sentenced to death are instead asked to “sincerely repent,” after which they will be forgiven. If they do not, they would be obligated to leave the country. Hossain’s contention and radical claim- making go even further, and she asserts that the only religion in Ladyland is one that is based on “Love and Truth.” As Sister Sara tells the narrator, “It is our religious duty to love one another and to be absolutely truthful.” It is clear from the story that nature is cherished and valued in Ladyland. As Sister Sara says, “Our noble Queen is exceedingly fond of botany; it is her ambition to convert the whole country into one grand garden.” In this radical new vision of the world, with the power that women wield, not only are they emancipated from gendered social and power restrictions, but they also use this power to reverse the ecological indifference characteristic of the colonial administration. With reversed and reimagined gender roles, Hossain attempts to present a world that is able to bring together scientific innovation, environmental sensitivity, and the emancipation of women and nature from oppressive social logics in Sultana’s Dream. Volume II, Issue 1 \| 11 Volume II, Issue 1 \| 11 © 2023 Labiba Rifah Nanjeeba 15 Hossain, Sultana’s Dream. 16 Bhattacharya, “Two Dystopian Fantasies,” 174. 17 Hossain, Sultana’s Dream. © 2023 Labiba Rifah Nanjeeba 15 Hossain, Sultana’s Dream. 16 Bhattacharya, “Two Dystopian Fantasies,” 174. 17 Hossain, Sultana’s Dream. The Columbia Journal of Asia The Columbia Journal of Asia science, and nature collaborate to build an eco-friendly and sustainable society, thereby making Sultana’s Dream a powerful case in the ecofeminist genre, even though the piece may be seen as overreaching and reductive in its generalizations of gender roles. By drawing closer connections between ecofeminism and environmental utopianism, this article attempts to show how Sultana’s Dream engenders a vision of society that inverts social logics, gender roles, and other normative axes to transcend networks of patriarchal and colonial oppression. Conclusion This article explored the depictions of British colonial authority and patriarchal influence on Indian ecology, its relations with gender roles, the intertwining of women’s and ecological “otherness,” and Hossain’s musings on sustainable futurism in Sultana’s Dream. In this work, Hossain blends utopian fiction, science fiction, and ecofeminist themes to show the agency women and nature can have if they are free from patriarchy and colonialism. She provides a dual criticism of patriarchal society and the colonial era by reversing the gender roles of men and women in conservative Bengali society. Hossain, through this piece, envisions a world in which women, Volume II, Issue 1 \| 12 Volume II, Issue 1 \| 12 © 2023 Labiba Rifah Nanjeeba © 2023 Labiba Rifah Nanjeeba BIBLIOGRAPHY Bhattacharya, Nilanjana. “Two Dystopian Fantasies.” Indian Literature, vol. 50, no. 1 (231), Sahitya Akademi, (2006): 172–77. JSTOR, www.jstor.org/stable/23346372. Bhattacharya, Nilanjana. “Two Dystopian Fantasies.” Indian Literature, vol. 50, no. 1 (231), Sahitya Akademi, (2006): 172–77. JSTOR, www.jstor.org/stable/23346372. Chatterjee, Partha. "The Nationalist Resolution of the Women’s Question (1989)." Empire and Nation. Columbia University Press, (2010): 116-135. Gaard, Greta, ed. Ecofeminism. Vol. 21. Temple University Press, 1993. Glazebrook, Trish. “Karen Warren’s Ecofeminism.” Ethics and the Environment 7, no. 2 (2002): 12–26. www.jstor.org/stable/40339034. Hossain, Rokeya Sakhawat. 1905. Sultana's Dream (digital edition transcribed from The Indian Ladies' Magazine, Madras). https://digital.library.upenn.edu/women/sultana/dream/dream.html Iqbal, Iftekhar. The Bengal Delta: Ecology, State and Social Change, 1840-1943, Palgrave Macmillan UK, 2010. Mookerjea-Leonard, Debali. 2017. "Futuristic technologies and purdah in the feminist utopia: Rokeya S. Hossain's 'Sultana's dream"." Feminist Review (Sage) 116 (Dystopias and utopias): 144-153. https://www.jstor.org/stable/44987318. Peers, Douglas M. and Nandini Gooptu (eds), India and the British Empire, Oxford History of the British Empire Companion Series, 2017. Plumwood, Val. "Ecofeminism: An overview and discussion of positions and arguments." Australasian Journal of Philosophy 64, no. sup1 (1986): 120-138. Pouchepadass, Jacques. “Colonialism and Environment in India: Comparative Perspective.” Economic and Political Weekly 30, no. 33 (1995): 2059–67. www.jstor.org/stable/4403103. © 2023 Labiba Rifah Nanjeeba Volume II, Issue 1 \| 13 Volume II, Issue 1 \| 13 © 2023 Labiba Rifah Nanjeeba The Columbia Journal of Asia The Columbia Journal of Asia Rahman, Md. Mominur & Md. Abdul Momen Sarker. ““Sultana’s Dream”: The Eco- Feminist Vision of Freedom” Global Journals, 18, no. 3, (2018): 37-40. globaljournals.org/GJHSS_Volume18/5-Sultanas-Dream-The-Eco- Feminist.pdf Rajan, Rajeswari Sunder. “Feminism’s Futures: The Limits and Ambitions of Rokeya’s Dream.” Economic and Political Weekly 50, no. 41 (2015): 39–45. www.jstor.org/stable/44002713. Satya, Laxman D. “British Imperial Railways in Nineteenth Century South Asia.” Economic and Political Weekly 43, no. 47 (2008): 69–77. www.jstor.org/stable/40278213. © 2023 Labiba Rifah Nanjeeba Volume II, Issue 1 \| 14
https://openalex.org/W4385957484	https://vspu.net/fks/index.php/fks/article/download/686/652	Ukrainian	null	GENERAL PARAMETERS OF THETRAINING WORK OF HIGHLYQUALIFIED FIELD HOCKEY PLAYERSIN THE COMPETITIVE PERIOD OF THEMACRO CYCLE	Fìzična kulʹtura, sport ta zdorovʼâ nacìï	2,023	cc-by	5,126	IІ. Науковий напрям IІ. Науковий напрям ЗАГАЛЬНІ ПАРАМЕТРИ ТРЕНУВАЛЬНОЇ РОБОТИ ВИСОКОКВАЛІФІКОВАНИХ ХОКЕЇСТІВ НА ТРАВІ У ЗМАГАЛЬНОМУ ПЕРІОДІ МАКРОЦИКЛУ Коннов Станіслав Вінницький державний педагогічний університет імені Михайла Коцюбинського GENERAL PARAMETERS OF THE TRAINING WORK OF HIGHLY QUALIFIED FIELD HOCKEY PLAYERS IN THE COMPETITIVE PERIOD OF THE MACRO CYCLE Актуальність теми дослідження. Основними компонентами тренувального процесу спортсменів командних ігрових видів спорту є засоби тренувальної роботи та навантаження різної спрямованості. Оптимальне їх поєднання обумовлює ефективність побудови тренувального процесу спортсменів на відповідному етапі (періоді) макроциклу. Мета дослідження – визначити величину та спрямованість тренувальних впливів на підготовленість висококваліфікованих хокеїстів на траві у змагальному періоді макроциклу. Методи дослідження: теоретичний аналіз і узагальнення літературних джерел; педагогічне спостереження; хронометраж тренувальної роботи; методи математичної статистики. Організація дослідження. У дослідженні брали участь спортсмени високої кваліфікації (п=24), гравці національної збірної команди України з хокею на траві. Дослідження проводилося у змагальному періоді другого циклу річної підготовки сезону 2021 року. Результати дослідження та висновки. Визначено загальні параметри тренувальної роботи висококваліфікованих хокеїстів на траві у змагальному періоді макроциклу. Встановлено, що загальний обсяг рухової тренувальної роботи у цьому періоді складає 140 год. Співвідношення вправ: загально-підготовчих – 49,5%, спеціально- підготовчих – 12,5%, підвідних – 15,8%, змагальних – 22,2%. Вплив тренувальних навантажень: аеробних – 57,9%, аеробно- анаеробних – 36,5%, анаеробно-алактатних – 2,9%, анаеробно-гліколітичних – 2,7%. Впродовж змагального періоду проводиться 99 тренувальних занять, 5 – контрольних та 20 календарних ігор. Konnov Stanislav Abstract Abstract Relevance of the research topic. The main components of the training process of athletes of team game sports are the means of training work and loads of various orientations. Their optimal combination determines the effectiveness of building the training process of athletes at the appropriate stage (period) of the macrocycle. The purpose of the study is to determine the magnitude and direction of training effects on the preparedness of highly qualified field hockey players in the competitive period of the macrocycle. Research methods: theoretical analysis and generalization of literary sources; pedagogical observation; timing of training work; methods of mathematical statistics. Research organization. Highly qualified athletes (n=24), players of the Ukrainian national field hockey team, participated in the study. The study was conducted in the competitive period of the second cycle of the annual training of the 2021 season. Research results and conclusions. The general parameters of the training work of highly qualified field hockey players in the competitive period of the macro cycle have been determined. It was established that the total amount of motor training work in this period is 140 hours. The ratio of exercises: general- preparatory – 49.5%, special-preparatory – 12.5%, training – 15.8%, competitive – 22.2%. The influence of training loads: aerobic – 57.9%, aerobic-anaerobic – 36.5%, anaerobic-lactate – 2.9%, anaerobic-glycolytic – 2.7%. During the competition period, 99 training sessions, 5 control sessions and 20 calendar games are held. Keywords: field hockey highly qualified Keywords: field hockey, highly qualified athletes, competitive, means of training work, loads of different orientation. Ключові слова: хокей на траві, висококваліфіковані спортсмени, змагання, засоби тренувальної роботи, навантаження різної спрямованості. Ключові слова: хокей на траві, висококваліфіковані спортсмени, змагання, засоби тренувальної роботи, навантаження різної спрямованості. 253 IІ. Науковий напрям Постановка проблеми. Побудова процесу підготовки спортсменів обумовлена цілеспрямованим плануванням тренувальної роботи з урахуванням завдань, що вирішуються у підготовчому, змагальному чи перехідному періодах макроциклу [1, 2, 4, 8, 9]. Особливо важливою є проблема планування тренувальних впливів у змагальному періоді тренувального циклу, в якому тренувальна робота спрямована на досягнення запланованого спортивного результату [6, 7, 10, 15]. Аналіз останніх досліджень та публікацій. З проблеми побудови тренувального процесу спортсменів на основі оптимального використання параметрів тренувальної роботи в окремих структурних утвореннях макроциклу опубліковані фундаментальні наукові праці Ц. Желязкова, В. Дашевої [2], В. Платонова [9, 11], T. Bompa [14], Harre [15], V. Platonov, M. Bulatova [17]. Abstract Фундаментальні праці науковців є підґрунтям для побудови тренувального процесу в окремих видах спорту, з урахуванням розвитку та календаря змагань. Зокрема, в командних ігрових видах спорту означена проблема досліджувалася Т. Вознюк зі співавт. [1], В. Костюкевичем [4], Г. Лисенчуком [8], І. Стасюком [12], В. Шамардіним [13], V. Kostiukevych et all [16], N. Shchepotina et all [18]. Що стосується хокею на траві, то визначення параметрів тренувальної роботи у різних періодах макроциклу залишається актуальним дослідженням відповідно до запитів теорії та практики цього виду спорту. Зв’язок дослідження з науковими темами, планами. Дослідження виконано в рамках плану науково-дослідної роботи кафедри теорії і методики спорту Вінницького державного педагогічного університету імені Михайла Коцюбинського на 2021-2025 рр. «Організаційно-методичні засади програмування тренувального процесу кваліфікованих та висококваліфікованих спортсменів» (номер державної реєстрації 0121U109550). Мета дослідження – визначити величину та спрямованість тренувальних впливів на підготовленість висококваліфікованих хокеїстів на траві у змагальному періоді макроциклу. Матеріал і методи дослідження. У дослідженні брали участь спортсмени високої кваліфікації (п=24), гравці національної збірної команди України з хокею на траві. Спортивна кваліфікація – майстри спорту України. Дослідження проводилося у змагальному періоді другого циклу річної підготовки спортивного сезону 2021 року. Науковий пошук здійснювався на основі таких методів дослідження: теоретичний аналіз та узагальнення літературних джерел, педагогічне спостереження, хронометраж тренувальної роботи, методи математичної статистики. Теоретичний аналіз літературних джерел дозволив визначити актуальність теми дослідження, розробити алгоритм наукового дослідження відповідно до поставленої мети роботи. Об’єктом педагогічного спостереження було планування, контроль і аналіз використання засобів тренувальної роботи з урахуванням режимів координаційної складності, тренувальних навантажень різної спрямованості та інтенсивності. 254 IІ. Науковий напрям Хронометраж тренувальної роботи використовувався з метою визначення величини та спрямованості тренувальних впливів. Зокрема, визначалися: тривалість вправ і інтервалів відпочинку між вправами; співвідношення засобів в структурних утвореннях тренувального процесу; співвідношення вправ у різних режимах координаційної складності. Математична обробка результатів дослідження здійснювалася на основі описової статистики [5, 19]. Результати дослідження. Результати наукового дослідження, що викладаються в статті стосуються програми наукового пошуку формувального етапу експерименту, на якому тренувальний процес висококваліфікованих хокеїстів на траві здійснювався на основі методів програмування. Результати констатувального експерименту за темою наукового дослідження опубліковано в статті «Структура та зміст тренувального процесу висококваліфікованих хокеїстів на траві у змагальному періоді макроциклу» [7]. На формувальному етапі педагогічного експерименту основним підґрунтям для побудови програм тренувальних занять, мікроциклів і мезоциклів стали програми тренувальних завдань, в яких цілеспрямовано реєструвалися основні компоненти тренувальних впливів: тривалість вправ, їх інтенсивність, режим координаційної складності, тривалість і інтенсивність навантажень різної спрямованості тощо. Abstract На етапі формувального експерименту у змагальному періоді макроциклу тренувальні впливи на рівень підготовленості висококваліфікованих хокеїстів на траві здійснювалися через використання засобів тренувальної роботи, навантажень різної спрямованості та, безумовно, безпосередньо змагальної діяльності. Загальний обсяг рухової діяльності гравців у змагальному періоді склав 8413 хв ( 140 год). Упродовж цього періоду було проведено 22 мікроцикли, на основі яких формувалися програми етапу безпосередньої підготовки до головних змагань і власне змагального періоду. Залежно від типу мікроциклу в тренувальному процесі виконувалися у різному співвідношенні загально-підготовчі (ЗПВ), спеціально-підготовчі (СПВ), підвідні (ПВ) та змагальні вправи (ЗВ) (табл. 1). У відновлювальних мікроциклах, а також у втягувальних мікроциклах застосовувалися лише ЗПВ, що було обумовлено завданнями, що вирішувалися в цей період. Насамперед, це відновлення спортивної працездатності гравців у відновлювальних мікроциклах і поступова адаптація до тренувальних навнтажень у втягувальному мікроциклі. В ударних, підвідних, міжігрових і змагальних мікроциклах використовувалися програми тренувальних завдань, в яких поєднувалися ЗПВ, СПВ, ПВ та ЗВ (рис. 1). Як видно з рисунка використання ЗПВ коливалося від 31,9% (міжігровий мікроцикл) до 75,5% (відновлювально-підвідний мікроцикл). Найбільша частка СПВ припадала на ударний мікроцикл (33,2%). Це, перш за все, обумовлено тим, що в цих мікроциклах переважно використовувалися програми 255 IІ. Науковий напрям тренувальних завдань, спрямовані на вдосконалення спеціальних швидкісних, швидкісно-силових якостей, швидкісної та спеціальної витривалості. Abstract Таблиця 1 Обсяг і співвідношення засобів тренувальної роботи висококваліфікованих хокеїстів на траві у змагальному періоді макроциклу (формувальний експеримент) Мікроцикли Кіль- кість Засоби тренувальної роботи, хв (%) Усього ЗПВ СПВ ПВ ЗВ 1 2 3 1 2 3 1 2 3 1 2 3 3-денний відновлю- вальний 3 462 (82,4) 99 (17,6)           561 3-денний відновлюва- льно- підвідний 5 480 (615) 300 (38,5)   35 (100)   125 (58,1) 90 (41,9)    1030 7-денний відновлю- вальний 1 265 (100)    54 (57,4) 40 (42,6)       359 4-денний утягувальний 1 179 (51,1) 159 (45,4) 12 (3,5)          350 4-денний ударний 2 384 (100)    352 (100)  18 (12,5) 124 (86,1) 2 (1,4) 6 (3,3) 92 (51,1) 82 (45,6) 1060 3-денний підвідний 1 81 (100)    42 (100)  3 (4,0) 38 (50,7) 34 (45,5)  36 (50,0) 36 (50,0) 270 4-денний підвідний 2 378 (94,9) 20 (511)   106 (77,9) 30 (22,1) 20 (9,2) 80 (36,7) 118 (59,1) 10 (5,9) 118 (69,4) 42 (24,7) 922 6-денний підвідний 1 228 (95,8) 10 (4,2)   69 (75,8) 15 (16,5) 7 (4,3) 106 (65,0) 50 (30,7) 28 (20,1) 57 (41,0) 54 (38,9) 624 5-денний міжігровий 1 165 (95,3) 8 (4,7)   111 (100)  13 (8,8) 89 (60,1) 46 (31,1) 7 (6,4) 72 (66,1) 30 (27,5) 541 4-денний змагальний 1 142 (94,7) 8 (5,3)   32 (100)   19 (45,2) 23 (54,8) 27 (15,0) 87 (48,3) 66 (36,7) 404 5-денний змагальний 3 522 (95,6) 24 (4,4)   114 (100)   78 (40,6) 114 (59,4) 108 (15,0) 348 (48,3) 264 (36,7) 1572 7-денний змагальний 1 232 (96,7) 8 (3,3)   51 (100)  6 (4,7) 52 (40,3) 71 (55,0) 45 (15,0) 145 (48,3) 110 (36,7) 720 Усього 22 3518 (84,4) 636 (15,2) 12 (0,4)  966 (91,9) 8,5 (8,1) 67 (5,1) 711 (53,6) 548 (41,3) 231 (12,3) 955 (51,1) 684 (36,6) 8413 4166 (49,5) 1051 (12,5) 1326 (15,8) 1870 (22,2) Примітки: ЗПВ – загально-підготовчі вправи; СПВ – спеціально-підготовчі вправи; ПВ – підвідні (допоміжні) вправи; ЗВ – змагальні вправи; 1 – перший режим координаційної складності; 2 – другий режим координаційної складності; 3 – третій режим координаційної складності. Abstract Обсяг і співвідношення засобів тренувальної роботи кваліфікованих хокеїстів на траві у змагальному періоді макроциклу (формувальний експеримент) Примітки: ЗПВ – загально-підготовчі вправи; СПВ – спеціально-підготовчі вправи; ПВ – підвідні (допоміжні) вправи; ЗВ – змагальні вправи; 1 – перший режим координаційної складності; 2 – другий режим координаційної складності; 3 – третій режим координаційної складності. У підвідних мікроциклах планувалося зменшення використання СПВ (16,1%) з одночасним збільшенням частки ПВ (25,1%) та ЗВ (19,3%). У цих мікроциклах переважно використовувалися програми тренувальних завдань, що сприяли вдосконаленню техніко-тактичної майстерності. 256 IІ. Науковий напрям 75,5 36,2 39,5 31,9 34,7 3,4 33,2 16,1 20,5 7,3 20,5 13,6 25,1 27,4 13,5 0 17,0 19,3 20,2 44,5 0 10 20 30 40 50 60 70 80 1 2 3 4 5 % 75,5 36,2 39,5 31,9 34,7 3,4 33,2 16,1 20,5 7,3 20,5 13,6 25,1 27,4 13,5 0 17,0 19,3 20,2 44,5 0 10 20 30 40 50 60 70 80 1 2 3 4 5 % 75,5 36,2 39,5 31,9 34,7 3,4 33,2 16,1 20,5 7,3 20,5 13,6 25,1 27,4 13,5 0 17,0 19,3 20,2 44,5 0 10 20 30 40 50 60 70 80 1 2 3 4 5 % Рис. 1. Співвідношення (середні значення) засобів тренувальної роботи в межах різних типів мікроциклів у змагальному періоді підготовки висококваліфікованих хокеїстів на траві (формувальний експеримент). 1 – відновлювально-підвідний мікроцикл; 2 – ударний мікроцикл; 3 – підвідний мікроцикл; 4 – міжігровий мікроцикл; 5 – змагальний мікроцикл. – загально-підготовчі вправи; – спеціально-підготовчі вправи; підвідні вправи; змагальні вправи 36,2 39,5 31,9 34,7 3,4 33,2 16,1 20,5 7,3 20,5 13,6 25,1 27,4 13,5 0 17,0 19,3 20,2 44,5 0 10 20 30 40 50 60 1 2 3 4 5 39,5 31,9 34,7 16,1 20,5 7,3 25,1 27,4 13,5 17,0 19,3 20,2 44,5 3 4 5 4 5 3 Рис. 1. Співвідношення (середні значення) засобів тренувальної роботи в межах різних типів мікроциклів у змагальному періоді підготовки висококваліфікованих хокеїстів на траві (формувальний експеримент). Рис. 1. Співвідношення (середні значення) засобів тренувальної роботи в межах різних типів мікроциклів у змагальному періоді підготовки висококваліфікованих хокеїстів на траві (формувальний експеримент). р (ф р у р ) 1 – відновлювально-підвідний мікроцикл; 2 – ударний мікроцикл; 3 – підвідний мікроцикл; 4 – міжігровий мікроцикл; 5 – змагальний мікроцикл. міжігровий мікроцикл; 5 змагальний мікроцикл. – загально-підготовчі вправи; – спеціально-підготовчі вправи; – підвідні вправи; – змагальні вправи. Abstract Найбільш збалансованим було використання засобів тренувальної роботи в міжігрових мікроциклах, в яких планувався комплексний вплив на рівень підготовленості висококваліфікованих хокеїстів на траві. Варто зазначити, що чим більша частка виконання у мікроциклі СПВ, ПВ та ЗВ по відношенню до ЗПВ, тим більш напруженим і більш енерговитратним буде тренувальний процес. У міжігровому мікроциклі СПВ, ПВ та ЗВ склали 68,1%, що більше на 4,3%, ніж в ударному (63,8%) та на 7,6% більше, ніж у підвідному (60,5%) мікроциклах. Що стосується змагальних мікроциклів, що можуть розглядатися як модель у співвідношенні використання різних засобів, то тут необхідно звернути увагу на достатньо велику частку використання ЗВ – 44,5%. Разом з СПВ та ПВ це складало 65,3%, що потребувало дещо більшого, ніж у міжігровому мікроциклі використання ЗПВ (34,7%), що застосовувалися, насамперед, для відновлення спортивної працездатності гравців після змагальної діяльності. Загалом, упродовж змагального періоду на етапі формувального етапу співвідношення різних засобів тренувальної роботи було таким: ЗПВ – 49,5%, СПВ – 12,5%, ПВ – 15,8% та ЗВ – 22,2% (рис. 2). Рис. 2. Співвідношення засобів тренувальної роботи висококваліфікованих хокеїстів на траві у змагальному періоді макроциклу (формувальний експеримент), % – загально-підготовчі вправи; – спеціально-підготовчі вправи; – підвідні вправи; – змагальні вправи. 49,5 12,5 15,8 22,2 12,5 15,8 22,2 257 IІ. Науковий напрям IІ. Науковий напрям Якщо розглядати використання різних засобів тренувального процесу у змагальному періоді підготовки висококваліфікованих хокеїстів на траві з урахуванням режимів координаційної складності (рис. 3), то можна стверджувати, що найбільш простими при виконанні були ЗПВ, а найбільш складними – СПВ, в яких частка вправ, що виконувалися у 2-му та 3-му РКС склала 100%. СПВ використовувалися з метою вдосконалення спеціальних фізичних якостей у поєднанні з технікою гри, а також при вдосконаленні координаційних здібностей. До 1-го РКС були віднесені вправи, що виконуються на місці або на зручній швидкості пересування, до 2-го РКС – вправи, що виконуються в русі з обмеженням простору та часу, до 3-го РКС – вправи, що виконувалися в умовах активної перешкоди з боку суперника. ЗПВ СПВ ПВ ЗВ Рис. 3. Співвідношення засобів тренувальної роботи з урахуванням режимів координаційної складності підготовки висококваліфікованих хокеїстів на траві у змагальному періоді макроциклу (формувальний експеримент), % ЗПВ – загально-підготовчі вправи; СПВ – спеціально-підготовчі вправи; ПВ – підвідні вправи; ЗВ – змагальні вправи. 1-й режим координаційної складності; 84,4 15,2 0,4 91,9 8,1 5,1 53,3 41,3 12,3 51,1 36,6 ЗПВ 84,4 15,2 0,4 ЗПВ 5,1 53,3 41,3 СПВ ЗВ 12,3 51,1 36,6 ЗПВ 5,1 Рис. 3. Співвідношення засобів тренувальної роботи з урахуванням режимів координаційної складності підготовки висококваліфікованих хокеїстів на траві у змагальному періоді макроциклу (формувальний експеримент), % ЗПВ – загально-підготовчі вправи; СПВ – спеціально-підготовчі вправи; ПВ – підвідні вправи; ЗВ – змагальні вправи.  1-й режим координаційної складності;  2-й режим координаційної складності;  3-й режим координаційної складності  3-й режим координаційної складності У тренувальному процесі та під час змагальної діяльності складніше виконувати вправи у 3-му РКС. Зазвичай, до цих вправ відносяться різні одноборства. У той же час, важливо підвищувати ефективність виконання таких вправ, особливо під час змагальної діяльності. В управлінні тренувальним процесом спортсменів важливою ланкою є планування, контроль і аналіз тренувальних навантажень різної спрямованості. 258 IІ. Науковий напрям Упродовж змагального періоду підготовки висококваліфікованих хокеїстів на етапі формувального експерименту вплив аеробних навантажень становив 57,9%, змішаних (аеробно-анаеробних) – 36,5%, анаеробно-алактатних – 2,9% і анаеробно-гліколітичних – 2,7% (табл. IІ. Науковий напрям 2) ц Обсяг і співвідношення тренувальних навантажень висококваліфікованих хокеїстів на траві у змагальному періоді макроциклу (формувальний експеримент) Мікроцикли Кіль- кість Тренувальні навантаження, хв (%) Усього аеробні аеробно- анаеробні (змішані) анаеробно- алактатні анаербно- гліколітичні 3-денний відновлювальний 3 546 (97,3) 15 (2,7)   561 3-денний відновлювально- підвідний 5 880 (85,4) 150 (14,6)   1030 7-денний відновлювальний 1 279 (77,7) 80 (22,3)   359 4-денний утягувальний 1 253 (79,3) 97 (277)   350 4-денний ударний 2 466 (43,9) 386 (36,4) 156 (14,7) 52 (5,0) 1060 3-денний підвідний 1 126 (46,7) 121 (44,8) 18 (6,7) 5 (1,8) 270 4-денний підвідний 2 570 (61,8) 246 (26,7) 36 (3,9) 70 (7,6) 922 6-денний підвідний 1 332 (53,2) 259 (41,5) 19 (3,0) 14 (2,3) 624 5-денний міжігровий 1 279 (51,6) 206 (38,1) 18 (3,3) 38 (7,0) 541 4-денний змагальний 1 179 (44,3) 215 (53,2)  10 (2,5) 404 5-денний змагальний 3 654 (41,6) 888 (56,5)  3,0 (1,9) 1572 7-денний змагальний 1 303 (42,1) 407 (56,5)  10 (1,4) 720 Усього 22 4867 (57,9) 3070 (36,5) 247 (2,9) 229 (2,7) 8413 Обсяг і співвідношення тренувальних навантажень висококваліфікованих хокеїстів на траві у змагальному періоді мак (формувальний експеримент) Тренувальні вправи на основі аеробних навантажень найбільшою мірою використовувалися у 3-денному відновлювальному (97,3%), 3-денному відновлювально-підвідному (85,4%), 7-денному відновлювальному (77,7%) та у 4- денному утягувальному (72,3%) мікроциклах. Найбільше використання змішаних (аеробно-анаеробних) навантажень було характерним для 4-денного ударного (36,4%), 3-денного підвідного (44,8%), 6- денного підвідного (41,5%) та змагальних (від 53,2 до 56,5%) мікроциклів. Найбільше використання змішаних (аеробно-анаеробних) навантажень було характерним для 4-денного ударного (36,4%), 3-денного підвідного (44,8%), 6- денного підвідного (41,5%) та змагальних (від 53,2 до 56,5%) мікроциклів. Що стосується анаеробних навантажень, то вони, зазвичай, планувалися при проведенні 4-денного ударного мікроциклу (19,7%), в якому переважно використовували програму тренувальних завдань щодо розвитку швидкісних, швидкісно-силових якостей, швидкісної та спеціальної витривалості. У підвідних мікроциклах частка анаеробних навантажень коливалася від 8,5 до 11,5%. 259 IІ. Науковий напрям Подібна тенденція планування аеробних навантажень стосувалася міжігрових мікроциклів (рис. 4.). У цих мікроциклах частка анаеробно-алактатних навантажень складала 3,3%, а анаеробно-гліколітичних – 7,0%. 89,6 85,4 74,3 43,9 56,6 51,6 42,1 10,4 14,6 27,7 36,4 34,5 38,1 56,0 0 0 0 14,7 4,0 3,3 2,0 0 0 0 5,0 4,9 7,0 4,9 0 20 40 60 80 100 1 2 3 4 5 6 7 % Рис. 4. IІ. Науковий напрям Співвідношення навантажень різної спрямованості в межах різних типів мікроциклів у змагальному періоді підготовки висококваліфікованих хокеїстів на траві (формувальний експеримент), % 89,6 85,4 74,3 43,9 56,6 51,6 42,1 10,4 14,6 27,7 36,4 34,5 38,1 56,0 0 0 0 14,7 4,0 3,3 2,0 0 0 0 5,0 4,9 7,0 4,9 0 20 40 60 80 100 1 2 3 4 5 6 7 % Рис. 4. Співвідношення навантажень різної спрямованості в межах різних типів мікроциклів у змагальному періоді підготовки висококваліфікованих хокеїстів на траві (формувальний експеримент), % Рис. 4. Співвідношення навантажень різної спрямованості в межах різних типів мікроциклів у змагальному періоді підготовки висококваліфікованих хокеїстів на траві (формувальний експеримент) % р ц у у р ф (формувальний експеримент), % (ф р у р ) 1 – відновлювальний мікроцикл; 2 – відновлювально-підвідний мікроцикл; 3 – утягувальний мікроцикл; 4 – ударний мікроцикл; 5 – підвідний мікроцикл; 6  міжігровий мікроцикл; 7 – змагальний мікроцикл р ц – аеробні навантаження; – змішані (аеробно-анаеробні) навантаження; – анаеробно-алактатні навантаження; – анаеробно-гліколітичні навантаження. Кількісні показники основних параметрів підготовки висококваліфікованих хокеїстів на траві у змагальному макроциклі представлено в табл. 3. Кількісні показники основних параметрів підготовки висококваліфікованих хокеїстів на траві у змагальному макроциклі представлено в табл. 3. Кількісні показники основних параметрів підготовки висококваліфікованих хокеїстів на траві у змагальному періоді макроциклу (формувальний експеримент) Кількість параметрів підготовки Мікроцикли Усього Утягува - льні Ударні Підвідні Міжігров і Змагальні Віновл ю- вальні Відновл ю- вально- підвідні Контрольних ігор   3 2    5 Календарних ігор     20   20 Тренувальних днів 4 8 17 5 26 14 15 89 З одноразовими заняттями 3 2 12 4 25 8 15 69 З двохразовими заняттями 1 6 5 1    13 Тренувальних занять 5 14 20 6 25 13 15 99 Відновлювальних   2  1 9 10 22 Підтримувальних 1     3  4 Розвивальних 4 14 18 6 24 2 5 73 Днів тестування   1 1    2 Теоретичних занять 1 4 12  18 2 10 52 Днів відпочинку      3 5 8 Обсяг тренувальних та змагальних впливів, хв (год) 350 (5,8) 1060 (17,7) 1816 (30,3 541 (9,0) 2696 (44,9) 920 (15,3) 1030 (17,2) 8413 (140) Кількісні показники основних параметрів підготовки висококваліфікованих хокеїстів на траві у змагальному періоді макроциклу (формувальний експеримент) 260 IІ. IІ. Науковий напрям Науковий напрям Отже, вплив тренувальних навантажень на рівень підготовленості гравців планувався відповідно до завдань, що ставилися у тому чи іншому мікроциклі. При створенні програм відновлювальних і втягувальних мікроциклів використовувалися програми тренувальних завдань із переважним аеробним фізіологічним забезпеченням рухової діяльності. В ударних, підвідних і міжігрових мікроциклах застосовувалися тренувальні програми з комплексним впливом аеробних, змішаних та анаеробних навантажень. Дискусія. Управління підготовкою спортсменів обумовлено цілеспрямованим плануванням тренувальних впливів через використання засобів тренувальної роботи та навантажень різної спрямованості [3, 4, 11]. У нашому дослідженні важливо було обрати науково-методичний підхід на основі якого здійснювався тренувальний процес висококваліфікованих хокеїстів на траві у змагальному періоді макроциклу. Системний аналіз літературних джерел дозволив прийти до висновку, що одним із ефективних таких підходів є побудова тренувального процесу спортсменів на основі методів програмування [1, 4, 6, 16]. Програмування тренувального процесу передбачає чітке планування та контроль навантажень з урахуванням їх величини, інтенсивності та координаційної складності [6, 18]. З цією метою в досліджені була використана методика контролю тренувальної роботи спортсменів командних ігрових видів спорту, що була розроблена В.М. Костюкевичем [4, 5]. Тобто, у тренувальних заняттях плануванню та контролю підлягали: види тренувальної роботи – загальна фізична, спеціальна фізична, техніко-тактична, ігрова та змагальна підготовки; неспецифічні та специфічні вправи, що використовувалися у трьох режимах координаційної складності – загально-підготовчі, спеціально-підготовчі, підвідні та змагальні; навантаження різної спрямованості – аеробні, змішані (аеробно- анаеробні), анаеробно-алактатні, анаеробно-гліколітичні; величина навантаження, що визначалася в балах залежно від витрат енергії; коефіцієнт інтенсивності навантаження, що визначався відношенням величини навантаження до тривалості виконання вправ. Отже, на основі вищевикладеного в дослідженні було визначено параметри тренувальної роботи висококваліфікованих хокеїстів на траві у змагальному періоді другого циклу річної підготовки, що відносився до формувального етапу експериментального наукового пошуку. У табл. 4 представлено загальні параметри тренувальної роботи висококваліфікованих хокеїстів на траві на етапах констатувального [7] та формувального експерименту. Як видно з табл. 4 на етапі формувального експерименту збільшилося співвідношення загально-підготовчих (13,0%) і спеціально-підготовчих (3,5%) вправ, з одночасним зменшенням частки підвідних (14,0%) і змагальних (2,3%) вправ. Це обумовлено тим, що при розробці програм тренувальних завдань більш цілеспрямовано планувалися стимуляційні та відновлювальні фази під час тренувальних впливів. У той же час, на етапі формувального експерименту на 1,7% збільшилася частка анаеробних навантажень, що дозволило підвищити рівень підготовленості висококваліфікованих хокеїстів на траві. 261 IІ. IІ. Науковий напрям Науковий напрям Таблиця 4 Таблиця 4 Параметри тренувальної роботи висококваліфікованих хокеїстів на траві у змагальному періоді макроциклу на етапах констатувального та формувального експерименту Параметри тренувальної роботи РКС Етап педагогічного експерименту Зміна констатувальний формувальний Засоби тренувальної роботи, % Загально-підготовчі вправи 1 36,5 87,4 49,5 13,0 2 15,2 3 0,4 Спеціально-підготовчі вправи 1 9,0  12,5 3,5 2 91,9 3 81,1 Підвідні вправи 1 29,8 5,1 15,8 +14,0 2 53,6 3 41,3 Змагальні вправи 1 24,5 12,3 22,2 +2,3 2 51,1 3 36,6 Навантаження, % Аеробні  56,0 57,9 1,9 Анаеробно-анаеробні  41,1 36,5 +5,1 Анаеробно-алактатні  1,9 2,9 1,0 Анаеробно-гліколітичні  1,0 2,7 0,7 Тренувальних днів  98 88 +10,0 Тренувальних занять  105 99 +6,0 Теоретичних занять  47 52 5,0 Днів відпочинку  9 8 +1 Контрольних ігор  13 5 +8,0 Календарних ігор  18 20 2,0 Днів тестування  4 2 +4,0 Загальний ігровий обсяг  12038 хв (201 год) 8413 хв (140 год) +61,0 год Варто зазначити, що наше дослідження було проведено з дотриманням основних положень теорії періодизації спортивного тренування [9, 10, 11, 15], що дозволяє розширити як теоретичну, так і практичну складову цієї теорії з урахуванням специфічних особливостей олімпійського виду спорту – хокею на траві. Висновки. Ефективне управління процесом підготовки спортсменів обумовлене цілеспрямованим плануванням і контролем тренувальних впливів, що здійснюються через використання засобів тренувальної роботи та навантажень різної величини, інтенсивності та спрямованості. У командних ігрових видах спорту, у т.ч. в хокеї на траві у тренувальному процесі використовуються в залежності від етапу річного макроциклу в різному співвідношенні загально-підготовчі, спеціально-підготовчі, підвідні, змагальні вправи, а також аеробні, аеробно-анаеробні, анаеробно-алактатні, анаеробно- гліколітичні навантаження. 262 References Список літературних джерел Список літературних джерел IІ. Науковий напрям IІ. Науковий напрям Встановлено, що у змагальному періоді макроциклу в тренувальному процесі висококваліфікованих хокеїстів на траві використання засобів тренувальної роботи характеризується таким співвідношенням: загально- підготовчих вправ – 49,5%, спеціально-підготовчих вправ – 12,5%, підвідних вправ – 15,8%, змагальних вправ – 22,2%. Частка впливу тренувальних навантажень у цьому періоді складає: аеробних – 57,9%, аеробно-анаеробних – 36,5%, анаеробно-алактатних – 2,9%, анаеробно- гліколітичних – 2,7%. У межах змагального періоду окремого макроциклу проводиться: 88 тренувальних днів, 99 тренувальних занять, 52 теоретичних занять, 5 контрольних ігор, 20 календарних ігор, днів відпочинку – 8, днів тестування – 2. Загальний обсяг рухової тренувальної роботи у змагальному періоді окремого макроциклу складає 140 год. Визначені загальні параметри тренувальної роботи можуть розглядатися як модельні при підготовці клубних і збірних команд з хокею на траві у змагальному періоді. 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Відомості про автора: Коннов С. Р.; orcid.org/0000-0002-2166-1735; konnovstas12345@gmail.com; Вінницький державний педагогічний університет імені Михайла Коцюбинського, вул. Острозького 32, м. Вінниця, 21001, Україна. 265
https://openalex.org/W4240145385	https://qmro.qmul.ac.uk/xmlui/bitstream/123456789/11470/2/Nelson%20The%20role%20of%20planetary%20formation%20and%20evolution%202015%20Published.pdf	English	null	The role of planetary formation and evolution in shaping the composition of exoplanetary atmospheres	Springer eBooks	2,014	cc-by	12,910	Exp Astron (2015) 40:501–522 DOI 10.1007/s10686-014-9401-6 Exp Astron (2015) 40:501–522 DOI 10.1007/s10686-014-9401-6 ORIGINAL ARTICLE M. Barbieri Centro Interdipartimentale di Studi e Attivit`a Spaziali CISAS, Universit`a di Padova, Via Venezia 15, 35131, Padova, Italy 1 Introduction The primary objective of space missions devoted to the characterization of extrasolar planets, like the ESA M3 mission candidate Exoplanet Characterization Observatory (EChO), is to accurately measure transmission spectra for extrasolar planets during primary transits, and day-side spectra from secondary eclipses. An operating wavelength range in the near infrared and mid infrared (respectively NIR and MIR in the following) covers key absorption signatures from the molecular species CH4, H2O, CO2 and CO, whose relative strengths scale with the abundances present in the planetary atmosphere. Chemical modelling shows that these abundances are particularly sensitive to the heavy-element content of the atmosphere (measured through the C/H and O/H ratios), and the C/O ratio (see [34], for example). Determination of the atmospheric elemental abundances from the measured spectra will provide important constraints on the formation, migration and enrichment history of the observed extrasolar planets. Spectroscopic observations of extrasolar planets using Hubble Space Telescope and Spitzer have already confirmed the existence of various elements and molecules such as sodium, water, methane, and carbon dioxide in the atmospheres of hot-Jupiters (e.g. [43, 46]). Recent observations of the transiting hot-Jupiter Wasp-12b suggest an atmosphere abundant in CO and deficient in H2O, consistent with an atmospheric C/O ratio >1, in contrast to the solar value C/O=0.54 [27]. Analysis of transmission and day-side spectra for the transiting 6.5 MEarth super-Earth GJ 1214b suggest a metal-rich atmosphere (e.g. [3]), in agreement with the general expectation that low mass planets will be well-endowed with heavy elements. A similar conclusion has been reached for the hot-Neptune GJ 436b, whose day-side spectrum lacks a clear signature of CH4 while displaying abundant CO and CO2 [28]. The derived carbon chemistry mixing ratios are consistent with chemical models that assume a heavy element abundance enhanced above solar by a factor >50 [34]. Although these and other data pertaining to extrasolar planetary atmospheres are tantalising, uncertainties originating in the relatively low signal to noise ratio, and low spectral resolution, mean that definitive conclusions concerning atmospheric abundances cannot be made. These data are not accurate enough to discriminate between different formation and migration scenarios for the observed planets. The spectral resolution and signal to noise ratio to be achieved by future space NIR/MIR missions similar to EChO will dramatically improve the situation and allow atmospheric compositions to be measured with unparalleled accuracy. The role of planetary formation and evolution in shaping the composition of exoplanetary atmospheres already known about the factors influencing the composition of planetary atmo- spheres, focusing on the case of gaseous giant planets, and what instead still need to be investigated. Keywords Planetary formation · Migration · Atmospheres · Giant planets · Space missions · Extrasolar planets Keywords Planetary formation · Migration · Atmospheres · Giant planets · Space missions · Extrasolar planets The role of planetary formation and evolution in shaping the composition of exoplanetary atmospheres D. Turrini ·R. P. Nelson·M. Barbieri Received: 20 January 2014 / Accepted: 12 June 2014 / Published online: 31 July 2014 © The Author(s) 2014. This article is published with open access at Springerlink.com Abstract Over the last twenty years, the search for extrasolar planets has revealed the rich diversity of outcomes from the formation and evolution of planetary systems. In order to fully understand how these extrasolar planets came to be, however, the orbital and physical data we possess are not enough, and they need to be comple- mented with information about the composition of the exoplanets. Ground-based and space-based observations provided the first data on the atmospheric composition of a few extrasolar planets, but a larger and more detailed sample is required before we can fully take advantage of it. The primary goal of a dedicated space mission like the Exoplanet Characterization Observatory (EChO) proposal is to fill this gap and to expand the limited data we possess by performing a systematic survey of extrasolar planets. The full exploitation of the data that space-based and ground-based facilities will provide in the near future, however, requires knowledge about the sources and sinks of the chemical species and molecules that will be observed. Luckily, the study of the past history of the Solar System provides several indications about the effects of processes like migration, late accretion and secular impacts, and on the time they occur in the life of planetary systems. In this work we will review what is D. Turrini () Istituto di Astrofisica e Planetologia Spaziali INAF-IAPS, Via Fosso del Cavaliere 100, 00133, Rome, Italy e-mail: diego.turrini@iaps.inaf.it R. P. Nelson School of Physics and Astronomy, Queen Mary University of London, Mile End Road, London, E1 4NS, U.K. R. P. Nelson School of Physics and Astronomy, Queen Mary University of London, Mile End Road, London, E1 4NS, U.K. M. Barbieri Centro Interdipartimentale di Studi e Attivit`a Spaziali CISAS, Universit`a di Padova, Via Venezia 15, 35131, Padova, Italy M. Barbieri Centro Interdipartimentale di Studi e Attivit`a Spaziali CISAS, Universit`a di Padova, Via Venezia 15, 35131, Padova, Italy 502 Exp Astron (2015) 40:501–522 already known about the factors influencing the composition of planetary atmo- spheres, focusing on the case of gaseous giant planets, and what instead still need to be investigated. 1 Introduction Combining these data with estimates of planetary bulk compositions from accurate measure- ments of their radii and masses will allow degeneracies associated with planetary Exp Astron (2015) 40:501–522 503 interior modelling to be broken (e.g. [1]), giving unique insight into the interior structure and elemental abundances of these alien worlds. interior modelling to be broken (e.g. [1]), giving unique insight into the interior structure and elemental abundances of these alien worlds. Understanding the link between the history of a planetary system and the atmos- pheric composition of its planets, however, is not only a problem of increasing and refining the sample of observational data. Also our comprehension of the formation and evolution of planetary systems needs to be expanded, and different open issues need to be addressed. The original view of the set of events and mechanisms involved in planetary formation, in fact, was derived from observations of the Solar System as it is today. The assumption derived from these observations was that planetary formation is a local, orderly process that produces regular, well-spaced and, above all, stable planetary systems and orbital configurations. However, with the discovery of increasing numbers of extrasolar planetary systems through ground- and space-based observations, it has become evident that planetary formation can result in a wide range of outcomes, most of them not obviously consistent with the picture derived from the observations of the Solar System. The orbital structure of the majority of the discovered planetary systems seems to be strongly affected by planetary migration. This can arise through the exchange of angular momentum with the circumstellar disk in which the forming planets are embedded (see e.g. [5, 38], and references therein), and through the so-called “Jumping Jupiters” mechanism [7, 29, 55], which invokes multiple planetary encoun- ters with a chaotic exchange of angular momentum and energy between the bodies involved. Each of these migration mechanisms has different implications for the chemical make-up of the planetary atmosphere, as migration through a disk allows the planet to accrete from regions with varying chemical abundances. This is not true for hot Jupiters reaching their present orbital location through planetary scattering. 1 Introduction The growing body of evidence that dynamical and collisional processes, often chaotic and violent, can dramatically influence the evolution of young planetary systems gave rise to the idea that our Solar System could have undergone the same kind of evolution and represent a “lucky” case in which the end result was a stable and regular planetary system. As we will discuss shortly, different attempts at modelling have been performed on this regard, but in the context of NIR/MIR observations the underlying and important idea is that the processes shaping the formation and evolution of planetary systems are general. As a consequence, on one hand there are lessons that can be drawn from the Solar System and used to shed light on the link between the history of a planetary system and the atmospheric composition of its giant planets. On the other hand, the insight that will be provided by future NIR/MIR space missions similar to EChO will prove invaluable to improve our understanding of our own Solar System and of the processes that made it a favourable environment for the rise of life. If we follow the description of the history of the Solar System by [11] and we generalize it, the life of planetary systems can be viewed as composed by three different phases (circumstellar disk, protoplanetary disk, fully formed planetary system), each characterized by different physical processes and different durations. This schematic view of the evolution of planetary systems is summarized in Fig. 1 (adapted and expanded from [11]), where we report the main events that can take 504 Exp Astron (2015) 40:501–522 Fig. 1 Timeline of the history of a generic planetary system following the division in three phases proposed by [11]. The events marking the transition between the different phases are in bold characters Fig. 1 Timeline of the history of a generic planetary system following the division in three phases proposed by [11]. The events marking the transition between the different phases are in bold characters place across the different phases. Giant planets must form during the circumstellar disk phase, since the gaseous component of the disk is required to provide the material for both the massive envelopes of Jupiter-like planets and the limited ones of Neptune-like planets. 1 Introduction Given that the processes that give rise to super-Earths are plausibly the same producing the cores of giant planets, also super-Earths can in principle complete their formation already during the circumstellar disk phase. Based on the case of the Solar System, terrestrial planets should instead complete their accretion after the dispersal of the gaseous component of the circumstellar disks. In the following we will not discuss the details of the mechanisms governing planetary formation: interested readers can find updated reviews on the formation of the terrestrial planets in [41] and [33], on the formation of the giant planets in [12], and on the subject of planetary migration in [38] and [5]. We will instead focus the discussion on the processes and events affecting the atmospheric composition of the giant planets, as they are the major players in shaping the evolution of planetary systems and they constitute the largest fraction of the targets that will be observed by future NIR/MIR instruments . 2 Planetary formation and composition The EChO mission proposal aimed to target super-Earths, Neptune-like and Jupiter- like exoplanets on relatively short period orbits. These broad classes of planets are all expected to have very different formation and migration histories that will be imprinted on their atmospheric and bulk chemical signatures. Within each of these planet taxonomic classes, the stochastic nature of planetary formation will be reflected in significant variations in the measured abundances, providing important information about the diverse formation and migration pathways experienced by planets that are members of the same broad class. Reconstructing formation histories Exp Astron (2015) 40:501–522 505 from spectral measurements presents a challenging inversion problem, but can nonetheless provide very useful constraints, as we detail below. from spectral measurements presents a challenging inversion problem, but can nonetheless provide very useful constraints, as we detail below. Formation processes and migration influence a planet’s composition in numerous ways. For example, we clearly expect gas giant planet formation via gravitational instability to result in very different bulk compositions and atmospheric abundances compared with planets that form through core accretion. In general one may expect planets formed via the former process to reflect the bulk composition of the nascent circumstellar disk, whereas planets formed through core accretion can display a range of abundance ratios that depend on the relative accretion rates for planetesimals and gas (see Fig. 2). Gas giant planets are expected to have atmospheric compositions very different from the presumably heavy element-rich atmospheres of super-Earths, if the examples of Uranus and Neptune in our Solar System provide a useful guide. Very little research has been done on this important question, mainly because the large uncertainties in current measurements of elemental abundances provide little in the way of discrimination between different models and scenarios. Future NIR/MIR missions similar to EChO will change this, stimulating in-depth analyses of the link between formation, migration, and post-formation enrichment. In the absence of existing detailed model results, we outline a number of different simplified formation and migration scenarios to illustrate how diverse atmospheric elemental abundances can arise. Formation of gas giants through gravitational instability during the earliest phases of a circumstellar disk evolution will lead initially to atmospheric abundances that are essentially the same as the central star’s (see Fig. 2). Recent studies show that rapid inward migration of planets formed in this way occurs on time scales ∼103 yr (e.g. 2 Planetary formation and composition [2, 60]), too short for significant dust growth or planetesimal formation to arise between formation and significant migration occurring. As such, migration and accompany- ing gas accretion should maintain the initial planetary abundances. As we describe later, post-formation enrichment may occur through bombardment from neighbour- ing planetesimals or long-period star-grazing comets, but this enrichment will occur in an atmosphere with abundances that are essentially equal to the stellar values and will take place at a time in which the population of planetesimals is reduced with respect to its initial value. In its simplest form, the core accretion model of planet formation begins with the growth and settling of dust grains, followed by the formation of planetesimals that accrete to form a planetary core. Growth of the core to a mass in excess of a few Earth masses allows settling of a significant gaseous atmosphere from the Formation by gravitational instability Formation by gravitational instability Planet is characterized by solar bulk composition Planet is characterized by solar bulk composition Formation by nucleated instability Formation by nucleated instability Planet is characterized by over- abundances in high-Z elements Planet is characterized by over- abundances in high-Z elements Fig. 2 Post-formation composition of a giant planet based on its formation process Formation by nucleated instability Formation by nucleated instability Planet is characterized by over- abundances in high-Z elements Planet is characterized by over- abundances in high-Z elements Formation by nucleated instability Formation by nucleated instability Formation by gravitational instability Formation by gravitational instability Planet is characterized by over- abundances in high-Z elements Planet is characterized by over- abundances in high-Z elements Planet is characterized by solar bulk composition Planet is characterized by solar bulk composition Planet is characterized by over- abundances in high-Z elements Planet is characterized by over- abundances in high-Z elements Fig. 2 Post-formation composition of a giant planet based on its formation proces Fig. 2 Post-formation composition of a giant planet based on its formation process 506 Exp Astron (2015) 40:501–522 surrounding nebula. Halting growth at this point results in a super-Earth or Neptune-like planet. Continued growth through accretion of planetesimals and gas can lead to runaway gas accretion, forming a Jupiter-like gas giant. 2 Planetary formation and composition 3 Locations of the ice-lines and their influence on the C/O ratios for the gas and solids (adapted from [37]) 507 Exp Astron (2015) 40:501–522 Various formation scenarios for gas giant planets are now assumed, based on Figs. 2 and 3, and their implications for the atmospheric C/O value are calculated. Various formation scenarios for gas giant planets are now assumed, based on Figs. 2 and 3, and their implications for the atmospheric C/O value are calculated. Scenario 1: A solid core forms at 5 au, undergoes type I migration inward, and only starts to accrete gas once it has moved interior to the H2O ice-line. Gas accretion is not accompanied by any accretion of solids, leading to an atmospheric C/O ratio ∼0.6. Such a scenario may apply when an earlier episode of planetary formation has occurred in the disc interior to 5 au, depleting the planetesimals that were orbiting there. Scenario 2: This is identical to scenario 1, except that gas accretion interior to the H2O ice-line is accompanied by accretion of solids, such that the C and O abundances of the accreted material equals the solar value. The atmospheric C/O ratio is ∼0.54. Here the inner disc would not have experienced an earlier episode of planet forma- tion, so planetesimals are available for accretion by the migrating giant as it migrates into this region and accretes gas. Scenario 3: A solid core forms at 5 au and accretes gas exterior to the H2O ice-line while migrating inward, without accreting any solids. Before crossing the H2O ice-line the planet opens a deep gap that prevents further accretion. The planet continues to migrate inward. The final C/O ratio is ∼0.86. In this scenario, the core of the giant planet must grow quickly as it forms at 5 au, allowing it to reach the critical core mass prior to migrating. Rapid gas accretion can then occur prior to gap formation and migration through the H2O ice-line. Scenario 4: A solid core forms at 15 au and accretes gas but no further solids from beyond the CO2 ice-line before forming a deep gap that prevents further gas accretion. The planet migrates inward to form a hot Jupiter and the final atmospheric C/O ratio is ∼1. In this scenario, the solid core of the planet needs to form and initiate gas accretion at quite large radius from the star. 2 Planetary formation and composition A key issue for determining the atmospheric abundances of a forming planet is the presence of ice-lines at various distances from the central star, where volatiles such as water, carbon dioxide and carbon monoxide freeze-out onto grains and are incorporated into planetesimals. Considering a typical protoplanetary disk orbiting a solar-type star, Fig. 3 shows that a H2O ice-line is expected at ∼2 au, a CO2 ice-line at ~10 au, and a CO ice-line at ∼40 au. The atmospheric abundances of a planet during formation will therefore depend on where it forms and the ratio of gas to solids accreted at late times (see Fig. 3). We recall that the EChO mission proposal focused on the study of planets with relatively short orbital periods, and these must have undergone large scale migration during their evolution: the inner regions of circumstellar disks contain too little solid material for in situ formation of planetary systems similar to those discovered by the Kepler mission (e.g. [26]) and radial velocity surveys (e.g. [31]). The final C/O ratio of the planet will therefore depend on how it accretes as it migrates through the disk. For the purpose of illustration, we now consider a number of highly simplified planetary accretion and migration scenarios and their influence on the atmospheric C/O ratio, which a space mission similar to EChO would measure for tens of giant planets (see [14] and, specifically, the “Origins” observational tier of EChO). The circumstellar disk is assumed to be of solar abundance, giving rise to an overall C/O ratio ∼0.54, as shown in Fig. 3. Interior to the H2O ice-line, carbon- and silicate- rich grains are condensed, leading to an increase of the gas-phase C/O ∼0.6 (due to the slight overabundance of oxygen relative to carbon in these refractory species). Water condenses between the H2O and CO2 ice-lines, increasing the gas-phase C/O ∼0.85 through removal of oxygen into the frozen-out H2O, and decreasing the solid phase C/O ∼0.26. Between the CO2 and CO ice-lines the CO2 freezes out, increasing the gas phase C/O ∼1 and moderately increasing the solid phase C/O ratio ∼0.31. Fig. 3 Locations of the ice-lines and their influence on the C/O ratios for the gas and solids (adapted from [37]) Fig. 2 Planetary formation and composition This may occur if planetary cores can migrate outward because of the strong influence of corotation torques, as considered by [39] and [20]. Rapid gas accretion leading to a gap-opening planet is likely to require the formation of a massive core out at 15 au. Scenario 5: A solid core forms at 15 au, migrates inward, and accretes equal amounts of gas from the region outside the CO2 ice-line, the region between the H2O and CO2 ice-lines, and the region interior the H2O ice-line. No solids are accreted. The final atmospheric C/O ratio is ∼0.77. This scenario can arise if outward migra- tion of planetary cores occurs, as described for scenario 4, but gas accretion occurs more gradually as the planet migrates inward. Here the core mass is likely to be smaller than in scenario 4, and/or the opacity of the gaseous envelope will be higher, leading to slower gas accretion. Scenario 6: A solid core forms at 15 au, migrates inward and accretes gas as in scenario 5, but also accretes 10 % of its atmosphere in the form of solids as it migrates. The final atmospheric C/O ratio is ∼0.73. The scenario arises when the 508 Exp Astron (2015) 40:501–522 conditions for scenario 5 are satisfied, and the inner disc regions retain a population of planetesimals that can be accreted by an incoming, migrating giant planet. All of the above scenarios assume gas-dominated accretion and lead to solar or super-solar atmosphere C/O ratios because of the tendency of O-rich compounds to freeze-out at higher temperatures. Sub-solar values of the C/O ratio can be obtained through substantial accretion of silicate-rich planetesimals as the planet migrates interior to the H2O ice-line. These examples simply serve to illustrate that a variety of formation, migration and accretion scenarios can lead to a broad distribution of C/O ratios. The final C/O value correlates with where and how the planet forms and migrates in a predictable manner, but this final value is not unique for all scenarios. Detailed predictions of the expected diversity of C/O ratios in planetary atmospheres require planetary formation models to be computed that account for the evolving chemistry of the protoplanetary disk and the chemical abundances of the accreted material. Finally, we note that gas disk-driven migration is only one plausible mechanism by which planets may migrate. 2 Planetary formation and composition As discussed previously, the large eccentricities (and obliquities) of the extrasolar planet population suggest that planet-planet gravitational scattering (“Jumping Jupiters”) may be important (e.g. [7, 29, 55]), and this is likely to occur toward the end of the gas disk lifetime when its ability to damp orbital eccen- tricities is diminished. When combined with tidal interaction with the central star, planet-planet scattering onto highly eccentric orbits can form short-period planets that have not migrated toward the central star while accreting from the circumstellar disk. These planets are likely to show chemical signatures that reflect this alternative formation history, being composed of higher volatile fractions if they form exterior to the H2O ice-line. 3 Post-formation evolution, late accretion and protoplanetary disks Immediately after (~105 years) they form, giant planets trigger a phase of intense remixing of solid material in the protoplanetary disk in which they are embedded [11, 42, 49, 50, 56, 57], which manifest as a bombardment on the other planetary bodies populating the forming system. In the Solar System this event has been named the Jovian Early Bombardment [11, 48–51] as Jupiter was likely the first planet to form [11, 42]. As a new phase of remixing and bombardment, likely of decreasing intensity, will be triggered by the formation of each giant planet in a planetary system hosting more than one, a more general name for this class of events is the Primordial Heavy Bombardments [11]. The duration of the phase of bombardment and remixing triggered by the formation of Jupiter in the Solar System was estimated to be of about 0.5–1 Ma [49, 50, 56]. The bombardment is caused by the interplay between the gravitational scattering of planetesimals near-by the newly formed giant planet and the appearance of the orbital resonances in regions farther away [42, 48–51, 56, 57]. From the point of view of future NIR/MIR observations by space missions simi- lar to EChO, the most important effect of this class of events is the reshuffling of the solid material present in the circumstellar disks: volatile-rich objects from beyond the H2O ice-line are injected into the inner, volatile-depleted regions of the disk Exp Astron (2015) 40:501–522 509 Fig. 4 Orbital distribution of the Solar Nebula 2×105 years after the beginning of the accretion of the nebular gas by Jupiter in the simulations performed by [50]. Planetesimals that formed between 2 au and 4 au are indicated in red, those that formed between 4 au and 7 au in light blue and those that between 7 au and 10 au in dark blue. The open circle is the position of Jupiter at the beginning of the gas accretion phase, the filled one is the position of Jupiter once fully formed Fig. 4 Orbital distribution of the Solar Nebula 2×105 years after the beginning of the accretion of the nebular gas by Jupiter in the simulations performed by [50]. Planetesimals that formed between 2 au and 4 au are indicated in red, those that formed between 4 au and 7 au in light blue and those that between 7 au and 10 au in dark blue. 3 Post-formation evolution, late accretion and protoplanetary disks standing on its initial orbit, hereafter labelled as the no migration case), • the giant planet not migrating (i.e. standing on its initial orbit, hereafter labelled as the no migration case), • the giant planet migrating to a semimajor axis of 0.7 AU with an e-folding time of 5×103 years (i.e. 99.4 % of the migration is completed in 2.5×104 years, hereafter labelled as the fast migration case), and • the giant planet migrating to a semimajor axis of 0.7 AU with an e-folding time of 5×103 years (i.e. 99.4 % of the migration is completed in 2.5×104 years, hereafter labelled as the fast migration case), and • the giant planet migrating to a semimajor axis of 0.7 AU with an e-folding time of 3×104 years (i.e. 99.4 % of the migration is completed in 1.5×105 years, hereafter labelled as the slow migration case). Migration, when occurring, always starts after 104 years from the beginning of the simulations. The migration scheme is implemented following [19]. As shown in Fig. 5, about 33 % to 50 % of the impacting particles are accreted by the giant planet extremely quickly in the first 104 years. Then, if the giant planet migrates, the late accretion phase slows down significantly for the first 2 e-folding times (i.e. the faster part of the migration, while the giant planet completes about 86 % of its displace- ment). Accretion starts again across the next 3 e-folding times (i.e. the slowest part of the migration, while the giant planet completes about 13 % of its displacement). If the giant planet does not migrate, the late accretion extends over about 5×105 years. Accretion in the no migration case is the most efficient, with the giant planet cap- turing about 4.8 % of the solid material in the circumstellar disk. The slow migration is more efficient accretion-wise than the fast migration case, with 3.8 % vs 3.3 % of the solid material of the disk captured by the giant planet. Assuming the disk is analo- gous to the Minimum Mass Solar Nebula and has a surface density profile governed by the relationship σ = 2700 r−3/2 g cm−2 [10] where r is the orbital distance in au, the mass of the disk we considered would be about 27.5 M⊕(where the symbol M⊕ indicates the mass of the Earth). 3 Post-formation evolution, late accretion and protoplanetary disks The open circle is the position of Jupiter at the beginning of the gas accretion phase, the filled one is the position of Jupiter once fully formed while rocky, metal-rich bodies are transferred from the latter to the former. The net effect is a change in the rock-ice, metal-ice and likely of the H2O-CO2 ice ratios in the different regions of the circumstellar disks, as can be seen by comparing Figs. 3 and 4. During this phase of remixing, the orbital regions of the giant planets are crossed by these fluxes of planetesimals and a fraction of the migrating material is captured by the giant planets themselves, as already pointed out by [56] for the case of Jupiter, thus enriching their atmospheric composition in high-Z elements. To illustrate the effects of these events and the implications for the interpretation of NIR/MIR observations, we will take advantage of the following toy model. We consider a Jupiter-sized giant planet embedded into a disk of massless particles representing the circumstellar disk. Dynamical friction between the bodies popu- lating the circumstellar disk and the effects of gas drag are ignored for simplicity. The giant planet is initially on a planar (i = 0◦) and circular (e = 0) orbit with the semimajor axis of Jupiter (a = 5.2 au). The massless particles are distributed between 1 and 10 au, leaving empty the region between 4.7 and 5.7 au to simu- late the gap created by the formation of the giant planet. The massless particles initially have eccentricities randomly distributed between 0 and 0.1 [54] and inclina- tions randomly distributed between 0◦and 1.7◦[50]. The H2O ice-line is assumed to be at 4.0 AU and the massless particles are divided into compositional classes according to their semimajor axes. Using the Solar System as a template, bodies inside 3 AU are considered composed of a mixture of rocks and metals analo- gous to ordinary chondrites. Bodies in the region between 3.0 AU and 4.0 AU are assumed transitional bodies depleted in metallic iron (most iron is in oxydized form) and enriched in carbon and water similarly to the carbonaceous chondrites. Finally, bodies from beyond 4.0 AU are assumed to be volatile-rich bodies similar to the comets. Exp Astron (2015) 40:501–522 510 We consider three scenarios: • the giant planet not migrating (i.e. 3 Post-formation evolution, late accretion and protoplanetary disks Assuming that the giant planet had a core of 5 M⊕, this leaves a total of 22.5 M⊕in the massless particles. The accreted masses would then translate in 1.1 M⊕(no migration scenario), 0.86 M⊕(slow migration scenario) and 0.75 M⊕(fast migration scenario). Fig. 5 Number of impacts on the giant planet as a function of time in the three scenarios considered. The vertical dashed line marks the beginning of the planetary migration in those scenario where it is present. The initial number of massless particles is 20000 Fig. 5 Number of impacts on the giant planet as a function of time in the three scenarios considered. The vertical dashed line marks the beginning of the planetary migration in those scenario where it is present. The initial number of massless particles is 20000 511 Exp Astron (2015) 40:501–522 As the dynamical and physical model underlying this toy model is quite simplis- tic, these numbers should be regarded just as more detailed back-of-the-envelope calculations. Yet, they provide a first indication of the effects of the post-formation accretion. While the overall accretion efficiency varies between the three scenarios, Fig. 6 shows that the relative importance of the different source regions in the cir- cumstellar disk varies little. This means that a newly formed giant planet can quickly accrete solid material from a vast feeding zone characterized by different composi- tions of the planetesimals. About 40 % of the accreted bodies originate from the inner (1 – 4 AU) region of the protoplanetary disk. ( ) g p p y If we use water and the most abundant elements (Si, C, N, S, Fe) as tracers of the composition of the accreted bodies, bodies originating between 1 – 3 AU would be composed (values derived from [22]) by Si (∼19 wt %), Fe (∼25 wt %), C (0.5 wt %), and S (1.8 wt %). Bodies coming from the 3 – 4 AU transition region would be composed (values derived from Jarosewich 1990) by Si (∼14 wt %), Fe (∼25 wt %), C (∼2 wt %), S (1.8 wt %), and H2O (∼10 wt %). 3 Post-formation evolution, late accretion and protoplanetary disks ∼20 % of the planetary mass, [18]) of initial solar composition in a homoge- nous way, so that the atmospheric composition is the same as that of the molecular shell. As a first approximation, we will ignore the effects of the chemistry induced by the temperature and pressure profiles of the atmosphere (e.g. if C is in the form of CO/CO2 it will reduce the amount of O available to form H2O, while the same is not true if C is in the form of CH4). As can be seen by comparing the values reported in Table 1 with the sensitivity of the three observational tiers of EChO (in order of increasing sensitivity: “Chemical Census”, “Origins” and “Rosetta Stones”; [14]), a space mission similar to EChO would be capable of detecting the species associated to the elements reported in Table 1 already in its lowest sensitivity tier (i.e. the “Chemical Census”, [14]). It is worth noting that, in principle, the sensitivity of the “Chemical Census” tier would be enough to detected enrichments respect to stellar abundances and to discriminate, for two otherwise similar planets, which one underwent migration and which one formed in situ: this indicates that a mission like EChO could provide us with a first look into the dynamical histories of hundreds of exoplanets during its operational lifetime. The intermediate sensitivity tier, the “Origins” tier [14], could then allow to study in more detail the past orbital evolution of tens of exoplanets. Primordial Heavy Bombardment, in Table 1 we show the final mixing ratios for a Jupiter-like planet in the no migration scenario and the differences between the mix- ing ratios in the no migration, fast migration and slow migration scenarios and, as a reference, the differences between the no migration scenario and solar abundances (data from [21]) using the previously reported compositions of the planetesimals. The accreted mass is assumed to be distributed into a molecular shell of 5000 km (i.e. ∼20 % of the planetary mass, [18]) of initial solar composition in a homoge- nous way, so that the atmospheric composition is the same as that of the molecular shell. As a first approximation, we will ignore the effects of the chemistry induced by the temperature and pressure profiles of the atmosphere (e.g. 3 Post-formation evolution, late accretion and protoplanetary disks Finally, half the mass of those bodies originating beyond the H2O ice-line (4 – 10 AU) will be assumed to have the same refractory-rich composition of carbonaceous chondrites, while the volatile- rich half of their mass will be mostly composed by H2O (∼74 wt %), C-bearing molecules (∼24 wt %), N-bearing molecules (∼0.7 %), and S-bearing molecules (∼1.2 wt %), where the wt % ratios are estimated from the abundances reported by [35]. Because of the condensation sequence of the different elements and chem- ical species in the protoplanetary disk [25], therefore, the post-formation accretion phase would bring to the giant planet high-Z materials with relative abundances of the different elements that are highly non-solar. As a consequence, the effects of this process should reflect into the C/O, S/O, N/O ratios and (possibly) in the content of silicates and metals in the atmospheres of the giant planets. Metals and sil- icates, however, had been observed by the Galileo spacecraft after the impact of the comet Shoemaker-Levy 9 on Jupiter only for a limited time [44], which could imply that they are removed efficiently (i.e. in a matter of months) from the observable regions of the atmosphere of a giant planet in contrast to water, for example (see also Section 4). As an example of the observable effects of late accretion during the Fig. 6 Fractions of the capture particles coming from the different regions of the disk of massless particles (red for the no migration scenario, green and blue for the fast and slow migration scenarios respectively) Fig. 6 Fractions of the capture particles coming from the different regions of the disk of massless particles (red for the no migration scenario, green and blue for the fast and slow migration scenarios respectively) 512 Exp Astron (2015) 40:501–522 Primordial Heavy Bombardment, in Table 1 we show the final mixing ratios for a Jupiter-like planet in the no migration scenario and the differences between the mix- ing ratios in the no migration, fast migration and slow migration scenarios and, as a reference, the differences between the no migration scenario and solar abundances (data from [21]) using the previously reported compositions of the planetesimals. The accreted mass is assumed to be distributed into a molecular shell of 5000 km (i.e. 3 Post-formation evolution, late accretion and protoplanetary disks if C is in the form of CO/CO2 it will reduce the amount of O available to form H2O, while the same is not true if C is in the form of CH4). As can be seen by comparing the values reported in Table 1 with the sensitivity of the three observational tiers of EChO (in order of increasing sensitivity: “Chemical Census”, “Origins” and “Rosetta Stones”; [14]), a space mission similar to EChO would be capable of detecting the species associated to the elements reported in Table 1 already in its lowest sensitivity tier (i.e. the “Chemical Census”, [14]). It is worth noting that, in principle, the sensitivity of the “Chemical Census” tier would be enough to detected enrichments respect to stellar abundances and to discriminate, for two otherwise similar planets, which one underwent migration and which one formed in situ: this indicates that a mission like EChO could provide us with a first look into the dynamical histories of hundreds of exoplanets during its operational lifetime. The intermediate sensitivity tier, the “Origins” tier [14], could then allow to study in more detail the past orbital evolution of tens of exoplanets. As mentioned above, the reshuffling process started by the Primordial Heavy Bombardment has a duration of about 1 Ma. At its end, however, it transitions (in the classical scenario for the formation of the Solar System) into a longer phase of reshuf- fling and dynamical clearing where the planetary embryos in the protoplanetary disk Table 1 Mixing ratios of a Jupiter-like planet in the no migration scenario and differences in the mixing ratios between the no migration. Fast migration and slow migration scenarios and between the no migration scenario and solar abundances (data from [21]), where the captured mass is homogeneously mixed into a 5000 km thick outer molecular shell of initial solar composition Table 1 Mixing ratios of a Jupiter-like planet in the no migration scenario and differences in the mixing ratios between the no migration. Fast migration and slow migration scenarios and between the no migration scenario and solar abundances (data from [21]), where the captured mass is homogeneously mixed into a 5000 km thick outer molecular shell of initial solar composition Table 1 Mixing ratios of a Jupiter-like planet in the no migration scenario and differences in the mixing ratios between the no migration. 3 Post-formation evolution, late accretion and protoplanetary disks The best estimate to date is the one done by [16], who assessed the capture efficiency of the four giant planets during the 100 Ma following their formation. The work of [16] was based on a simplified model similar to the toy model we used to illustrate the effects of the Primordial Bombardment. The disk of massless particles extended from 4 AU to 35 AU and planetary embryos were not included in the simulations. The cumulative capture efficiency of the four giant planets was found to be about 4 %, i.e. of the same level as the one found with our toy model for Jupiter alone. It would therefore appear that the combined perturbations of the giant planets, once they are all present in the planetary system, and the effects of concurrent accretion make late accretion very inefficient after the first few Ma (ejection from the planetary system is favoured). This is confirmed also by [16], who in a second set of simulations show that the accretion efficiency of Jupiter alone could rise up to 7–8 % in a disk extending between 4 AU and 13 AU, so twice as much as that of the four giant planets cumulatively. Extensive migration of the giant planets, both due to the interaction with the disk or to planet-planet scattering (i.e. the Jumping Jupiter mechanism, [7, 29, 55]) can supply an alternate evolutionary path to the previously described picture derived from the Solar System. The dynamical effects of the migrating planet on the protoplanetary disk would replace the slow erosion due to the interplay between planetary embryos and orbital resonances with the giant planets, but the end result would be analogous: the reshuffling of material from different regions of the protoplanetary disk and the depletion of the population of planetesimals with a consequence capture of part of the removed population by the giant planets. As our toy model shows for a very simple configuration of the planetary system, the migrating giant planet would still capture material from a wide range of orbital distances. Another, more complex example is constituted by the so-called “Grand Tack” scenario [52, 53], where the four giant planets of the Solar System are suggested to have migrated inward, then outward and to get locked in the resonant configuration they were suggested to be before the Late Heavy Bombardment [24]. 3 Post-formation evolution, late accretion and protoplanetary disks Fast migration and slow migration scenarios and between the no migration scenario and solar abundances (data from [21]), where the captured mass is homogeneously mixed into a 5000 km thick outer molecular shell of initial solar composition Table 1 Mixing ratios of a Jupiter-like planet in the no migration scenario and differences in the mixing ratios between the no migration. Fast migration and slow migration scenarios and between the no migration scenario and solar abundances (data from [21]), where the captured mass is homogeneously mixed into a 5000 km thick outer molecular shell of initial solar composition No migration No migration No migration Slow Element/ No migration vs Solar vs Fast vs Slow Migration vs molecule Mixing ratios Abundances Migration Migration Fast migration Fe 7.7 × 10−3 6.6 × 10−3 7.5 × 10−4 5.3 × 10−4 2.2 × 10−4 Si 2.2 × 10−3 1.5 × 10−4 4.8 × 10−4 3.4 × 10−4 1.3 × 10−4 C 2.6 × 10−3 4.7 × 10−4 1.5 × 10−4 9.8 × 10−5 5.0 × 10−4 N 6.6 × 10−4 4.2 × 10−5 1.3 × 10−5 8.7 × 10−6 4.5 × 10−4 S 8.3 × 10−4 4.9 × 10−4 6.6 × 10−5 4.6 × 10−5 2.0 × 10−4 H2O 9.0 × 10−3 5.4 × 10−3 1.2 × 10−3 7.6 × 10−4 4.0 × 10−4 Exp Astron (2015) 40:501–522 513 scatter planetesimals inside the now-depleted locations of the orbital resonances with the giant planets. This phase has been studied, in the Solar System, to investigate the mass depletion of the asteroid belt [6, 36, 40, 58]. During this phase, the pop- ulation of planetesimals in the affected regions decays exponentially, decreasing by about two orders of magnitude in about 100 Ma (see e.g. [36]. Across these 100 Ma the giant planets continues to capture part of the solid material that is expelled by the resonances: in principle, this process could possibly be even more efficient in the case of giant planets on inner orbits (e.g. less than 1 AU) if they did not completely dispersed the planetary bodies populating the regions they crossed while migrating. Limited data are currently available on the implications of the phase of dynamical clearing for the composition of the giant planets taking into account the role of planetary embryos. 3 Post-formation evolution, late accretion and protoplanetary disks During their extensive migration, the giant planets would scatter and redistribute the primordial planetesimals in the Solar System and likely capture a fraction of them. While it has been argued that this scenario has a low prob- ability of reproducing the actual configuration of the Solar System [13], the richness of orbital configurations of the known extrasolar planets can imply that several of the 514 Exp Astron (2015) 40:501–522 other planetary systems differing from our Solar System can be the outcome of the “failed” cases of this kind of evolutionary path. The results of [16] on the concurrent accretion and of our toy model for the accretion efficiency of migrating planets sug- gest, however, that in such a scenario the fraction of captured material (i.e. the late accretion) would be low. A Jumping Jupiters evolution can also take place at a later time as has been hypothesized in the case of the Solar System by the so-called Nice Model [15, 32, 47]. The Nice Model is a Jumping Jupiter scenario formulated to link the event known as the Late Heavy Bombardment [45], assumed to have occurred about 600–800 Ma after the formation of the Solar System (see Fig. 1) to a migration event involving all the giant planets. In the Nice Model, the giant planets of the Solar System are postulated to have been initially located on a more compact orbital configuration than their present one and to interact with a massive primordial trans-neptunian region. The gravitational perturbations among the giant planets are initially mitigated by the trans-neptunian disk, whose population in turn is eroded. Once the trans-neptunian disk becomes unable to mitigate the effects of the interactions among the giant planets, the orbits of the latter become excited and a series of close encounters takes place. The end result of the Jumping Jupiters mechanism in the Nice Model is a small inward migration of Jupiter and marked outward migration of Saturn, Uranus and Neptune [24, 47]. Due to the late time at which this migration and the associated bombardment take place and the depletion previously occurred in the population of planetesimals, however, the amount of solid material captured by the giant planets across the Late Heavy Bombardment would be limited. [30] estimated that the accreted material would amount to 0.15 M⊕for Jupiter, 0.08 M⊕for Saturn and ∼0.05 M⊕for Uranus and Neptune. 3 Post-formation evolution, late accretion and protoplanetary disks In the case of Jupiter, the material accreted during such a late event would be about an order of magnitude lower than the one accreted immediately after its formation. These results collectively seem to indicate that the late accretion phase can in principle extend over a long temporal interval (500 Ma – 1 Ga) but that the magnitude of its effects decreases quickly with time, so that the main role is played by the first few Ma after the formation of a giant planet. Late accretion then turns into a slow, secular contamination process whose temporary effects, however, can have implica- tions for future NIR/MIR observations and their interpretation, as will be illustrated in the next section. 4 Secular contamination of planetary atmospheres Once they complete the most active and violent phases of their evolution, planetary systems enter a stationary phase governed by secular processes. During this phase, the main processes affecting the atmospheric composition of the planets are impacts, atmospheric chemistry and, in the case of short-period planets, the stellar radiation and wind. Impacts, in particular, allow the transfer of material between different planetary bodies and between different orbital regions, continuing the remixing process that acted across the early phases of the evolution of the planetary system but at a much lower rate. An example of this process in the Solar System is represented 515 Exp Astron (2015) 40:501–522 by the flux of comets impacting Jupiter, the most famous (and studied) of which is the impact of comet Shoemaker-Levy 9 (SL9 in the following) in 1994. Across the last 17 years the giant planet has been hit by five impactors: SL9 itself in 1994 (∼5 km in diameter), then a sub-km impactor in 2009 (∼10 m in diameter), two in 2010 (∼500 m in diameter the first and undetermined the second) and one in 2012 (<10 m in diameter). A sixth impact of a meteoroid was observed by Voyager 1 in 1979 (estimated mass of 11 kg, i.e. ∼10 cm in diameter, [9]). It must be stressed that these impact rates do not represent the real flux on Jupiter, but they are more likely a reflection of the observational coverage of the giant planet. g g p Recent results from the Herschel mission [4] indicate that the spatially-resolved distribution of stratospheric water for Jupiter cannot be explained by local processes or a steady state flux of interplanetary dust particles, and are instead a reflection of the impact of comet SL9. In particular, about 95 % of the stratospheric water con- tent of the giant planet as been reported to be due to this cometary impact. Similar but non-spatially resolved results were previously obtained for both water, CO and CO2, supporting the case for an external source for these molecules as the thermal and pressure profiles in the atmosphere of Jupiter create a transport barrier between troposphere and stratosphere (see [4] and reference therein), where water is expected to condense. The mixing ratio of stratospheric water modelled from the measure- ments of Herschel is 1.7×10−8 [4]. 4 Secular contamination of planetary atmospheres The coupling between the long persistence of water in the stratosphere of Jupiter and its limited mixing ratio is of particular importance, because the detection of such small mixing ratios requires NIR/MIR observations over long timescales, comparable to the persistence of water itself. To link the atmospheric composition of exoplanets to their formation it is there- fore mandatory to be able to discriminate the (plausibly) transient contribution in high-Z elements due to external sources from the constant one due to the bulk com- position of the planet. To get a zero-order estimate of the implications of secular, cometary impacts for the observations we can use a simple toy model. We consider the planet HD 189733 b as our test case. For this planet atmospheric water has been detected with a model mixing ratio of 5×10−4 [46], which is well inside the obser- vational capabilities of EChO of the “Chemical Census” tier described in the EChO Yellow Book [14]. Orbital and physical parameters for the host star and the planet were obtained from the Extrasolar Planets Encyclopaedia (www.exoplanet.eu). If we consider an atmospheric shell analogous in size to the Jovian stratosphere, i.e. about 300 km and with a average density of 2x10−8 g cm−3(from the geometric mean of the extreme values reported by [59] for the Jovian stratosphere), the water content of this shell would be about 2.5×1017 g. As possible impactors, we consider a population of star-grazing exocomets mod- elled after the 1265 Sun-grazing comets observed by SOHO since 1996: their orbital parameters were obtained from the JPL Small Bodies Database Search Engine (http:// ssd.jpl.nasa.gov/sbdb query.cgi). Sun-grazing comets observed by SOHO have high orbital inclination values (i.e. ∼140◦) therefore our preliminary estimate gives 1 impact on our test planet every 200 years. Assuming an ecliptic population of star- grazing exocomets, our preliminary estimate gives the larger flux of 1 impact every 20 years. The 100 comets for which we have estimates of the diameter range in size between 0.5 km to 60 km. If we consider the whole sample, the average diameter is 516 Exp Astron (2015) 40:501–522 about 5 km, i.e. the estimated size of comet Shoemaker-Levy 9, while if we ignore comets larger than 10 km, to compensate for the observational bias favouring larger objects with respect to smaller ones, the average diameter is about 3 km. We will consider this value as the reference one. 4 Secular contamination of planetary atmospheres Assuming a density of 1 g cm−3, the mass of these bodies would range between 5×1014 g (1 km) and 6.5×1016 g (5 km), with our reference case (3 km) being 1.4×1016 g. From [35], we can assume an average water content of 74 % of their mass. Therefore, each of these impactors would bring, on average, 1016 g. This implies that it would take the cumulative water budget of about 25 cometary impacts to reproduce the water content of the atmospheric shell we considered. Equiva- lently, the water content delivered by cometary impactors should be able to survive in said shell between 5×102 years (1 impact every 20 years) and 5×103 years (1 impact every 200 years). These values obviously assume that all the cometary water is released in the stratosphere of the exoplanet, which is not necessarily the case. Note that also this toy model should be regarded just as a more sophisticated back- of-the-envelope calculation, as the uncertainties on both the observational constrains and the assumptions can plausibly affect the order of magnitude of the values here considered. As an example of the uncertainties affecting these estimates, [46] report that changing the mixing ratio of water in the atmosphere of HD 189733 b by plus or minus one order of magnitude does not significantly affect the fit to the observational data. This uncertainty on the mixing ratio has significant implications for the time required to accumulate the expected water content. In the most favourable case (i.e. a mixing ration of 5×10−5), which would still be detectable in the “Chemical Census” tier described in the EChO Yellow Book [14], it would take only a couple of impacts to provide the required water budget or, alternatively, water would need to survive in the atmospheric shell only about 50 years (consider that water from the SL9 impact resided in the Jovian stratosphere for at least 14 years at the time of the observations used by [4]). In the most unfavourable case, cometary water would need about 5×104 years to accumulate. From an observational perspective, the effects of the secular contamination can be expressed in terms of the mixing ratio that cometary impactors would produce. 5 Planetary processes affecting atmospheric composition after cometary or asteroidal impacts As we mentioned above, the study of the impacts of small bodies with giant planets took a great advantage of the impact of SL9 on Jupiter in 1994. In the following years other small bodies impacted with Jupiter, confirming the idea that these events are not so rare in the history of the Solar System. The analysis of the SL9 impact data and the application of the theory of atmospheric entry of meteors on Earth made it possible to understand the physical behaviour of the Jovian atmosphere after the impact. Each fragment of SL9 reached a different depth inside the planet, before exploding and releasing all his energy to the surrounding atmosphere. The maximum depth reached depend only on the physical properties of the impactor as: size, density, velocity and angle of entry. The larger impactors, about 3 km in size and mainly composed of water ice, reached maximum depths of about 500 km (approximatively 1 Kbar). At the maximum depth the fragments exploded and liberated all their energy. In a time- scale of few seconds the hot gases generated by the explosion have risen to the higher parts of the Jovian atmosphere characterized by lower opacities, where they reached a luminosity of the order of 1025erg/s in the near-infrared. The sudden release of this large amount of energy created a shock wave that propa- gated at high velocity trough the atmosphere, with the shocked material reaching very high temperatures and pressures. In this condition it is possible to form new chemi- cal species and transport them over great distances in the atmosphere. In the case of SL9 impact the most surprising report was that of S2 at the impact site of G fragment. Newly detected or enhanced molecular species resulting from impacts included: CS, CS2, OCS, H2S, SO2, HCN, CO, H2O, NH3, C2H4, PH3. All new and enhanced species were detected in Jupiter’s stratosphere. A general rule of shocked chemistry is that CO forms until either C or O is exhausted. If O > C, the other products are oxidized, and excess O goes to H2O. If C > O, the other products are reduced, and excess C goes to HCN, C2H2 and a wide variety of more complex organics. The dark ejecta debris is probably composed in part of carbonaceous particles generated by the shocks. 4 Secular contamination of planetary atmospheres From the previous calculations, we can derive the following empirical relationship for the mixing ratio produced by a cometary impactor in the stratosphere of an exogiant planet: mr = 2.98 × 10−6χ Dcomet 1km ρatmosph. 2 × 10−5kg m−3 −1 R 100km −1 where mr is the mixing ratio of atmospheric water contributed by the impactor, χ is the fraction of the impactor dissolved in the considered atmospheric shell (i.e. the effective amount of water delivered in the observable region) and R is the thick- ness of the atmospheric shell considered. This relationship can be used to obtain a rough estimate of the effects of cometary impactors for what it concerns water, but similar relationships can be derived for other high-Z elements and populations of impactors. 517 Exp Astron (2015) 40:501–522 5 Planetary processes affecting atmospheric composition after cometary or asteroidal impacts The observed O/S ratio was reasonably consistent with cometary abun- dances, but the O/N ratio was much larger, suggesting that another N species was formed (presumably N2) but probably remained undetected. Convective motions in the upper part of the atmosphere and atmospheric circulation then provided a rapid mixing of the newly formed species over short time scales (few days) in the plane- tary atmosphere, but of the observed species OCS, H2S and NH3were found to be transient on a time scale of months. CS2 and CO were observed to weaken over time and to be still present in the atmosphere 1 year after the impact. In contrast, spectral signatures of HCN and CS remained strong also 1 year after the impact, indicating that their abundances remained constant or even increased with time. Despite the great changes induced by the impact of SL9 on Jupiter it will be dif- ficult to observe with the same details a similar event on an extrasolar planet and to infer the nature of changes in its atmospheric chemical composition as induced by a single impact. However, assuming that the phases underwent by the Solar System in the earliest epochs of its life are common to all planetary systems in the Galaxy, there is a series of event that could alter in a secular way the chemical composition of an 518 Exp Astron (2015) 40:501–522 extrasolar planet. As we also mentioned before, the migration of massive bodies like Jupiter is associated to large perturbations on smaller bodies present in the extrasolar analogues of the asteroid belt or of the Oort clouds, displacing them from their orig- inal orbits and injecting them in more eccentric orbits that could intersect also the central star. During the migration, the planet could be hit many times by these small bodies: even if the exogenous material delivered in the exoplanetary atmosphere can be limited, especially during late events as illustrated in the previous sections, the changes induced by the energy input associated to the impacts can be relevant. If the planet has a radiative zone in the upper part of its atmosphere, as suggested by [17] in the case of Jupiter, it can extend from 500 to 1000 km (see Fig. 7). 5 Planetary processes affecting atmospheric composition after cometary or asteroidal impacts This radiative zone act as a barrier between the planet interior and the upper atmosphere, impeding any exchange of material between the two convective regions, preventing in this way a full mixing of the internal material with the external zones. The same Fig. 7 Temperature gradients inside Jupiter from the models of [17]. The gradients are: in red the adiabatic, in green the radiative, and in blue the total gradient. Each panel presents the gradients as function of different physical variables Fig. 7 Temperature gradients inside Jupiter from the models of [17]. The gradients are: in red the adiabatic, in green the radiative, and in blue the total gradient. Each panel presents the gradients as function of different physical variables Exp Astron (2015) 40:501–522 519 situation happens in the main sequence stars of spectral type F where a radiative zone separates the external atmosphere from the inner region where convection is active and nuclear burning products are well mixed. In the case of an impactor with size of about 10 km on Jupiter, with velocity and impact angle similar to those of SL9, the maximum depth reached by the body could be larger than 1000 km, well below the radiative zone. In this case the fireball and the shock wave created by the impact will expand in a region with different chemical composition (more He rich) with respect to the upper atmosphere. At the same time the snowball effect created by the shock wave will be able to bring He rich material above the radiative zone, hence altering the atmospheric chemical composition of the planet. The material below the radiative zone will be brought to the surface, while the upper atmospheric material is depleted in heavier elements and chemical species. These events have a strong similarity with the dredge-up events in low mass stars evolving along the Red Giant Branch: in the stellar case the convection acts as dredge and bring to the surface the product of nuclear burning altering the chemical com- position of the star. In the planetary case, instead, it is the shock wave that acts as a dredge. The results of such a large impact can alter the chemical composition of the planet for longer time-scales than smaller events like SL9. A repeated series of col- lisions of large impactor (i.e. 5 Planetary processes affecting atmospheric composition after cometary or asteroidal impacts with size > 10 km) can therefore permanently alter the chemical structure of the atmosphere of an extrasolar planet. 6 Toward the investigation of exoplanetary atmospheres in NIR/MIR In view of the amounts of data that will be provided by future EChO-like space missions devoted to the systematic investigation of the atmospheric composition of extrasolar planets, in the next years it will be necessary to focus part of our attention on the strategies to link the atmospheric composition of an extrasolar planet to its past evolution and its formation region. As an example, Fig. 8 illustrates the steps Assume single planet and disk-driven migration Assume single planet and disk-driven migration Reconstruct disk T & P profiles for host stars Reconstruct disk T & P profiles for host stars Determine source regions of molecules Determine source regions of molecules Test whether molecules can be delivered by secular impacts over the life of the planet Test whether molecules can be delivered by secular impacts over the life of the planet Determine whether molecules can be produced in situ in the atmospheres Determine whether molecules can be produced in situ in the atmospheres If not, assess the lifetime of these molecules once in the atmospheres If not, assess the lifetime of these molecules once in the atmospheres Assess whether we can constrain the formation region of the planets or the sources of these molecules Assess whether we can constrain the formation region of the planets or the sources of these molecules Fig. 8 A possible schematic approach to interpret the origins of molecular species in exoplanetary atmospheres Assume single planet and disk-driven migration Assume single planet and disk-driven migration Reconstruct disk T & P profiles for host stars Reconstruct disk T & P profiles for host stars Determine whether molecules can be produced in situ in the atmospheres Determine whether molecules can be produced in situ in the atmospheres Test whether molecules can be delivered by secular impacts over the life of the planet Test whether molecules can be delivered by secular impacts over the life of the planet Determine source regions of molecules Determine source regions of molecules If not, assess the lifetime of these molecules once in the atmospheres If not, assess the lifetime of these molecules once in the atmospheres Assess whether we can constrain the formation region of the planets or the sources of these molecules Assess whether we can constrain the formation region of the planets or the sources of these molecules Fig. 6 Toward the investigation of exoplanetary atmospheres in NIR/MIR 8 A possible schematic approach to interpret the origins of molecular species in exoplanetary atmospheres 520 Exp Astron (2015) 40:501–522 that need to be undertaken to unveil the sources and sinks of the molecular species detected in the exoplanetary atmosphere of a giant planet (assuming it is the only one present in the planetary system). First, we would need to be able to assess whether the detected molecular species can form in situ due to the thermodynamical conditions in the atmosphere of the planet: if not, what we need to assess is whether they are stable or transient and, in the latter case, how long they can reside in the atmosphere before being destroyed or reprocessed (the “atmospheric” line of investigation identified by the orange arrows in Fig. 8). If the species are exogenous and transient, we need to assess whether secular processes like the impacts of cometary or asteroidal bodies have a reasonable chance of producing the observed abundances based on plausible fluxes and size frequency distributions of the impactors (the “secular contamination” line of investigation identified by the green arrows in Fig. 8). If the species are long- lived and/or secular effects cannot account for the observed abundances, then we need to reconstruct the composition of the gas in the circumstellar disk and identify if the regions that could represent the sources of said species are compatible with plausible dynamical histories of the giant planet (the “primordial accretion” line of investigation identified by the light blue arrows in Fig. 8). As the previous discussion highlights, our present understanding of the formation and evolution of planetary systems, both around the Sun and other stars, allows us to have a more or less clear picture (from a qualitative point of view) of what are the processes and factors that can affect the composition of the atmospheres of giant planets. The same is not necessarily true, however, for the outcomes of said processes (especially from a quantitative point of view) and, consequently, for our capability to constrain the past histories of the planetary systems from the observations of the composition of planetary atmospheres. As schematically illustrated by Fig. 9 for what it concerns the problem of the migration of giant planets, we can track the past dynamical evolution of planetary systems in those cases where the planets did not migrate significantly (e.g. 6 Toward the investigation of exoplanetary atmospheres in NIR/MIR they accreted the gas while the disk was dissipating or got locked in resonant configurations that prevented migration) or where a single No Migration No Migration Planet formed where we see it now Planet formed where we see it now Composition reflects formation environment Composition reflects formation environment Migration Migration Simple Case: Single Planet, Disk-Planet Interactions Simple Case: Single Planet, Disk-Planet Interactions Complex Case: Multiple Planets, Jumping Jupiters Complex Case: Multiple Planets, Jumping Jupiters Linear migration, can track/constrain Linear migration, can track/constrain Chaotic migration, cannot track Chaotic migration, cannot track Fig. 9 Schematic representation of the possible dynamical paths of planetary systems and of how they affect our capability to identify their signatures in the atmospheric composition of the exoplanets No Migration No Migration Migration Migration Planet formed where we see it now Planet formed where we see it now Simple Case: Single Planet, Disk-Planet Interactions Simple Case: Single Planet, Disk-Planet Interactions Complex Case: Multiple Planets, Jumping Jupiters Complex Case: Multiple Planets, Jumping Jupiters Composition reflects formation environment Composition reflects formation environment Chaotic migration, cannot track Chaotic migration, cannot track Linear migration, can track/constrain Linear migration, can track/constrain Fig. 9 Schematic representation of the possible dynamical paths of planetary systems and of how they affect our capability to identify their signatures in the atmospheric composition of the exoplanets Exp Astron (2015) 40:501–522 521 giant planet was present and its migration was induced solely by its interactions with the gas in the circumstellar disk (as assumed implicitly in the example of Fig. 8). More complex cases, like those where the final orbital configuration of the planetary system is created by the Jumping Jupiters mechanism, are much more difficult (if not impossible) to disentangle due to the intrinsic degeneracy of the problem. In the coming years, therefore, we will also need to devote several efforts to improve our comprehension of the atmospheric evolution of exoplanets and of how the past evolution of the extrasolar systems could have shaped them, in order to be prepared for the challenges that the new data will present. Acknowledgments D.T. would like to thanks Alberto Adriani, Francesca Altieri, Maria Teresa Capria, Davide Grassi and Roberto Peron for the helpful discussions. D.T. and M.B. acknowledge the financial contribution from agreement no. 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https://openalex.org/W4226064230	https://www.researchsquare.com/article/rs-486469/latest.pdf	English	null	Effect of droplet transition on the dynamic behavior of the keyhole during 6061 aluminum alloy laser-MIG hybrid welding	The international journal of advanced manufacturing technology/International journal, advanced manufacturing technology	2,021	cc-by	8,052	Abstract The simulation method in laser-MIG hybrid welding, which involves two heat sources and multiple welding parameters, is beneficial to reveal the complex physical phenomena and dynamic behavior of molten pool keyhole during welding process. In this investigation, laser-MIG hybrid welding for 6-mm-thick 6061 aluminum alloy was performed under different heat input by the high-power disc laser, MIG welding system and KUKA Robot. The high-speed camera system was used to observe the droplet transition phenomenon in the welding process. Besides, a thermal-fluid coupling model was established to simulate the temperature field and flow field, which were changed by the droplet transfer during laser-MIG hybrid welding. The experimental and simulated results showed that the droplet transition behavior affected the formation of the keyhole. The keyhole was the smallest when the droplet contacted the molten pool. In addition, the droplet transition brought external momentum and energy to the molten pool, which was conducive to the increase of the flow rate of the molten pool. Keywords Droplet ‧ Keyhole ‧ Thermal-fluid coupling simulation ‧ Laser-MIG hybrid welding aluminum alloy laser-MIG hybrid welding Yue Lia, Yanqiu Zhaoa, Xudong Zhoua, Xiaohong Zhana a College of Materials Science and Technology, Nanjing University of Aeronautics and Astronautics, Nanjing 211106, China Corresponding author: Xiaohong Zhan, xiaohongzhan_nuaa@126.com Yue Lia, Yanqiu Zhaoa, Xudong Zhoua, Xiaohong Zhana a College of Materials Science and Technology, Nanjing University of Aeronautics and Ast 211106, China Yue Lia, Yanqiu Zhaoa, Xudong Zhoua, Xiaohong Zhana a College of Materials Science and Technology, Nanjing University of Aeronautics and Astronautics, Nanjing 211106, China Corresponding author: Xiaohong Zhan, xiaohongzhan_nuaa@126.com Research Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at The International Journal of Advanced Manufacturing Technology on November 11th, 2021. See the published version at https://doi.org/10.1007/s00170-021-08270-1. 1 Introduction Contemporary aerospace is based on the goal of improving the carrying capacity through weight reduction[1, 2]. Aluminum alloys have been widely used in the aerospace field owing to their small density, high specific strength and corrosion resistance[3, 4]. At present, there are numerous problems in the traditional arc welding, such as low welding efficiency, large heat input and large deformation after welding, which seriously limits its application[5]. Although aluminum alloy has high reflectivity to laser, a keyhole is still generated at a high energy density, i.e. greater than 106 W/cm2, which greatly improves the laser absorption rate of aluminum alloy[6, 7]. Laser welding for aluminum alloy is gradually used in aerospace, vehicle, rail transportation and other fields due to its advantages of obvious weight loss, slight deformation and high welding efficiency[8, 9]. However, laser welding exists the weaknesses of poor bridging ability and strict requirement of the joint gap[10]. As an applicative and emerging deep penetration connection technology, laser-arc hybrid welding technology, which developed in the 1990s, has been widely applied in civil passenger aircraft, automobiles, construction machinery, rail transportation and equipment manufacturing fields[11, 12]. In the laser-arc hybrid welding process, the dual heat sources synergy and interact effect between laser and arc produces narrow and deep welds, thereby greatly improving production efficiency[13]. Laser-arc hybrid welding with the characteristics of high welding energy, strong gap bridging ability and high joint quality[14], makes up for the deficiencies in the single processing heat source, meanwhile, effectively combines the advantages of the two welding methods[15]. Many researches have been carried out to observe the morphology of the molten pool during the laser-MIG hybrid welding process by high-speed cameras. Faraji et al. [16] used laser-MIG hybrid to fabricate 3-mm-thick 2198 aluminum alloy, and the mechanical property of the welded joint was compared and analyzed. It was found that comparing with a single laser heat source, the keyhole with the deep penetration was easy to obtained even at higher welding speed. Moreover, the micro-hardness and microstructure of high laser power with low arc power were better. Liu et al. [17] observed the droplet transfer and the keyhole in the molten pool during laser-arc hybrid welding by high speed camera. It revealed that the laser had a compressive effect on the arc pressure with the surface of the molten pool. Zhang et al. 1 Introduction [18] performed laser-MAG hybrid welding of large thickness steel and investigated the influence of the ratio of laser power to arc power on the weld morphology. What is more, the image of the molten droplet and arc morphology were observed with the help of a high-speed camera system. It was revealed that as the ratio of laser to arc energy increases, the aspect ratio of the weld exhibits a linear increase. In recent years, the research method of simulation was used to explain complex physical phenomena, which promoted the research on the mechanism level. Lu et al. [19] proposed a three-dimensional transient model coupled with fluid flow, bubble motion and solidification to study the process of keyhole-induced porosity. The simulation results showed that the number of porosity was mainly determined by the frequency of keyhole collapse. As the laser power increased, as well as the welding speed and spot diameter decreased, the keyhole tended to collapse more easily. Guo et al. [20] performed laser-GMAW hybrid welding of 6 mm aluminum alloy, and simulated the formation of keyhole-induced pores with the help of two-dimensional flow field calculations. It was found that under the action of the arc force, relatively stable counterclockwise vortices appeared in the middle and rear of the composite molten pool, which tended to prevent the bubbles from moving to the low temperature area at the rear of the molten pool, thus helping the bubbles to float upward. However, the research on the droplet transfer behavior in the laser-MIG hybrid welding process is still insufficient. It is not enough to study on the effect of droplet transition on the temperature field distribution of the molten pool. In this paper, with the help of high-speed camera and the establishment of a thermal-fluid coupling model, the droplet transition behavior is studied. 1.1 Experimental material and setup The base metal used in this paper is 6061-T6 aluminum alloy, its chemical composition is shown in Table 1. The size of aluminum alloy plate is 150 mm×30 mm×6 mm, with a Y-shaped groove, as shown in Fig. 2(a). The selected filler wire is ER4047 with a diameter of 1.2 mm whose main chemical composition is shown in Table 1. The laser-MIG hybrid welding experiment equipment is adopted TruDisk-12003 disc laser with a maximum laser output of 12000 W, KR60HA robot, TPS 5000 Fronius welding machine and self-designed installation fixture. Besides, CP70 high-speed camera is performed to take pictures of the molten pool morphology and droplet transition during the welding process. The experimental equipment is shown in Fig. 1. Table 1 Chemical composition of 6061 aluminum alloy and ER4047 (wt.%) Composition Si Fe Cu Mn Mg Zn Ti Cr Al 6061 0.56 0.70 0.30 0.89 0.93 0.25 0.15 0.04 Balance ER4047 12 0.8 0.03 0.15 0.1 0.2 - - Balance Table 1 Chemical composition of 6061 aluminum alloy and ER4047 (wt.%) Fig. 1 Experimental setup of 6061 aluminum alloy laser-MIG hybrid welding. (a) Equipment and work table. (b) Disc laser. (c) Fronius welder Fig. 1 Experimental setup of 6061 aluminum alloy laser-MIG hybrid welding. (a) Equipment and work table. (b) Disc laser. (c) Fronius welder 1.2 Experimental process During the laser-MIG hybrid welding of 6061 aluminum alloy, the aluminum alloy plate is connected to the negative electrode effectively, considering the dense oxide film on the surface of aluminum alloy. The cathode crushing effect in arc welding is instrumental to remove the oxide film and facilitate the droplet transition. The schematic diagram of laser-MIG hybrid welding is as shown in Fig. 2. In order to prevent the damage of laser head by the vertical laser irradiation, the angle between the laser beam and the workpiece is 86.5°. At the same time, the angle between the MIG welding torch and the aluminum alloy plate is 45° considering the interference of the experimental equipment. Argon with purity of 99% is used as welding shielding gas and its flow rate is 15 L/min. The welding parameters adopted in this experiment is as shown in Table 2. Fig. 2 Schematic diagram of laser-MIG hybrid welding. (a) Welding. (b)Molten pool morphology Fig. 2 Schematic diagram of laser-MIG hybrid welding. (a) Welding. (b)Molten pool morphology Table 2 The welding parameters in laser-MIG hybrid welding process Case Laser power (kW) Arc current (A) Welding speed (m/min) Filling speed (m/min) 1 4.0 90 1.2 5.3 2 4.0 100 1.2 5.7 3 4.5 120 1.2 7.0 Table 2 The welding parameters in laser-MIG hybrid welding process 3.1 Governing equations The flow of the molten pool and the behavior of metal vapor during the laser-MIG hybrid welding process are both complex physical phenomena[21]. It is followed the law of conservation of mass, energy and momentum, which govern the thermodynamics and kinetics of the molten pool owing to the fluid characteristics at the welding process[22]. The formulas of the three laws are as follows. Continuity equation ) ( ) ( ) (     Continuity equation Continuity equation 0 ) ( ) ( ) (             z w y v x u t     (1) Continuity equation 0 ) ( ) ( ) (             z w y v x u t     (1) (1) especially for incompressible fluids, 0    t  . especially for incompressible fluids, 0    t  . Energy equation Energy equation net q z T k z y T k y x T k x z H w y H v x H u u t H                             ) ( ) ( ) ( ] ) ( [ 0  (2) (2) where H is the mixing enthalpy, T is the temperature, k is the heat conductive coefficient. qnet is the heat input during laser-MIG hybrid welding process. The heat balance equation is: The heat balance equation is: C R E D L A net q q q q q q q       (3) (3) where qA is the heat input from the electric arc; qL is the heat input from the laser; qD is the heat input from droplet; qE is the evaporation heat loss from the molten metal; qR is the heat loss by radiation; qC is the heat loss by convective heat exchange with the environment. The unite of these parameters is J/m2/s. 3.1 Governing equations Momentum equation m S I u T u u p u u u t                       ] 3 2 ) [( ) ( ) (    (4) (4) where u=(u, v, w), Sm is the source term for momentum conservation. where u=(u, v, w), Sm is the source term for momentum conservation. 3.2 Driving forces The vapor recoil pressure (PR) of the gas is an important reason for the keyhole, which is mainly driven by the plasma generated at the bottom of the keyhole, and the liquid metal is expelled to both sides of the molten pool[23,24]. The equation of recoil pressure is expressed as follows: ) exp( 0 b b v R RTT T T L P P    (5) (5) where P0 is the ambient air pressure, Lv is latent heat of vaporization, Tb is the boiling temperature, and R is the gas constant value. where P0 is the ambient air pressure, Lv is latent heat of vaporization, Tb is the boiling temperature, and R is the gas constant value. The surface tension is one of the driving forces to increase the width of the molten pool. The temperature coefficient of the surface tension with the aluminum alloy liquid metal is negative[25]. The molten metal on the surface of the molten pool flows around the keyhole, resulting in a wide and shallow cross-section of the molten pool. Marangoni force has great effect on the width of molten pool. Especially, when the temperature gradient coefficient of surface tension is negative, the surface tension of the liquid metal increases as the temperature decreases. Thus, the surface tension is low at the center of the molten pool, and it is high near the molten pool edge, which contributes to an outward flow and consequently a wider and shallower molten pool. The equation of Marangoni force is expressed as follows: dy dT dT d  (6) (6) where τ is the Marangoni stress, dγ/dT is temperature coefficient of surface tension and dT/dy is the temperature gradient. The buoyancy force is caused by the density variations. The variation of density in the molten pool induces the fluid rising in the hotter and less-dense region. At the same time, the fluid sinks in the cooler and denser region. The force is expressed by the following equation: ) ( ref b T T g F     (7) (7) where ρ is the density of liquid metal, β is the thermal expansion coefficient, and Tref is where ρ is the density of liquid metal, β is the thermal expansion coefficient, and Tref is arbitrarily selected reference temperature. 3.2 Driving forces Electromagnetic force is named as Lorentz force, which effects on the heat and mass transfer in the molten pool and contributes to smaller width and deeper penetration. The equation is expressed as: as: as: B J Fm     (8) (8) where J is the current density and B is the Magnetic field. where J is the current density and B is the Magnetic field. Arc pressure contributes to smaller width and larger penetration. On the contrary, the plasma flow force leads to larger width and shallower penetration. The scheme diagram of driving force in the molten pool is as shown in Fig. 3. Fig. 3 The schematic diagram of driving force during laser-MIG hybrid welding. (a) In cross section. (b) In longitudinal section ce during laser-MIG hybrid welding. (a) In cross section. (b) In Fig. 3 The schematic diagram of driving force during laser-MIG hybrid welding. (a) In cross section. (b) In longitudinal section Convection heat transfer coefficient Dynamic viscosity 3.3 Condition of computational domain Considering the gas-liquid two-phase flow under the laser-MIG hybrid welding process, two domains of air and aluminum alloy are established, where the thickness of the air is 3 mm and the thickness of the aluminum alloy is 6 mm, as shown in Fig. 4. The top of the air domain is selected as the inflow, the velocity is set to 0.1m/s, and other three sides of the air domain as the outflow are set as an atmosphere. The remaining faces are set as walls. The geometric model is meshed to 487,978 tetrahedron elements. The thermo-physical properties of 6061 aluminum alloy and plasma are as shown in Table 3and Table 4, respectively. Fig. 4 The schematic diagram of computational domain Table 3 Thermo-physical properties of 6061 aluminum alloy for simulation Fig. 4 The schematic diagram of computational domain Table 3 Thermo-physical properties of 6061 aluminum alloy for simulation Fig. 4 The schematic diagram of computational domain Table 3 Thermo-physical properties of 6061 aluminum alloy for simulation Table 3 Thermo-physical properties of 6061 aluminum alloy for simulation Physical property Symbol Value Unit Mass density ρ 2630 Kg/m3 Thermal expansion coefficient β 3.02×10-5 1/K Heat conductivity λ 204 W/(m·K) Solid temperature Tm 858.13 K Liquid temperature Tl 923.15 K Evaporation temperature Tb 2790 K Latent heat of fusion Lm 5.03×105 J/kg Latent heat of evaporation Lv 1.07×107 J/kg Special heat capacity of solid Cp 900 J/(kg·K) Special heat capacity of liquid Cl 1200 J/(kg·K) Surface tension σ 0.99 N/m Temperature coefficient of surface tension Aσ -1.3×10-4 N/(m·K) Dynamic viscosity η 0.0045 Pa·s Surface emissivity ε 0.1 / Ambient temperature Tref 300 K Magnetic permeability μ 1.26×10-6 H/m Table 4 Thermo-physical properties of plasma for simulation Physical property Symbol Value Unit Mass density ρg 0.06 Kg·m-3 Special heat capacity Cg 49 J·Kg-1 Convection heat transfer coefficient 3.4 Heat source Considering the laser and MIG heat sources during the welding process, a hybrid heat source model of a Gaussian rotating body heat source combination and a double ellipsoid heat source are selected for simulation, as shown in Fig. 5. The heating range of the MIG heat source is wider and the width of the weld seam is larger, thus the double ellipsoid heat source model is better reflect the shape of the molten pool[26]. The laser heat source acts on the surface of the workpiece and produces a keyhole effect, which plays a leading role in the welding process[27]. Fig. 5 The model of laser-MIG hybrid heat source. (a) Gaussian rotating body combined heat source model. (b) Double ellipsoid heat source model The model of laser-MIG hybrid heat source. (a) Gaussian rotating body combined heat source model. Fig. 5 The model of laser-MIG hybrid heat source. (a) Gaussian rotating body combined heat source model. (b) Double ellipsoid heat source model Double ellipsoid heat source model Since the weld under the laser heat source is nail-shaped in cross section, in order to better fit the simulation effect, the Gaussian rotating body heat source is selects as the laser heat source model, which mainly includes Gaussian surface heat source and Gaussian body heat source. The distribution functions of heat flux density are described as:              2 2 2 2 exp , s s s s r y x b r bQ y x q  (9)                     z t H y x e t H R Q z v ) ( lg 9 exp 1 ) ( 9 2 2 3 0  (10) (9) (10) where qs and qv are the heat flux distributions of Gaussian surface heat source and bulk heat where qs and qv are the heat flux distributions of Gaussian surface heat source and bulk heat source respectively, b is the heat flux concentration coefficient, Qs and Qv represent the power of surface heat source and bulk heat source, rs and R0 are the effective radius of action of the surface heat source and bulk heat source, respectively , H(t) is the effective depth of action at which the body heat source changes over time. 3.4 Heat source And the relationship between Qs and Qv is expressed as: P Q Q L S V    (11) (11) where P is the laser power and ηL is the thermal efficiency of the laser. The double ellipsoid heat source model is described as: The double ellipsoid heat source model is described as:       0 3 3 3 exp 3 6 , , 2 2 2 2 0 2 2               y c z b t v y a x bc a IU f z y x q f f A f f ，    (12)       0 3 3 3 exp 3 6 , , 2 2 2 2 0 2 2               y c z b t v y a x bc a IU f z y x q f f A f f ，    (12)       0 3 3 3 exp 3 6 , , 2 2 2 2 0 2 2             y c z b t v y a x bc a IU f z y x q r r A r r ，    (13) (12) (13) 2   r f f f (14) (14) In the formula, qr and qf are the heat source densities of the two ellipsoids in the double ellipsoid heat source model, ff and fr are the heat distribution coefficients of the two ellipsoids, and I, U and v0 are the arc current, voltage and Welding speed, af, ar, b and c are heat source model parameters. ηA is the arc thermal efficiency. 3.6 Droplet transition model The mass, energy and momentum brought by the droplet transition behavior, which impacts on the temperature field and flow field of the molten pool as well as the dynamic evolution behavior of the keyhole. In this model, the boundary at the entrance in the geometric model is set as the position where the droplet grows up gradually. As the droplet grows up completely, it falls into the molten pool by free fall. Based on the results observed by the high-speed camera, the droplet transition period of 0.06s was set, and the wire moving speed was set the same as the laser heat source moving speed. The geometric model and boundary conditions of the droplet transition are shown in Fig. 7. Fig. 7 Geometric and boundary conditions of droplet transition model Fig. 7 Geometric and boundary conditions of droplet transition model 3.5 Experiment validation According to the heat-fluid coupling solution process of the laser-MIG hybrid welding process, the numerical simulation calculation is carried out. After the relevant parameters of the model are corrected, the results of heat source verification result for 1# is shown in Fig. 6. Comparing the weld cross section with the weld simulation results, it is seen that the macroscopic appearances of 1# is basically symmetrical, so the modified hybrid heat source model can be used for the subsequent simulation calculation of the temperature field and flow field of laser-MIG hybrid welding. Fig. 6 Comparison of the experimental result and simulation result i d l Fig. 6 Comparison of the experimental result and simulation result 3.1 High-speed camera results In order to facilitate the analysis of the droplet transfer behavior in the laser-MIG hybrid welding process, the high-speed camera results of 1# parameter (P=4.0 kW, I=90 A, v=1.2 m/min) were taken, and a picture was extracted every 2 ms for observation and analysis, as shown in Fig. 8. At t0, the droplet just started to contact the molten pool. Due to inertia, the center of gravity of the molten droplet shifted along the side opposite to the welding direction. The contact surface gradually increased under the action of the surface tension of the molten pool and gravity, the molten droplet was completely separated from the end of the welding wire at t0+6 ms. With the falling of the molten droplet, the molten droplet gradually merged into the molten pool. At t0+10 ms, the molten droplet completely merged into the molten pool. The front of the molten pool formed a keyhole under the action of the laser. Owing to the impact and momentum brought by the droplet drop, a depression appeared in the front of the liquid molten pool at t0+12 ms, and the diameter of the keyhole was also significantly reduced. It is seen that the droplet transition behavior has a very important influence on the stability of the molten pool and the volatility of the keyhole. The external momentum and energy brought by the droplet transition promote the flow of the molten pool, which is beneficial for the beneficial elements of the molten droplet. The elements are fully mixed with the molten pool and promote the escape of bubbles. In the process of droplet transition, the back wall of the keyhole is squeezed due to the impact force of the droplet on the molten pool. The opening of the keyhole is suppressed, meanwhile, the laser energy is affected to the bottom of the keyhole, which results a smaller keyhole. Fig. 8 High-speed camera photos in the droplet transition process Fig. 8 High-speed camera photos in the droplet transition process Fig. 8 High-speed camera photos in the droplet transition process 3.2 Effect of droplet transition on thermal-fluid coupling field distribution In order to study the effect of droplet transition on the temperature field and flow field of the molten pool, under 2# parameter (P=4.0 kW, I=100 A, v=1.2 m/min), the droplet between 64.8 ms and 68.8 ms is selected. The results of the transition simulation were analyzed, and the method of adding and not adding the droplet transition was used to analyze the influence of the droplet transition behavior on the morphology of the keyhole. The simulation results of the temperature field are shown in Fig. 9 and Fig. 10, respectively. The droplet transition process causes pressure on the back wall of the keyhole, so the angle of the keyhole back wall changes, as shown in Fig. 9. At t=64.8 ms, the molten droplet was contracted and hung above the molten pool under the action of electromagnetic force, the back of the molten pool was slightly dented by the action of the arc force. At t=65.6 ms, the droplet contacted the back of the molten pool, the momentum by the falling droplet caused an impact on the molten pool, and the back wall of the keyhole was squeezed by the liquid at the back of the molten pool. At t=67.2 ms, the back wall of the keyhole bulged due to the squeezing effect of the droplets falling into the molten pool, and the direction pointed to the inside of the keyhole. At t=68.0 ms, owing to the squeezing of the molten metal in the rear molten pool and the filling of the molten pool, the angle of the lower part of the back wall of the keyhole was changed, which facilitated the laser irradiation into the keyhole and brought a greater depth of the keyhole. At t=68.8 ms, the back wall of the keyhole continued to be affected by the fusion of the molten droplets to the back of the molten pool, and the lower part of the back wall of the keyhole had a boss pointing to the inside, which was not conducive to the laser irradiation to the bottom of the keyhole, thus affected the absorption of laser energy in molten pool. Compare with added droplet transfer, the surface morphology of the back of the molten pool without droplet is smoother, as shown in Fig. 10. It is found that the depth of the keyhole is the same, but the back wall of the keyhole changes greatly. 3.2 Effect of droplet transition on thermal-fluid coupling field distribution The increase in the volume of the rear part causes a slight depression in the lower part of the rear wall of the keyhole. Fig. 9 Distribution of temperature field in molten pool during droplet transition. (a) t=64.8 ms. (b) t=65.5 ms. (c) t=66.4 ms. (d) t=67.2 ms. (e) t=68.0 ms. (f) t=68.8ms Fig. 9 Distribution of temperature field in molten pool during droplet transition. (a) t=64.8 ms. (b) t=65.5 ms. (c) t=66.4 ms. (d) t=67.2 ms. (e) t=68.0 ms. (f) t=68.8ms Fig. 10 Distribution of temperature field in molten pool without droplet transition. (a) t=64.8 ms. (b) t=65.5 ms. (c) t=66.4 ms. (d) t=67.2 ms. (e) t=68.0 ms. (f) t=68.8ms Fig. 10 Distribution of temperature field in molten pool without droplet transition. (a) t=64.8 ms. (b) t=65.5 ms. (c) t=66.4 ms. (d) t=67.2 ms. (e) t=68.0 ms. (f) t=68.8ms (c) t=66.4 ms. (d) t=67.2 ms. (e) t=68.0 ms. (f) t=68.8ms Because the molten droplet brings external momentum to the molten pool, the flow field distribution of the molten pool also changes. The simulation results of the flow field are shown in Fig. 11 and Fig. 12, respectively. It is seen from Fig. 11 that the flow velocity inside the molten pool is very small, the flow velocity on the surface of the molten pool and the wall of the keyhole is relatively large. The flow velocity on the surface of the molten pool varies from 0.7 m/s to 1.1 m/s, the flow velocity at the bottom of the keyhole varies greatly, about 0.4 m/s to 1.4 m/s. At t=64.8 ms, the molten droplet had not yet fallen into the molten pool. At this time, the fluid in the front of the molten pool flowed upward along the front wall of the keyhole, and the fluid at the rear of the molten pool flowed down in a clockwise direction in front of the molten pool. It flowed upward along the back wall of the keyhole and extends backward under the action of Marangoni circulation on the surface of the molten pool. A clockwise vortex appeared in the fluid below the back of the molten pool. At t=65.6 ms, the transition of the droplet brought an impact to the back of the molten pool, so the flow direction of the fluid in the bottom area of the keyhole was from the back to the front of molten pool. 3.2 Effect of droplet transition on thermal-fluid coupling field distribution Meanwhile, the droplet completely fell into the molten pool, the impact force brought by the droplet was relatively large, and the maximum velocity reached 2.05 m/s. For the flow field distribution of the molten pool without added droplet transition, the flow field distribution is basically the same as that of the added droplet transition, but its peak velocity at different times is less than the added droplet transition, mainly because the droplet transition brings about External momentum[28]. The change of the shape of the keyhole by the droplet transition is mainly achieved by promoting the downward flow of the fluid at the back of the molten pool to fill the depression below the back wall of the keyhole, thus squeezing the back wall of the keyhole. When convex shape is appeared on the back wall of the keyhole, which affects the heating effect of the laser on the molten pool, the back wall of the keyhole will adjust the angle under the thermal capillary force to maintain the absorption of the laser by the molten pool. Fig. 11 Distribution of flow field in molten pool during droplet transition. (a) t=64.8 ms. (b) t=65.5 ms. Fig. 11 Distribution of flow field in molten pool during droplet transition. (a) t=64.8 ms. (b) t=65.5 ms. (c) t=66.4 ms. (d) t=67.2 ms. (e) t=68.0 ms. (f) t=68.8ms (c) t=66.4 ms. (d) t=67.2 ms. (e) t=68.0 ms. (f) t=68.8ms (c) t=66.4 ms. (d) t=67.2 ms. (e) t=68.0 ms. (f) t=68.8ms Fig. 12 Distribution of flow field in molten pool without droplet transition. (a) t=64.8 ms. (b) t=65.5 ms. (c) t=66.4 ms. (d) t=67.2 ms. (e) t=68.0 ms. (f) t=68.8ms 12 Distribution of flow field in molten pool without droplet transition. (a) t=64.8 ms. (b) t=65.5 ms. Fig. 12 Distribution of flow field in molten pool without droplet transition. (a) t=64.8 ms. (b) t=65.5 ms (c) t=66.4 ms. (d) t=67.2 ms. (e) t=68.0 ms. (f) t=68.8ms (c) t=66.4 ms. (d) t=67.2 ms. (e) t=68.0 ms. (f) t=68.8ms 3.3 Effect of droplet on keyhole induced pore Fig. 13 shows that the trajectory of the bubble formed during the droplet transition under 2# parameter (P=4.0 kW, I=100 A, v=1.2 m/min). It is seen that the droplet transition has an important influence on the stability of the keyhole, and it is easy to bring external bubbles when falling into the molten pool. Before the molten droplet fell into the molten pool, the maximum speed of the free fall reached 2.262 m/s, which brought external momentum and energy to the molten pool. At t=66.4 ms, the molten droplet fell into the molten pool forming a gap, and the movement direction of the surrounding fluid was a counterclockwise vortex, the vortex velocity was 0.39~0.78 m/s; at t=67.2 ms, an irregular bubble formed on the surface at the back of the molten pool. The flow direction of the fluid was still a counterclockwise vortex, and the moving speed is reduced; at t=67.6 ms, the liquid bridge above the bubble was broken, and the bubble escaped from the molten pool. The flow direction of the fluid around the bubble was clockwise circulation, and the maximum surface velocity caused by the liquid bridge fracture reached 2.805 m/s. It can be seen that the droplet transition brings smaller bubbles to the surface of the molten pool, and escapes the surface of the molten pool under the action of the surrounding circulation. The circulation velocity is slightly higher than that of the surrounding fluid, and the bubbles are easy to escape. Fig. 13 The trajectory of the bubble formed during the droplet transition. (a) t=65.6 ms. (b) t=66.4 ms. (c) t=67.2 ms. (d) t=67.6 ms Fig. 13 The trajectory of the bubble formed during the droplet transition. (a) t=65.6 ms. (b) t=66.4 ms. (c) t=67.2 ms. (d) t=67.6 ms (c) t=67.2 ms. (d) t=67.6 ms The forces acting on the bubbles in the molten pool are buoyancy force Fb, gravity G, the fluid driving force Fd and viscous force FV, as shown in Fig. 14(a). Because most of the bubbles are very small with the diameter of tens of microns, Fd is the main force. The bubbles move in the molten pool under the driving force of the molten pool fluid[29]. Fig. 3.3 Effect of droplet on keyhole induced pore 14(b) shows that the smaller bubbles move downward along the solidification interface under the action of fluid flow in the middle of the molten pool, and upward along the back wall of keyhole under the action of Marangoni circulation at the back of the molten pool. Finally, the smaller bubbles escape from the molten pool. For the large bubbles formed in the molten pool, which are mainly affected by Fb and easy to escape from the molten pool. Fig. 14 (a) The schematic diagram of the bubble trajectory during the droplet transition. (b) The schematic diagram of the bubbles trajectory in different positions of the molten pool Fig. 14 (a) The schematic diagram of the bubble trajectory during the droplet transition. (b) The schematic diagram of the bubbles trajectory in different positions of the molten pool Fig. 14 (a) The schematic diagram of the bubble trajectory during the droplet transition. (b) The of the bubbles trajectory in different positions of the molten pool Consent for publication All authors and their institutions agree to publish this article in The International Journal of Advanced Manufacturing Technology. Consent to participate All authors in this article have informed consent to participate in this study. Code availability Not applicable. 4. Conclusions In this investigation, high-speed camera was used to observe the droplet transition of laser-MIG hybrid welding for aluminum alloy. The thermal-fluid coupling model was established to study the effect of droplet transfer on the shape of the molten pool. Based on the experimental and simulation results, the conclusions are drawn as follows: (1) The process of the droplet falling into the molten pool hinders the formation of the keyhole, and the keyhole opening is the smallest when the droplets are in contact with the molten pool. (2) The mass, energy and momentum brought by the droplet transition behavior, which changes the temperature field and flow field of the molten pool. In addition, the maximum velocity of molten pool is bigger than that without droplet. (3) The changes of the keyhole morphology are mainly behaved as the squeezing of the back wall of the keyhole. Additionally, the appearance of a boss on the keyhole wall affects the absorption of laser energy by the molten pool, thus affects the depth of keyhole. (4) The droplet transition is easy to bring bubbles, which are drawn into the molten pool by the counterclockwise vortex formed on the upper surface of the molten pool. After that, under the (4) The droplet transition is easy to bring bubbles, which are drawn into the molten pool by the counterclockwise vortex formed on the upper surface of the molten pool. After that, under the combined action of the thermal buoyancy and the clockwise vortex formed on the upper surface of the molten pool, the bubbles is easy to escape the molten pool. combined action of the thermal buoyancy and the clockwise vortex formed on the upper surface of the molten pool, the bubbles is easy to escape the molten pool. Availability of data and material All data generated or analyzed during this study are included in this published article. Authors' contributions Yue Li: investigation, experiment, data analysis, original manuscript writing, manuscript revising. Yanqiu Zhao: validation, supervision, funding acquisition. Xudong Zhou: investigation, experiment. Xiaohong Zhan: manuscript revising. Ethics approval The content in this article does not violate the ethics. 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[16] Faraji AH, Moradi M, Goodarzi M, Coluccid P, Maletta C (2017) An investigation on capability of hybrid Nd:YAG laser-TIG welding technology for AA2198 Al-Li alloy. Opt Lasers Eng 96 :1-6. [17] Liu S, Zhang F, Dong S, Zhang H, Liu F (2018) Characteristics analysis of droplet transfer in laser-MAG hybrid welding process. Int J Heat Mass Transf 121:805-811. [18] Zhang F, Liu S, Liu F, Zhang H (2019) Stability evaluation of laser-MAG hybrid welding process. Opt Laser Technol 116:284-292. [19] Lu FG, Li XB, Li ZG, Tang XH, Cui CH (2015) Formation and influence mechanism of keyhole-induced porosity in deep-penetration laser welding based on 3D transient modeling. Int J Heat Mass Transf 90:1143-1152. [20] Xu G, Li L, Wang H, Li P, Guo Q, Hu Q, Du B (2019) Simulation and experimental studies of keyhole induced porosity in laser-MIG hybrid fillet welding of aluminum alloy in the horizontal position. Opt Laser Technol 119:105667. [21] Cunningham R, Zhao C, Parab N, Kantzos C, Pauza J, Fezzaa K, Sun T, Rollett AD (2019) Keyhole threshold and morphology in laser melting revealed by ultrahigh-speed x-ray imaging. Science 363:849-852. [22] Ye XH, Chen X (2002) Three-dimensional modelling of heat transfer and fluid flow in laser full-penetration welding. J Phys D: Appl Phys 35:1049-1056. [23] Semak V, Matsunawa A (1997) The role of recoil pressure in energy balance during laser materials processing. J Phys D: Appl. Phys 30:2541-2552. [23] Semak V, Matsunawa A (1997) The role of recoil pressure in energy balance during laser materials processing. J Phys D: Appl. Phys 30:2541-2552. [24] Pang S, Chen X, Shao X, Gong S, Xiao J (2016) Dynamics of vapor plume in transient keyhole during laser welding of stainless steel: Local evaporation, plume swing and gas entrapment into porosity. Opt Lasers Eng 82:28-40. [25] Cho WI, Na SJ, Cho MH, Lee SJ (2010) Numerical study of alloying element distribution in CO2 laser–GMA hybrid welding. Comput Mater Sci 49:792-800. [26] Wang Y, Tsai HL (2001) Impingement of filler droplets and weld pool dynamics during gas [26] Wang Y, Tsai HL (2001) Impingement of filler droplets and weld pool dynamics during gas metal arc welding process. Int J Heat Mass Transf 44:2067-2080. 66:1467-1473. [27] Lankalapalli KN, Tu JF, Gartner M (1996) A model for estimating penetration depth of laser welding processes. Phys D: Appl Phys 29:1831-1841. [27] Lankalapalli KN, Tu JF, Gartner M (1996) A model for estimating penetration depth of laser welding processes. Phys D: Appl Phys 29:1831-1841. [27] Lankalapalli KN, Tu JF, Gartner M (1996) A model for estimating penetration depth of laser welding processes. Phys D: Appl Phys 29:1831-1841. [28] Liu S, Zhang F, Dong S, Zhang H, Liu F (2018) Characteristics analysis of droplet transfer in laser-MAG hybrid welding process. Int J Heat Mass Transf 121:805-811. [28] Liu S, Zhang F, Dong S, Zhang H, Liu F (2018) Characteristics analysis of droplet transfer in laser-MAG hybrid welding process. Int J Heat Mass Transf 121:805-811. [29] Zhang C, Gao M, Wang DZ, Yin J, Zeng XY (2017) Relationship between pool characteristic and weld porosity in laser arc hybrid welding of AA6082 aluminum alloy. J Mater Proc Technol 240:217-222. [29] Zhang C, Gao M, Wang DZ, Yin J, Zeng XY (2017) Relationship between pool characteristic and weld porosity in laser arc hybrid welding of AA6082 aluminum alloy. J Mater Proc Technol 240:217-222. Figures Figure 1 Experimental setup of 6061 aluminum alloy laser-MIG hybrid welding. (a) Equipment and work table. (b) Disc laser. (c) Fronius welder Figure 1 Experimental setup of 6061 aluminum alloy laser-MIG hybrid welding. (a) Equipment and work table. (b) Disc laser. (c) Fronius welder Figure 2 Schematic diagram of laser-MIG hybrid welding. (a) Welding. (b)Molten pool morphology Figure 2 Schematic diagram of laser-MIG hybrid welding. (a) Welding. (b)Molten pool morphology Schematic diagram of laser-MIG hybrid welding. (a) Welding. (b)Molten pool morphology Figure 3 The schematic diagram of driving force during laser-MIG hybrid welding. (a) In cross section. (b) In Figure 3 The schematic diagram of driving force during laser-MIG hybrid welding. (a) In cross section. (b) In longitudinal section The schematic diagram of driving force during laser-MIG hybrid welding. (a) In cross section. (b) In longitudinal section g Figure 4 The schematic diagram of computational domain Figure 4 The schematic diagram of computational domain The schematic diagram of computational domain The schematic diagram of computational domain Figure 5 The model of laser-MIG hybrid heat source. (a) Gaussian rotating body combined heat source model. (b) Double ellipsoid heat source model Figure 5 Figure 5 The model of laser-MIG hybrid heat source. (a) Gaussian rotating body combined heat source model. (b) Double ellipsoid heat source model The model of laser-MIG hybrid heat source. (a) Gaussian rotating body combined heat source model. (b) Double ellipsoid heat source model Figure 6 Comparison of the experimental result and simulation result Figure 6 Comparison of the experimental result and simulation result Figure 7 Geometric and boundary conditions of droplet transition model Figure 7 Geometric and boundary conditions of droplet transition model Figure 8 High-speed camera photos in the droplet transition process Figure 8 Figure 8 High-speed camera photos in the droplet transition process Figure 9 Distribution of temperature ¦eld in molten pool during droplet transition. (a) t=64.8 ms. (b) t=65.5 ms. (c) t=66.4 ms. (d) t=67.2 ms. (e) t=68.0 ms. (f) t=68.8ms Figure 9 Distribution of temperature ¦eld in molten pool during droplet transition. (a) t=64.8 ms. (b) t=65.5 ms. (c) t=66.4 ms. (d) t=67.2 ms. (e) t=68.0 ms. (f) t=68.8ms (c) t 66. s. (d) t 6 . s. (e) t 68.0 s. ( ) t 68.8 s Figure 10 Distribution of temperature ¦eld in molten pool without droplet transition. (a) t=64.8 ms. (b) t=65.5 ms. (c) t=66.4 ms. (d) t=67.2 ms. (e) t=68.0 ms. (f) t=68.8ms Figure 10 Distribution of temperature ¦eld in molten pool without droplet transition. (a) t=64.8 ms. (b) t=65.5 ms. (c) t=66.4 ms. (d) t=67.2 ms. (e) t=68.0 ms. (f) t=68.8ms Distribution of temperature ¦eld in molten pool without droplet transition. (a) t=64.8 ms. (b) t=65.5 ms. (c) t=66.4 ms. (d) t=67.2 ms. (e) t=68.0 ms. (f) t=68.8ms Figure 11 Distribution of §ow ¦eld in molten pool during droplet transition. (a) t=64.8 ms. (b) t=65.5 ms. (c) t=66.4 (d) t 67 2 ( ) t 68 0 (f) t 68 8 Figure 11 Distribution of §ow ¦eld in molten pool during droplet transition. (a) t=64.8 ms. (b) t=65.5 ms. (c) t=66.4 ms. (d) t=67.2 ms. (e) t=68.0 ms. (f) t=68.8ms Figure 12 Distribution of §ow ¦eld in molten pool without droplet transition. (a) t=64.8 ms. (b) t=65.5 ms. (c) t=66.4 ms. (d) t=67.2 ms. (e) t=68.0 ms. (f) t=68.8ms Figure 12 Figure 12 Distribution of §ow ¦eld in molten pool without droplet transition. (a) t=64.8 ms. (b) t=65.5 ms. (c) t=66.4 ms. (d) t=67.2 ms. (e) t=68.0 ms. (f) t=68.8ms Figure 13 The trajectory of the bubble formed during the droplet transition. (a) t=65.6 ms. (b) t=66.4 ms. (c) t=67.2 (d) t 67 6 Figure 13 The trajectory of the bubble formed during the droplet transition. (a) t=65.6 ms. (b) t=66.4 ms. (c) t=67.2 ms (d) t=67 6 ms The trajectory of the bubble formed during the droplet transition. (a) t=65.6 ms. (b) t=66.4 ms. (c) t=67.2 ms. (d) t=67.6 ms Figure 14 (a) The schematic diagram of the bubble trajectory during the droplet transition. Figure 7 (b) The schematic diagram of the bubbles trajectory in different positions of the molten pool Figure 14 Figure 14 (a) The schematic diagram of the bubble trajectory during the droplet transition. (b) The schematic diagram of the bubbles trajectory in different positions of the molten pool (a) The schematic diagram of the bubble trajectory during the droplet transition. (b) The schematic diagram of the bubbles trajectory in different positions of the molten pool (a) The schematic diagram of the bubble trajectory during the droplet transition. (b) The schematic diagram of the bubbles trajectory in different positions of the molten pool
https://openalex.org/W4388000637	https://www.nature.com/articles/s42004-023-01025-x.pdf	English	null	Simulating the structural phase transitions of metal-organic frameworks with control over the volume of nanocrystallites	Communications chemistry	2,023	cc-by	9,443	1 Ruhr-Universität Bochum, Faculty of Chemistry and Biochemistry, Computational Materials Chemistry Group, Universitätsstr. 150, 44801 Bochum, Germany. ✉ il h h id@ b d Simulating the structural phase transitions of metal-organic frameworks with control over the volume of nanocrystallites https://doi.org/10.1038/s42004-023-01025-x OPEN Larissa Schaper1 & Rochus Schmid 1✉ Flexible metal-organic frameworks (MOFs) can undergo structural transitions with signiﬁcant pore volume changes upon guest adsorption or other external triggers while maintaining their porosity. In computational studies of this breathing behavior, molecular dynamics (MD) simulations within periodic boundary conditions (PBCs) are commonly performed. However, to account for the ﬁnite size and surface effects affecting the phase transition mechanism, the simulation of non-periodic nanocrystallite (NC) models without the constraint of PBCs is an important alternative. In this study, we present an approach allowing the analysis and control of the volume of ﬁnite-size structures during MD simulations by a tetrahedral tessellation of the (deformed) NC’s volume. The method allows for deﬁning the current NC’s volume during the simulation and manipulating it regarding a particular reference volume to compute free energies for the phase transformation via umbrella sampling. The application on differently sized DMOF-1 and DUT-128 NCs reveals ﬂexible pore closing mechanisms without signiﬁcant biasing of the transition pathway. The concept provides the theoretical foundation for further research on ﬂexible materials regarding targeted initialization of the structural phase behavior to elucidate the underlying mechanism, which can be used to improve the applications of ﬂexible materials by targeted controlling of the phase transition. 1 Ruhr-Universität Bochum, Faculty of Chemistry and Biochemistry, Computational Materials Chemistry Group, Universitätsstr. 150, 44801 Bochum, Germany. ✉email: rochus.schmid@rub.de 1 Ruhr-Universität Bochum, Faculty of Chemistry and Biochemistry, Computational Materials Chemistry Group, Univer ✉email: rochus.schmid@rub.de COMMUNICATIONS CHEMISTRY \| (2023) 6:233 \| https://doi.org/10.1038/s42004-023-01025-x \| www.nature.com/commschem 1 ARTICLE COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-023-01025-x O approach is hampered by two difﬁculties: (i) An appropriate model for the surface termination of the NC is needed, and (ii) the application of pressure and the control of the system’s volume of an arbitrarily deformed NC is complex. O ne peculiar feature of metal-organic frameworks (MOFs), which differentiates them from other porous systems like zeolites, is the ability of speciﬁc systems to undergo sig- niﬁcant volume changes upon guest molecule adsorption or other external triggers like hydrostatic pressure, temperature, or mag- netic ﬁelds1,2. This “breathing effect" has attracted great interest because of its potential use in various areas, like improving the working capacity in gas separation3. The surface termination of a MOF, which also represents a defect concerning the bulk system, needs to be better investi- gated in experimental and theoretical studies. Simulating the structural phase transitions of metal-organic frameworks with control over the volume of nanocrystallites https://doi.org/10.1038/s42004-023-01025-x OPEN However, assuming a saturation of dangling bonds by modulators, often used in synthesis, allows staging an approximate and idealized surface termination16,17. More problematic is the second point, namely that a clear deﬁnition of the NC’s volume needs to be included in contrast to PBC simulations. In this context, it has to be considered how hydrostatic pressure can be typically simulated. In experiments, hydrostatic pressure acts as a force on the surface of the crystal exerted by a medium, i.e., the surrounding gas, which is adsorbed simultaneously. Therefore, pressure is connected to adsorption. In a theoretical simulation, disentangling this can be advantageous since pressure can be simulated in the absence of an adsorbing gas as a force acting on the cell parameters. As a result, the system’s volume is always well-deﬁned by the unit cell. In a PBC simulation, there is no surface on which a medium could act. In contrast, there is no such well-deﬁned volume for a deformed NC during a phase transition. In this context, the so-called “negative gas adsorption" was recently reported for DUT-49 and related MOFs, where the crystal cell volume is reduced at a limiting pressure, reducing the gas loading at higher pressure4,5. All these breathing and gate-opening effects are accompanied by a structural transition and deformation of the MOF, which is possible due to the ﬂexibility of the organic linkers or their connection to the inorganic secondary building blocks (SBUs). These 3rd generation MOFs are called soft porous materials6. Although these breathing phase transitions are inten- sively investigated both experimentally and by theoretical methods, the phenomenon still needs to be fully understood, and a prediction of, e.g., limiting gate pressures is still difﬁcult7. Recently, experi- mental evidence for a dependence of the breathing effect on the crystallite size has been found, indicating an inﬂuence of the system-to-volume ratio8. Also, the inﬂuence of defects and disorder on the ﬂexibility of MOFs is still being determined9,10. From a theoretical point of view, most studies employ artiﬁcial periodic boundary conditions (PBCs) to simulate crystalline materials with relatively small unit cells and ideal, non-defective MOF systems leading to a well-deﬁned cell volume, which allows pressure control by barostats11. It has been recently shown that large unit cells and a numerically effective GPU-based simulation approach allow studying the ﬁrst-order phase transitions with the presence of both phases within PBCs12,13. Simulating the structural phase transitions of metal-organic frameworks with control over the volume of nanocrystallites https://doi.org/10.1038/s42004-023-01025-x OPEN These meso-sized systems have a crystal size of ~100 nm side length. Figure 1 gives an overview of our various concepts to handle ﬁnite-size systems during simulation. In the NC simulations of MOF NCs, a distance restraint between SBUs on the edges of the system was used to exert a mechanical force on the system, mimicking the effect of pressure and inducing the structural phase transition14. This mechanical force has, of course, nothing in common with hydrostatic pressure and is derived from the expected reaction coordinate, at the same time enforcing a par- ticular mechanism of pore closing. g In contrast to such PBC simulations, the structural phase transition of DMOF-1 and derivatives was investigated as non- periodic nanocrystallites (NCs) with an approximated surface termination model14,15. The simulations conﬁrm a ﬁrst-order phase transition behavior with an interface between the open and the closed pore phase moving through the system. Already for the small NCs of ~1 nm side length, size effects on the free energy barrier could be observed, indicating a surface hindrance on the phase transition. This would explain that smaller crystallites typically are less ﬂexible or do not switch in experimental studies. The current results corroborate that the simulation of NCs beyond PBCs is a necessary route to fully reveal the atomistic details of the structural phase transition of MOFs. However, the To alleviate this bias, we recently considered the pressure bath method, where a ﬁctitious medium is added, actually exerting pressure on the surface of the NC18. This method was inspired by the experimental mercury porosimetry, where a non-wetting ﬂuid like mercury is used, which cannot enter the MOF nanopores. Therefore, in the absence of adsorption, the compression of a MOF crystallite could be experimentally triggered by pressure. In the simulation, spherically large par- ticles that strongly attract themself but interact only repulsive with the MOF are used, leading to considerable surface tension. Using a PBC simulation cell with the NC embedded in the pressure bath medium, the structural phase transition could be investigated in a constant pressure (NPT) ensemble. In contrast Fig. 1 Handling of ﬁnite size systems in MD simulation. The ﬁrst approach introduces a mechanical force to initialize the pore closing mechanism by lowering the distance between opposite-corner SBUs. The pressure bath concept returns to PBCs: A ﬁnite-sized system is set up in a ﬂuid. This system as a whole is simulated using PBCs. Methodology A ll i These derivatives are in turn used to compute the forces acting on the actual atoms of the paddle-wheels by the chain rule, since the vertices are deﬁned as their COMs. The COM of a paddle-wheel is calculated by taking into account both metal atoms and the four carboxylates. For all vertices in the interior of the NC, the volume derivatives of the eight neighboring unit-cells will always exactly compensate. Thus, for the total volume derivative of the entire NC, it is sufﬁcient to compute derivatives only for the vertices (paddle-wheel units) at the surface of the NC. For analysis, the volume is computed on a per unit-cell basis. These derivatives are in turn used to compute the forces acting on the actual atoms of the paddle-wheels by the chain rule, since the vertices are deﬁned as their COMs. The COM of a paddle-wheel is calculated by taking into account both metal atoms and the four carboxylates. For all vertices in the interior of the NC, the volume derivatives of the eight neighboring unit-cells will always exactly compensate. Thus, for the total volume derivative of the entire NC, it is sufﬁcient to compute derivatives only for the vertices (paddle-wheel units) at the surface of the NC. For analysis, the volume is computed on a per unit-cell basis. A collective variable for the NC volume. In order to overcome the problem that the NC’s volume is not well-deﬁned by the simulation cell as in PBC, the individual pores of ﬁnite-size MOF NCs are described by tetrahedra to calculate the volume and its derivatives. To clarify this strategy, a MOF pore is decomposed to its geometrical basics. g Figure 2 shows the DMOF-1 unit-cell corresponding to only one pore of the NC. The pore is deﬁned by the positions of eight paddle-wheel units at the corners and can be treated as a polyhedron. The corners (black dots in Fig. 2) represent the vertex positions of the paddle-wheels connected by the linkers (black lines). However, if deformed pores are considered, the geometrical description of an orthorhombus is not sufﬁcient, which is why the volume has to be determined by a more suitable geometric object. In order to develop a general approach, which can be used for an arbitrary pore shape and, therefore, for different MOFs, the unit-cell is divided into ﬁve tetrahedrons as shown in Fig. 2. ARTICLE ARTICLE COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-023-01025-x The volume of one tetrahedron with the vertices O, A, B, and C can be calculated by to the previous simulations with a ﬁctitious mechanical force, various mechanisms could be observed, indicating the absence of a bias on the pore closing path. Vtet ¼ 1 6 a! b!´ c! ¼ 1 6 ax ðby cz bz cyÞ þ ay ðbz cx bx czÞ þ az ðbx cy by cxÞ ; ð1Þ Despite the intriguing similarity of the pressure bath method to the actual experimental conditions, the method has several drawbacks. First, the free volume of the sufﬁciently large simu- lation cell must be densely packed with the pressure bath med- ium, adding a substantial numerical overhead to the simulation since the overall number of particles is substantially increased. Furthermore, simulations of negative pressure for reopening a system are impossible due to the purely repulsive potential between the pressure bath and MOF. Because of the high surface tension, ﬁrst pore closing events are observed at the corners and edges of the NC, where the curvature of the NC surface is highest. This is a realistic effect, but it is possibly artiﬁcially exaggerated for the case of very small NCs, as in the simulations. Most importantly, however, due to signiﬁcant pressure ﬂuctuations in the pressure bath itself, the phase transition of the NC is not triggered at a ﬁxed global pressure, and integration of the free energy is not possible. ð1Þ with the vectors a!, b!, and c!, pointing from O to A, B, and C, respectively. Note that labeling the vertices is arbitrary and must be done once in the beginning for each of the ﬁve tetrahedrons, forming one of the two representations. The derivative of the volume with respect to the Cartesian coordinates of the vertices can be determined via the derivatives with respect to the components of the vectors a!, b!, and c!. ARTICLE ∂V ∂ax ¼ 1 6 by cz bz cy ∂V ∂ay ¼ 1 6 bz cx bx cz ∂V ∂az ¼ 1 6 bx cy by cx ∂V ∂bx ¼ 1 6 ay cz az cy ∂V ∂by ¼ 1 6 az cx ax cz ∂V ∂bz ¼ 1 6 ax cy ay cx ∂V ∂cx ¼ 1 6 by az bz ay ∂V ∂cy ¼ 1 6 bz ax bx az ∂V ∂cz ¼ 1 6 bx ay by ax ; ð2Þ gy p Due to these drawbacks, a general approach is ultimately needed in order to access the volume of MOF NCs and to be able to manipulate it during the simulation while keeping the ﬂex- ibility of the pore closing pathway, as observed in the pressure bath studies. A sufﬁciently accurate approximation of the deformed unit-cell volume is necessary to do this for an arbi- trarily shaped NC. We propose to use a tetrahedron 3D tessel- lation of the individual pore and the summation of the individual pore volumes to achieve this. The following section discusses the general approach of the volume control method, including the necessary forces resulting from a harmonic restraining potential term. The approach is demonstrated for two different MOF NCs with the underlying topology pcu, namely the DMOF-1 (Zn2(bdc)2(dabco))19 and DUT-128 (Zn) (Zn2(4,4’- bpdc)2(dabco); 4,4’-bpdc: 4,4’-biphenyldicarboxylate)20 investi- gated already in previous studies as ﬁnite size systems. ð2Þ Simulating the structural phase transitions of metal-organic frameworks with control over the volume of nanocrystallites https://doi.org/10.1038/s42004-023-01025-x OPEN The third approach, introduced in this study, focuses on the volume of the ﬁnite size system, i.e., to get access to the pore volume and inﬂuence it. Fig. 1 Handling of ﬁnite size systems in MD simulation. The ﬁrst approach introduces a mechanical force to initialize the pore closing mechanism by lowering the distance between opposite-corner SBUs. The pressure bath concept returns to PBCs: A ﬁnite-sized system is set up in a ﬂuid. This system as a whole is simulated using PBCs. The third approach, introduced in this study, focuses on the volume of the ﬁnite size system, i.e., to get access to the pore volume and inﬂuence it COMMUNICATIONS CHEMISTRY \| (2023) 6:233 \| https://doi.org/10.1038/s42004-023-01025-x \| www.nature.com/commschem COMMUNICATIONS CHEMISTRY \| (2023) 6:233 \| https://doi.org/10.1038/s42004-023-01025-x \| www.nature.com/commschem 2 Methodology A ll i b The cuboidal volume can be tessellated by tetrahedrons spanned by the vectors a!, b!, and c!. In order to describe an arbitrary pore shape, the pore volume can be obtained by the sum of the volume of ﬁve tetrahedrons: One inner tetrahedron (c) and four tetrahedrons built by the sides of the pore and one face of the inner tetrahedron (d). The two possibilities to tessellate the pore illustrated on left and right in (c) and (d). Computational details. All force ﬁeld calculations were per- formed with the LAMMPS molecular mechanics program package23,24. The volume constraint has been implemented in Python using the LAMMPS ﬁx python/invoke in order to per- form calculations of the NC’s volume and the forces resulting from the external potential Uext (Eq. (3)) in each timestep. For numerical efﬁciency, the computation is done in parallel, dis- tributing over the pores, and the core routines are accelerated by a just-in-time compiler Numba25. The implementation was tested to give energy conserving dynamics in a microcanonical simulation (see further details in Supplementary Note 1.2 and Supplementary Fig. 1). distribution. The system was coupled to the Nosé-Hoover chain thermostat31 with a relaxation time of 0.05 ps. All subsequent simulations were carried out with a relaxation time of 0.1 ps and a temperature of 300 K, whereby the temperature is only a reference for the kinetic energy in the system. gy y To apply the volume restraining external potential, a proper choice of the force constant k is needed since it controls the strength of the potential to bias the volume. For this purpose, a screening experiment was performed using DMOF-1 NCs in the op form as a test system in order to determine its magnitude. Further details are given in the Supplementary Note 1.1. Supplementary Table 1 lists the tested force constants and the difference between the mean volume of the respective NC during the simulation and the reference volume for the respective system. As a result, a force constant k = 0.002 kcal ⋅mol−1 ⋅Å−6 was used in all further SMD calculations to maintain a difference between the mean volume of the respective NC during the simulation and the reference volume of approximately ΔV = 10 Å3 up to ΔV = 80 Å3 for all investigated sizes, while the ﬂuctuation of the volume is ΔΔV ≥±50 Å3. Methodology A ll i pp y g The open pore form of the differently sized DMOF-1 and DUT-128 NCs was constructed by the reverse topological approach (RTA)26–28. The procedure of generating the cubic NCs is described in detail in reference14: First, the blueprints for the differently sized nanocrystallites were prepared by slicing the respective supercells of the pcu topology and extending them by vertices for the stubs. Then, the BBs were assigned to their respective vertices. The NCs were named after the number of paddle-wheel units in the x-, y-, and z-direction, e.g., the 5 × 5 × 5 NC has ﬁve paddle-wheel units and four pores in each spatial direction. In this study, the 3 × 3 × 3 up to the 6 × 6 × 6 DMOF-1 NCs and the 3 × 3 × 3 up to the 9 × 9 × 9 DUT-128 NCs were investigated. The interactions of the DMOF-1 NCs were described by the ﬁrst principles parameterized MOF-FF force ﬁeld29,30. The heated systems were ﬁrst equilibrated for 100ps without any restraints. Then, the external potential for volume control was switched on. First, the reference volume was kept constant to the current volume for 10 ps. Then, the reference volume was linearly decreased from 1170 Å3 ⋅npores to 610 Å3 ⋅npores for DMOF-1 and from 2200 Å3 ⋅npores to 8200 Å3 ⋅npores for DUT- 128, where n refers to the number of pores in the respective NC, with a speed of 0.1 Å3 per time step. For the 5 × 5 × 5 DMOF-1 NC, reversed calculations were performed, in which the reference volume was linearly increased from 610 Å3 ⋅npores to 1170 Å3 ⋅npores to simulate the reopening from the closed pore form to the open pore form. The structures were structurally optimized using the conjugate gradient minimization method and, in addition, the steepest descent algorithm to relax the cell. First, the atomic positions were optimized with an energy convergence criterion of 0.15 kcal ⋅mol−1 ⋅Å−1. Then, the cell and atomic positions were changed by the steepest descent algorithm until the forces acting are lower than 0.1 kcal ⋅mol−1 ⋅Å−1. Finally, the atomic posi- tions were optimized once more with an energy convergence criterium of 0.05 kcal ⋅mol−1 ⋅Å−1. The free energy needed to transform the differently sized NCs from the open to the closed pore form was determined by US simulations32 using the volume as CV. Methodology A ll i There is one tetrahedron inside the pore (colored in blue and orange, respectively) and four tetrahedrons built by the sides of the pore and one of the faces of the centered tetrahedron. Four paddle-wheel vertices deﬁne each tetrahedron. There are two possibilities to insert these ﬁve tetrahedrons. For an orthorhombic pore, both possibilities are equivalent, but for a deformed system, the resulting volume depends on how the tetrahedrons are placed. To avoid this ambiguity, both possibilities are considered and the resulting total volumes are averaged. In order to control the individual pore volume, a volume restraining potential is applied to the MOF NCs, which modiﬁes the acting forces by a volume-dependent harmonic part. The external potential has the form UðrÞext ¼ 1 2 k V rð Þ Vref 2 ; ð3Þ ð3Þ where k is the force constant, Vref is the reference volume, V is the total volume of the NC, and r represents the position of the atoms in space. The quadratic term adds a penalty proportional to the deviation from the reference volume. Note, that only the atoms of the paddle-wheel unit (metal and carboxylate atoms) deﬁning the tetrahedron vertices via their COM are contributing and are affected by forces from the restraint. The introduction of this external potential allows for deﬁning a collective variable (CV) and performing umbrella sampling (US)21,22 to get a free energy proﬁle. COMMUNICATIONS CHEMISTRY \| (2023) 6:233 \| https://doi.org/10.1038/s42004-023-01025-x \| www.nature.com/commschem 3 ARTICLE COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-023-01025-x Fig. 2 Geometrical consideration of porous materials. a The smallest possible DMOF-1 NC with only one pore, shown without stubs and H atoms, can be treated as a cuboid, which is deﬁned by the eight paddle-wheel vertices. b The cuboidal volume can be tessellated by tetrahedrons spanned by the vectors a!, b!, and c!. In order to describe an arbitrary pore shape, the pore volume can be obtained by the sum of the volume of ﬁve tetrahedrons: One inner tetrahedron (c) and four tetrahedrons built by the sides of the pore and one face of the inner tetrahedron (d). The two possibilities to tessellate the pore illustrated on left and right in (c) and (d). Fig. 2 Geometrical consideration of porous materials. a The smallest possible DMOF-1 NC with only one pore, shown without stubs and H atoms, can be treated as a cuboid, which is deﬁned by the eight paddle-wheel vertices. Results and discussion Phase transformation of the model system DMOF-1. The volume restraining potential is applied to DMOF-1 NCs to initiate the pore closing mechanism from an open to a closed form via SMD. As previously observed for the pillared-layer MOF NCs in the pressure bath medium, the pore closing propagates through the x-y-plane for all z-layers of the NC. Hence, the process is again analyzed within this plane. In the following, the phase transition pathways of DMOF-1 NCs with the sizes 3 × 3 × 3 up to 6 × 6 × 6 are discussed concerning their ﬂexibility to end up in the closed pore form. Nevertheless, the results demonstrate that the method main- tains the ﬂexibility to correct a (partially-)closed pore form to a more favored one. This allows two conclusions: First, the chosen force constant seems suitable to ﬁx the system in a given conformation on the potential energy surface, but it allows enough ﬂuctuation in the volume of the NCs to reopen closed pores. Second, it is validated that only one phase boundary in the transition structure and the diamond-shaped closed pore form are the favored formations of the NCs. p p Figure 3 shows the open and closed pore, as well as selected intermediate structures. All investigated systems end up in the diamond-shaped closed pore form. Transition structures with one phase boundary are the most probable intermediate structures, but also, two phase boundaries are observed during the phase transformation. To discuss the transition path in detail, the individual pore sizes are averaged along the z-axis to show the view on the xy-plane (Fig. 4). The center of the pores are colored according to the respective volume. Blue-colored dots represent closed pores with a volume of ~610 Å3, whereas a red-colored symbol indicates open pores with a volume of ~1170 Å3. Umbrella sampling calculations of the model system DMOF-1. In the next step, the approximate total volume of the NCs, deter- mined by the tetrahedron tessellation, was chosen as a CV to compute the Helmholtz free energy proﬁle of the phase trans- formation by US. The initial structures for the US windows were obtained from snapshots of the SMD discussed in the previous section. Hence, e.g., for the 5 × 5 × 5 NC and the 6 × 6 × 6 NC, some windows start with the transition structures containing two phase boundaries. COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-023-01025-x Fig. 3 Structural transition of differently sized DMOF-1 NCs. The open, partially closed, and closed pore forms are obtained by applying the extended volume restraining potential during simulation. All system sizes end up in a diamond-shaped closed pore form. Fig. 3 Structural transition of differently sized DMOF-1 NCs. The open, partially closed, and closed pore forms are obtained by applying the extended volume restraining potential during simulation. All system sizes end up in a diamond-shaped closed pore form. Fig. 3 Structural transition of differently sized DMOF-1 NCs. The open, partially closed, and closed pore forms are volume restraining potential during simulation. All system sizes end up in a diamond-shaped closed pore form. and a force constant of 0.001 kcal ⋅mol−1 ⋅Å−6 for the 3 × 3 × 3 and the 4 × 4 × 4 DMOF-1 NC, and a force constant of 0.0001 kcal ⋅mol−1 ⋅Å−6 for the 5 × 5 × 5 and the 6 × 6 × 6 DMOF-1 NC as well as for all DUT-128 NCs. The CV was recorded every 100 steps for DMOF-1 NCs and every 500 steps for DUT-128 NCs. The overlap of the respective windows was checked by visualization of the distributions of the CV (see Supplementary Note 1.3 and Supplementary Fig. 2 for DMOF-1 as well as Supplementary Note 2.1 and Supplementary Fig. 4 for DUT-128). The free energy proﬁle was determined by the Weighted Histogram Analysis Method (WHAM) using the wham code33 and a convergence threshold of 0.0001 kcal ⋅mol−1. For each system size, three independent runs of SMD and consecutive US calculations with different initial conditions were carried out and the resulting free energies were averaged. a diamond-shaped structure of the entire NC. The 5 × 5 × 5 and 6 × 6 × 6 NC show a slightly different transition mechanism: The (centered) inner layer closes ﬁrst, whereas no outer layers or corners are affected initially. For the 5 × 5 × 5 NC, a reopening of closed pores is observed after one of the outer layers is entirely closed. Then, the known layer-by-layer pore closing proceeds, ending in the diamond-shaped closed pore form. In the case of the 6 × 6 × 6 NC, the central and a neighboring layer close ﬁrst, resulting in two phase boundaries. COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-023-01025-x The fact that the outer layers are still open, whereas the mid-layers are entirely closed, causes structural tension in the NC and explains the reopening of one of the middle layers and the spontaneous pore closing of the outer layer at t = 480 ps. In the end, the diamond-shaped structure is formed again by a layer-by-layer closing. The free energy proﬁle was determined by the Weighted Histogram Analysis Method (WHAM) using the wham code33 and a convergence threshold of 0.0001 kcal ⋅mol−1. For each system size, three independent runs of SMD and consecutive US calculations with different initial conditions were carried out and the resulting free energies were averaged. These pore closing mechanisms are selected results from several SMDs of each investigated system. They demonstrate trends and allow for testing of the volume control approach. However, the SMD settings, like the speed of change of the reference volume or the chosen force constant, inﬂuence the individual processes. In addition, they depend on the initial conditions, and in order to elucidate the pore closing mechanism as statistically meaningful, more than three performed SMD simulations for each system size would be needed. Methodology A ll i For this purpose, snapshots were taken from steered Molecular Dynamics (SMD) simulation every ΔV0 = 100 Å3 for the 3 × 3 × 3 and the 4 × 4 × 4 DMOF-1 NC, and every ΔV0 = 200 Å3 for the 5 × 5 × 5 and the 6 × 6 × 6 DMOF-1 NC as well as for all DUT-128 NCs. For each window, a 100 ps simulation was carried out with a ﬁxed reference volume The optimized structures were heated up from 10 K to 300 K within 50 ps in the NVT ensemble with a time step of 1 fs. The initial velocities were generated by a Maxwell-Boltzmann COMMUNICATIONS CHEMISTRY \| (2023) 6:233 \| https://doi.org/10.1038/s42004-023-01025-x \| www.nature.com/commschem 4 ARTICLE COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-023-01025-x COMMUNICATIONS CHEMISTRY \| (2023) 6:233 \| https://doi.org/10.1038/s42004-023-01025-x \| www.nature.com/commschem COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-023-01025-x ARTICLE Fig. 4 Pore size analysis of differently sized DMOF-1 NCs. The pores are colored according to their size. The pore closing is induced by the harmonic restraining potential. For the 3 × 3 × 3 and the 4 × 4 × 4 NC, the common layer-by-layer pore-closing mechanism is observed. In contrast, the pore-closing mechanism of the 5 × 5 × 5 and the 6 × 6 × 6 NC starts at the centered layers and propagates to the edges. Fig. 4 Pore size analysis of differently sized DMOF-1 NCs. The pores are colored according to their size. The pore closing is induced by the harmonic restraining potential. For the 3 × 3 × 3 and the 4 × 4 × 4 NC, the common layer-by-layer pore-closing mechanism is observed. In contrast, the pore-closing mechanism of the 5 × 5 × 5 and the 6 × 6 × 6 NC starts at the centered layers and propagates to the edges. Fig. 4 Pore size analysis of differently sized DMOF-1 NCs. The pores are colored according to their size. The pore closing is induced by the harmonic restraining potential. For the 3 × 3 × 3 and the 4 × 4 × 4 NC, the common layer-by-layer pore-closing mechanism is observed. In contrast, the pore-closing mechanism of the 5 × 5 × 5 and the 6 × 6 × 6 NC starts at the centered layers and propagates to the edges. Fig. 5 Chronological snapshots from the trajectory of the 6 × 6 × 6 NC with the reference volume of V0 = 1 ×105 Å3. a t = 0 ns, b t = 0.03 ns, and c t = 0.1 ns. While the reference volume is kept constant, the system closes the outer layer pores (from (a) to (b)) and reopens its inner pores (from (b) to (c)), avoiding two phase boundaries present in (a). Fig. 5 Chronological snapshots from the trajectory of the 6 × 6 × 6 NC with the reference volume of V0 = 1 ×105 Å3. a t = 0 ns, b t = 0.03 ns, and c t = 0.1 ns. While the reference volume is kept constant, the system closes the outer layer pores (from (a) to (b)) and reopens its inner pores (from (b) to (c)), avoiding two phase boundaries present in (a). In Fig. Results and discussion Interestingly, it is found that these structures reopen during the simulation with a constant reference volume y p p The closing mechanism of the 3 × 3 × 3 NC and the 4 × 4 × 4 NC is the archetypical mechanism for the phase transformation: The process starts at one corner and propagates asymmetrical through the NC, i.e., the pore closing occurs in one direction ﬁrst, which induces the closing of the neighbored layer, and ends up in COMMUNICATIONS CHEMISTRY \| (2023) 6:233 \| https://doi.org/10.1038/s42004-023-01025-x \| www.nature.com/commschem 5 COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-023-01025-x COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-023-01025-x ARTICLE Fig. 6 Free energy curves for DMOF-1. a The total Free energy ΔA curves of the structural phase transition of the differently sized DMOF-1 NCs reveal that the open pore form is lower in energy. b The free energy ΔA proﬁle of the 5 × 5 × 5 NC based on US simulations using initial structures obtained by an SMD from the open to the closed pore form (dark green) and from the closed to the open pore form (light green) has a similar shape. open and closed form becomes higher when the system size is increased. A similar trend was observed for the unnormalized free energy curves in reference14, where a simple mechanical force initiate the phase transition. This was explained by the increasing total barrier due to the larger number of pores, which have to be closed simultaneously, which also appears valid for the here presented results. For even larger NCs, the barrier is expected to converge to a particular value since only a limited fraction of the pores will have to undergo a structural transition for the pore closing. This will allow us to extrapolate the limiting barrier for arbitrarily large systems. This proof-of-concept study demonstrates that US using the NC’s total volume as a CV results in a similar transition pathway as the US using a restrained distance between SBUs on opposite edges of the NC14. Since the new volume-based CV allows for more ﬂexibility concerning the pore closing and reopening, it can be concluded that this represents a realistic scenario for the mechanism of the phase transformation. In addition, the proposed method solves the problems of the pressure bath method, e.g., the reversed structural transition from closed to open pore form can be mechanistically investigated. Impact of an Extended Linker: DUT-128 NCs. In order to generalize the volume control method, it is also applied to DUT- 128, which has an experimentally proven stable closed pore form. The DUT-128 NCs are related to DMOF-1, i.e., they have the same underlying topology but the linker in x- and y-direction is extended, leading to a larger dispersive interaction in the closed form. In the ﬁrst step, the DUT-128 NCs with the sizes 3 × 3 × 3 up to 9 × 9 × 9 were investigated. COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-023-01025-x This is the dominant closing mechanism for all investigated systems and sizes. In Fig. 8a, several pore closing mechanisms of the 6 × 6 × 6 NC are displayed from individual SMD simulations. The most signiﬁcant differences are visible at the beginning of the process. The formation of one phase boundary, propagating through the system, can be initialized from different sides and edges of the NC. Despite the different orientations of the phase boundary and the consecutive direction of the propagation, they end up all in the same diamond-shaped structure. An exception is observed in the trajectories for the 4 × 4 × 4 NC: Figure 8b reveals that the partially closed pore form differs in one case. Here, no distinct phase boundary can be identiﬁed, and the pore closing starts randomly in several pores, resulting in a non-diamond-shaped form. This demonstrates that the volume control method does not enforce a speciﬁc mechanism and allows to explore the potential energy landscape of the structural change mechanism in an unbiased way. To validate the RC chosen for the US approach and to check convergence, the SMD to obtain the initial structures was repeated for the 5 × 5 × 5 NC. In contrast to the initial run, ramping the reference volume up enforced the reversed transition from the closed to the open pore form. In Fig. 6b, both free energy curves obtained via SMD in opposite directions are given, showing a slight deviation. This indicates the remaining statistical error due to insufﬁcient convergence. Umbrella sampling calculations of DUT-128 NCs. Due to the enlarged linkers, the number of atoms in the systems increases compared to DMOF-1 NCs with an equal number of pores. This results in a larger numerical effort especially for the US simula- tions, and thus we focused on the NCs up to a size of 5 × 5 × 5. Figure 9 shows the total free energy curves of the pore closing mechanism of the 3 × 3 × 3 up to the 5 × 5 × 5 DUT-128 NCs. As expected and before observed for DMOF-1 NCs, they reveal a However, the shape of the two curves is similar with an energetic minimum at approximately 4 × 104 Å3 for the closed pore form and at approximately 7.3 × 104 Å3 for the open pore form. COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-023-01025-x Similar to DMOF-1 NCs, the pore closing is triggered via SMD and observed along the x-y- plane for all z-layers of the NCs. Supplementary Note 1.4 and Supplementary Fig. 3). Second, the closed pore form is an energetic minimum structure of the DMOF- 1 NCs. Thus, the calculations based on MOF-FF predict a stable structure for the closed pore form. However, it is substantially higher in energy than the open pore form, meaning that the open pore form is the more favored structure. Experimentally, DMOF-1 is a non-ﬂexible MOF, which tends to amorphize under hydrostatic pressure34. However, bond breaking can not be simulated for a non- reactive model like MOF-FF, and a ﬁctitious stable but high-energy Fig. 6 Free energy curves for DMOF-1. a The total Free energy ΔA curves of the structural phase transition of the differently sized DMOF-1 NCs reveal that the open pore form is lower in energy. b The free energy ΔA proﬁle of the 5 × 5 × 5 NC based on US simulations using initial structures obtained by an SMD from the open to the closed pore form (dark green) and from the closed to the open pore form (light green) has a similar shape. Fig. 6 Free energy curves for DMOF-1. a The total Free energy ΔA curves of the structural phase transition of the differently sized DMOF-1 NCs Fig. 6 Free energy curves for DMOF-1. a The total Free energy ΔA curves of the structural phase transition of the differently sized DMOF-1 NCs reveal that the open pore form is lower in energy. b The free energy ΔA proﬁle of the 5 × 5 × 5 NC based on US simulations using initial structures obtained by an SMD from the open to the closed pore form (dark green) and from the closed to the open pore form (light green) has a similar shape. Figure 7 shows the open and closed pore form, as well as selected transition structures. Most of the investigated systems end up in the diamond-shaped closed pore form. The transition structures for the 3 × 3 × 3 and the 6 × 6 × 6 up to the 9 × 9 × 9 NC reveal that the phase transition occurs subsequently via propagation of a one phase boundary through the system from one edge to the opposite one. COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-023-01025-x 6a, the total free energies are plotted against the RC. All curves have roughly the same shape. In all cases, the energetically lowest structure is the open pore form, which has been taken as zero. When the total volume of the NC is decreased, the energy rapidly increases until it reaches a local maximum. For small volumes, the energy curve has a local minimum representing the closed pore form of the NC of the respective size. The free energy proﬁles allow two essential conclusions: First, for all sizes, the phase transformation implies overcoming a free energy barrier between the open and the completely closed pore form, which is substantially smaller than for the PBC case14,34 after normalizing per pore (see and transform into a conﬁguration with only one phase bound- ary, which is expected to reduce the strain energy resulting from such a phase boundary. Figure 5 shows snapshots from the trajectory of the 6 × 6 × 6 NC, biased by a reference volume of V0 = 1 × 105 Å3, as an example. In contrast, a structure with only one phase boundary does not reopen during the simulation, corroborating that structures with one phase boundary are energetically preferred. p y g y p Figure 6 displays the averaged free energy proﬁles for the phase transition of all four investigated system sizes determined from the US calculation concerning the volume restraint. COMMUNICATIONS CHEMISTRY \| (2023) 6:233 \| https://doi.org/10.1038/s42004-023-01025-x \| www.nature.com/commschem 6 COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-023-01025-x COMMUNICATIONS CHEMISTRY \| (2023) 6:233 \| https://doi.org/10.1038/s42004-023-01025-x \| www.nature.com/commschem COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-023-01025-x Thus, the method gives qualitatively correct results, but an even longer simulation time would be needed to improve the quantitative results. Figure 9 shows the total free energy curves of the pore closing mechanism of the 3 × 3 × 3 up to the 5 × 5 × 5 DUT-128 NCs. As expected and before observed for DMOF-1 NCs, they reveal a When comparing energy proﬁles of different system size, it is evident that both the energy barrier and the difference between 7 ARTICLE COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-023-01025-x substantial size effect i e the energy difference between closed computational experiment however we are able to observe the Fig. 7 Structural transition of differently sized DUT-128 NCs. The open, partially closed, and closed pore forms are obtained by applying the extended volume restraining potential during simulation. Although the transition structures are different, all systems end up in a diamond-shaped closed pore form. Fig. 7 Structural transition of differently sized DUT-128 NCs. The open, partially closed, and closed pore forms are obtained by applying the extended volume restraining potential during simulation. Although the transition structures are different, all systems end up in a diamond-shaped closed pore form. substantial size effect, i.e., the energy difference between closed and open pore form, as well as the free energy barrier between the states, is increasing for larger system sizes. A plot of the free energy normalized per pore can be found in Supplementary Fig. 3 (Supplementary Note 1.4). computational experiment, however, we are able to observe the process disentangled from adsorption and focus on the properties of the pure MOF NC. A further feature of the free energy curves is that the local minima in the plateau region between the closed and open pore form increase in number and depth with growing system size. For DMOF-1, they are much less pronounced. Each minimum corresponds to an entirely closed row of pores. For the 5 × 5 × 5 NC with four rows of pores, exactly three such minima are observed before the system is closed. Such minima for the intermediate system with phase coexistence have previously been observed for simulations in PBC when large supercells are used12,13. pp y However, in contrast to DMOF-1, for DUT-128, the closed pore form is now lower in energy than the open pore form due to the stabilizing dispersive interactions for the biphenyl bridge in the linker. COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-023-01025-x This also conﬁrms the experimentally observed stable closed pore form of DUT-128. It is instructive to compare the energetics of phase transformation for the 5 × 5 × 5 NCs of DMOF-1 with DUT-128: For the smaller DMOF-1, the closed pore minimum is about 250 kcal ⋅mol−1 above to open form, but with a small barrier of only about 25 kcal ⋅mol−1, whereas the larger DUT-128 the closed form is more stable by about 150 kcal ⋅mol−1 and with a substantially larger barrier of about 75 kcal ⋅mol−1. Thus, also the activated open pore form of DUT- 128 can be observed. Note that under experimental conditions, phase transformations are usually triggered by guest molecule adsorption, which will alter the free energy curves. In our COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-023-01025-x Fig. 8 Structural phase transition pathway of DUT-128 NCs with different sizes. a For the 6 × 6 × 6 NC, all phase transitions end up in the diamond- shaped closed pore form. Signiﬁcant differences during the phase transition are observable at the beginning of the mechanism. For smaller reference volumes, a phase boundary between open and closed pore forms travels through the system, independent of how the pore closing starts. b For the 4 × 4 × 4 NC, a different closed pore form than the diamond-shaped one is observed. COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-023-01025-x ARTICLE Fig. 8 Structural phase transition pathway of DUT-128 NCs with different sizes. a For the 6 × 6 × 6 NC, all phase transitions end up in the diamond- shaped closed pore form. Signiﬁcant differences during the phase transition are observable at the beginning of the mechanism. For smaller reference volumes, a phase boundary between open and closed pore forms travels through the system, independent of how the pore closing starts. b For the 4 × 4 × 4 NC, a different closed pore form than the diamond-shaped one is observed. mechanism without bias. In addition, the direct volume control is a suitable CV and can be used as a bias potential to perform US to calculate the free energy proﬁles of the structural phase transition. In contrast to DMOF-1, for the DUT-128 NCs with an extended linker, the closed pore form is computed to have lower free energy than the open pore form. However, the latter should also be a metastable phase because of a larger barrier. volumes are calculated individually and summed up. The pro- cedure is repeated for every pore of the NC. A volume restraining potential is employed to control the NC’s total volume without enforcing an individual pore volume and to avoid any bias on the phase transformation mechanism. The volume control method was applied to differently sized DMOF-1 and DUT-128 nanocrystallites to investigate their structural transformation and compare it to the formerly obtained pressure bath results18. For this purpose, MD simula- tions on the FF level of the structural transition from the open to the closed pore form were performed. The non-synchronous phase transformation results in differently shaped closed pore forms revealing various possible transition pathways, especially for larger-sized linkers. COMMUNICATIONS CHEMISTRY \| (2023) 6:233 \| https://doi.org/10.1038/s42004-023-01025-x \| www.nature.com/commschem Conclusion The methods for computational investigation of structural phase transitions of MOFs beyond PBCs using NCs have been extended by developing and testing an approach to control the volume of irregularly shaped objects accurately. To determine the volume of an arbitrarily shaped pore, it is tessellated by tetrahedrons, whose COMMUNICATIONS CHEMISTRY \| (2023) 6:233 \| https://doi.org/10.1038/s42004-023-01025-x \| www.nature.com/commschem 8 ARTICLE COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-023-01025-x COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-023-01025-x The total Free energy ΔA curves of the structural phase transition of the differently sized DUT-128 NCs reveal that the closed pore form is lower in energy. 12. Rogge, S. M. J., Waroquier, M. & Van Speybroeck, V. Unraveling the thermodynamic criteria for size-dependent spontaneous phase separation in soft porous crystals. Nat. Commun. 10, 4842 (2019). 13. Vandenhaute, S., Rogge, S. M. J. & Van Speybroeck, V. Large-scale molecular dynamics simulations reveal new insights into the phase transition mechanisms in MIL-53(Al). Front. Chem. 9, 718920 (2021). ratios, the direct volume control method will make such inves- tigations computationally more tractable since size effects are observed experimentally for synthesized MOF powders, whose sizes are still one or two orders of magnitude larger than the here studied systems. In addition, the method could be combined with the explicit simulation of guest molecules surrounding the NC, which allows to study transport and adsorption in a joint fashion. 14. Keupp, J. & Schmid, R. Molecular dynamics simulations of the “breathing" phase transformation of MOF nanocrystallites. Adv. Theory Simul. 2, 1900117 (2019). 15. Keupp, J., Dürholt, J. P. & Schmid, R. Inﬂuence of ﬂexible side-chains on the breathing phase transition of pillared layer MOFs: a force ﬁeld investigation. Faraday Discuss. 225, 324–340 (2021). y 16. Zacher, D., Schmid, R., Wöll, C. & Fischer, R. A. Surface chemistry of metal- organic frameworks at the liquid-solid interface. Angew. Chem. Int. Ed. 50, 176–199 (2011). The results illustrate that the phase transition of MOFs can follow various possible pathways, which can only be simulated properly in the absence of the constraint of periodic boundary conditions. Consequently, it requires appropriate methods to sample the phase space, especially in low probability regions. In this context, the direct volume control is the ideal collective variable to study such processes. Its application to MOF NCs will lead to a more detailed understanding of the underlying mechanism, i.e., to elucidate the surface and transport effects of guest molecules inducing structural transition processes by determining the limiting pressure of closing, which can be used to improve the applications of ﬂexible materials by the targeted design of the phase transition behavior. 17. Amirjalayer, S., Taﬁpolsky, M. & Schmid, R. Surface termination of the metal- organic framework HKUST-1: a theoretical investigation. J. Phys. Chem. Lett. 5, 3206–3210 (2014). 18. Schaper, L., Keupp, J. & Schmid, R. COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-023-01025-x The results agree with those obtained previously by invoking hydrostatic pressure on the NC by the pressure bath method, demonstrating that direct volume control allows similar ﬂexibility to explore the phase transition The approach has three main advantages compared to the pressure bath method. First, it requires a substantially lower numerical effort because the pressure mediating particles are not needed. Second, it solves the main problems of the pressure bath method, namely that the pressure ﬂuctuations in the pressure bath prevent the determination of pressure-volume curves and, thus, the computation of free energy curves. Moreover, third, the reversed process of a structural transition from the closed to the open pore form can also be investigated. Especially for the simulation of substantially larger NCs, e.g., with different aspect 9 ARTICLE COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-023-01025-x Fig. 9 Free energy curves of DUT-128. The total Free energy ΔA curves of the structural phase transition of the differently sized DUT-128 NCs reveal that the closed pore form is lower in energy. 5. Krause, S. et al. Impact of defects and crystal size on negative gas adsorption in DUT-49 analyzed by in situ 129 Xe NMR spectroscopy. Chem. Mater. 32, 4641–4650 (2020). 6. Kitagawa, S., Kitaura, R. & Noro, S.-i Functional porous coordination polymers. Angew. Chem. Int. Ed. 43, 2334–2375 (2004). 7. Vandenhaute, S., Cools-Ceuppens, M., DeKeyser, S., Verstraelen, T. & Speybroeck, V. V. Machine learning potentials for metal-organic frameworks using an 7. Vandenhaute, S., Cools-Ceuppens, M., DeKeyser, S., Verstraelen, T. & Speybroeck, V. V. Machine learning potentials for metal-organic frameworks using an incremental learning approach. npj Comput. Mater. 9, 19 (2023). incremental learning approach. npj Comput. Mater. 9, 19 (2023) 8. Ehrling, S., Miura, H., Senkovska, I. & Kaskel, S. From macro- to nanoscale: ﬁnite size effects on metal-organic framework switchability. Trends Chem. 3, 291–304 (2021). 9. Bennett, T. D., Cheetham, A. K., Fuchs, A. H. & Coudert, F.-X. Interplay between defects, disorder and ﬂexibility in metal-organic frameworks. Nat. Chem. 9, 11–16 (2017). 10. Bechis, I. et al. 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TopoFF: MOF structure prediction using speciﬁcally optimized blueprints. Faraday Discuss. 211, 79–101 (2018). y The Python code for the volume restraint can be found in the github repository: https:// github.com/cmc-rub/supporting_data/tree/master/109-Schaper-commschem-2023. y The Python code for the volume restraint can be found in the github repository: https:// github.com/cmc-rub/supporting_data/tree/master/109-Schaper-commschem-2023. 27. Bureekaew, S., Balwani, V., Amirjalayer, S. & Schmid, R. Isoreticular isomerism in 4,4-connected paddle-wheel metal-organic frameworks: structural prediction by the reverse topological approach. CrystEngComm 17, 344–352 (2015). Received: 14 April 2023; Accepted: 9 October 2023; 28. Impeng, S., Cedeno, R., Dürholt, J. P., Schmid, R. & Bureekaew, S. Computational structure prediction of (4,4)-connected copper paddle-wheel- based MOFs: inﬂuence of ligand functionalization on the topological preference. Cryst. Growth Des. 18, 2699–2706 (2018). Acknowledgements Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. This work has been funded by the Deutsche Forschungsgemeinschaft (DFG) by grants (SCHM1389/10-1 and SCHM1389/10-2) within research unit FOR2433. This work has been funded by the Deutsche Forschungsgemeinschaft (DFG) by grants (SCHM1389/10-1 and SCHM1389/10-2) within research unit FOR2433. Author contributions 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/. R.S. designed the model and carried out the implementation. L.S. performed the calcu- lations and analyzed the data. Both authors wrote the manuscript. Funding Funding Open Access funding enabled and organized by Projekt DEAL. References 29. Bureekaew, S. et al. MOF-FF - A ﬂexible ﬁrst-principles derived force ﬁeld for metal-organic frameworks. Phys. Status Solidi B Basic Res. 250, 1128–1141 (2013). References 1. Coudert, F.-X. Responsive metal-organic frameworks and framework materials: under pressure, taking the heat, in the spotlight, with friends. Chem. Mater. 27, 1905–1916 (2015). 2. Dürholt, J. P., Jahromi, B. F. & Schmid, R. Tuning the electric ﬁeld response of MOFs by rotatable dipolar linkers. ACS Cent. Sci. 5, 1440–1448 (2019). 3. Mason, J. A. et al. Methane storage in ﬂexible metal-organic frameworks with intrinsic thermal management. Nature 527, 357–361 (2015). 4. Evans, J. D., Bocquet, L. & Coudert, F.-X. Origins of negative gas adsorption. Chem 1, 873–886 (2016). 1. Coudert, F.-X. Responsive metal-organic frameworks and framework materials: under pressure, taking the heat, in the spotlight, with friends. Chem. Mater. 27, 1905–1916 (2015). 1. Coudert, F.-X. Responsive metal-organic frameworks and framework materials: under pressure, taking the heat, in the spotlight, with friends. Chem. Mater. 27, 1905–1916 (2015). 30. Dürholt, J. P., Fraux, G., Coudert, F.-X. & Schmid, R. Ab initio derived force ﬁelds for zeolitic imidazolate frameworks: MOF-FF for ZIFs. J. Chem. Theory Comput. 15, 2420–2432 (2019). 2. Dürholt, J. P., Jahromi, B. F. & Schmid, R. Tuning the electric ﬁeld response of MOFs by rotatable dipolar linkers. ACS Cent. Sci. 5, 1440–1448 (2019). y p 3. Mason, J. A. et al. Methane storage in ﬂexible metal-organic frameworks with intrinsic thermal management. Nature 527, 357–361 (2015). y p 3. Mason, J. A. et al. Methane storage in ﬂexible metal-organic frameworks with intrinsic thermal management. Nature 527, 357–361 (2015). 31. Hoover, W. G. Canonical dynamics: equilibrium phase-space distributions. Phys. Rev. A 31, 1695–1697 (1985). 4. Evans, J. D., Bocquet, L. & Coudert, F.-X. Origins of negative gas adsorption. Chem 1, 873–886 (2016). 4. Evans, J. D., Bocquet, L. & Coudert, F.-X. Origins of negative gas adsorption. Chem 1, 873–886 (2016). 32. Kästner, J. Umbrella sampling: umbrella sampling. WIREs Comput. Mol. Sci. 1, 932–942 (2011). COMMUNICATIONS CHEMISTRY \| (2023) 6:233 \| https://doi.org/10.1038/s42004-023-01025-x \| www.nature.com/commschem 10 ARTICLE COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-023-01025-x Correspondence and requests for materials should be addressed to Rochus Schmid. Correspondence and requests for materials should be addressed to Rochus Schmid. 33. Grossﬁeld, A. "WHAM: The weighted histogram analysis method", version 2.0.10.2. http://membrane.urmc.rochester.edu/content/wham. Peer review information Communications Chemistry thanks Claire Hobday and the other, anonymous, reviewers for their contribution to the peer review of this work. A peer review ﬁle is available. 34. Vervoorts, P. et al. Conﬁgurational entropy driven high-pressure behaviour of a ﬂexible metal–organic framework (MOF). Angew. Chem. Int. Ed. 60, 787–793 (2020). 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https://openalex.org/W1596947964	https://www.nature.com/articles/srep11476.pdf	English	null	Improving prediction of secondary structure, local backbone angles and solvent accessible surface area of proteins by iterative deep learning	Scientific reports	2,015	cc-by	7,900	Improving prediction of secondary structure, local backbone angles, and solvent accessible surface area of proteins by iterative deep learning received: 04 March 2015 accepted: 19 May 2015 Published: 22 June 2015 Rhys Heffernan1, Kuldip Paliwal1, James Lyons1, Abdollah Dehzangi1,2, Alok Sharma2,3, Jihua Wang4, Abdul Sattar2,5, Yuedong Yang6 & Yaoqi Zhou4,6 Direct prediction of protein structure from sequence is a challenging problem. An effective approach is to break it up into independent sub-problems. These sub-problems such as prediction of protein secondary structure can then be solved independently. In a previous study, we found that an iterative use of predicted secondary structure and backbone torsion angles can further improve secondary structure and torsion angle prediction. In this study, we expand the iterative features to include solvent accessible surface area and backbone angles and dihedrals based on Cα atoms. By using a deep learning neural network in three iterations, we achieved 82% accuracy for secondary structure prediction, 0.76 for the correlation coefficient between predicted and actual solvent accessible surface area, 19° and 30° for mean absolute errors of backbone ϕ and ψ angles, respectively, and 8° and 32° for mean absolute errors of Cα-based θ and τ angles, respectively, for an independent test dataset of 1199 proteins. The accuracy of the method is slightly lower for 72 CASP 11 targets but much higher than those of model structures from current state-of-the-art techniques. This suggests the potentially beneficial use of these predicted properties for model assessment and ranking. Three-dimensional structures for most proteins are determined by their one-dimensional sequences of amino acid residues. How to predict three-dimensional structures from one-dimensional sequences has been an unsolved problem for the last half century1. This problem is challenging because it demands an efficient technique to search in astronomically large conformational space and a highly accurate energy function to rank and guide the conformational search, both of which are not yet available2. As a result, it is necessary to divide the structure prediction problem into many smaller problems with the hope that solving smaller problems will ultimately lead to the solution of the big problem. g p y g p One of those smaller or sub-problems is the prediction of one-dimensional structural properties of proteins from their sequences. The most commonly predicted one-dimensional structural property of a protein is secondary structure. Secondary structure describes each amino residue in a number of discrete states3 for which three state description (helix, sheet and coil) is the most common. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports \| 5:11476 \| DOI: 10.1038/srep11476 www.nature.com/scientificreports/ www.nature.com/scientificreports/ has been a slow but steady improvement of secondary structure prediction to above 81% when homolo- gous sequences are not utilised for training (ab initio prediction)4,5. The steady improvement is due to a combination of improved machine-learning algorithms, improved features and larger training datasets. Other methods have also been developed to go beyond 81% by including homologous sequences in train- ing6–9. Secondary structure directly predicted from sequence was shown more accurate than secondary structure of the models predicted by protein structure prediction techniques for template-free modelling targets in critical assessment of structure prediction (CASP 9)4. g Secondary structure, however, is a coarse-grained description of local backbone structure because ideal helical and strand conformations do not exist in protein structures and the boundary between coil states and helical/strand states is not well defined10. This leads to development of backbone torsion angle prediction (φ and ψ ) in discontinuous11,12 and in real, continuous values13–15. More recently, a method for predicting angles based on Cα atoms (the angle between Cα i−1− Cα i− Cα i+1 (θ ) and a dihedral angle rotated about the Cα i− Cα i+1 bond (τ)) was also developed16. These local structure descriptors are complementary with each other because torsion angles (φ and ψ ), Cα − atom based angles (θ and τ), and secondary structure involve amino acid residues at different sequence separation: neighbouring residues for φ and ψ , 3–4 residues for θ and τ, and 4 for 310 helix, 5 for α -helix, and an undefined number of residues for sheet residues. Another important one-dimensional structure property is solvent Accessible Surface Area (ASA). ASA measures the level of exposure of an amino acid residue to solvent (water) in a protein. This is an important structural property as active sites of proteins are often located on their surfaces. Multistate prediction of earlier methods17–19 have been replaced by continuous real value prediction14,20–23.t p p y p One interesting observation is that predicted secondary structure is often utilized to predict other one-dimensional structural properties but rarely the other way around. Several studies, however, indi- cated that other predicted structural properties can be utilized to improve secondary structure predic- tion such as predicted torsion angles4,13 and predicted solvent accessible surface area24. www.nature.com/scientificreports/ In particular, we have shown that the accuracy of secondary structure and torsion angle prediction can be substantially improved by iteratively adding improved prediction of torsion angles and secondary structure4.i p y y g p p g y Artificial neural networks have been widely employed in predicting structural properties of proteins due to the availability of large datasets25. Deep neural networks26, referring to artificial neural networks with more than two hidden layers, have been explored in prediction of local and nonlocal structural properties of proteins27–30. For example, Qi et al.29 developed a unified multi-task, local-structure pre- dictor of proteins using deep neural networks as a classifier. They trained a single neural network using sequential and evolutionary features to predict a number of protein properties including protein sec- ondary structure and solvent accessibility. Spencer et al.30 developed an iterative deep neural network for protein secondary structure prediction. The method utilized one deep neural network to predict sec- ondary structure by using physicochemical and evolutionary information in their first step and another deep neural network to predict their final secondary structure prediction based on predicted secondary structures in addition to the same input used in the first step. These methods achieved secondary struc- ture prediction with accuracy that is slightly higher than 80%.hf p y g y g The goal of this paper is to develop an iterative method that predicts four different sets of structural properties: secondary structure, torsion angles, Cα − atom based angles and dihedral angles, and solvent accessible surface area. That is, both local and nonlocal structural information were utilized in iterations. At each iteration, a deep-learning neural network is employed to predict a structural property based on structural properties predicted in the previous iteration. We showed that all structural properties can be improved during the iteration process. The resulting method provides state-of-the-art, all-in-one accu- rate prediction of local structure and solvent accessible surface area. The method (named SPIDER2) is available as an on-line server at http://sparks-lab.org. Improving prediction of secondary structure, local backbone angles, and solvent accessible surface area of proteins by iterative deep learning In recent years, there 1Signal Processing Laboratory, School of Engineering, Griffith University, Brisbane, Australia. 2Institute for Integrated and Intelligent Systems, Griffith University, Brisbane, Australia. 3School of Engineering and Physics, University of the South Pacific, Private Mail Bag, Laucala Campus, Suva, Fiji. 4Shandong Provincial Key Laboratory of Functional Macromolecular Biophysics, Dezhou University, Dezhou, Shandong, China. 5National ICT Australia (NICTA), Brisbane, Australia. 6Institute for Glycomics and School of Information and Communication Technique, Griffith University, Parklands Dr. Southport, QLD 4222, Australia. Correspondence and requests for materials should be addressed to Y.Y. (email: yuedong.yang@griffith.edu.au) or Y.Z. (email: yaoqi.zhou@griffith.edu.au) Scientific Reports \| 5:11476 \| DOI: 10.1038/srep11476 1 Methods Th This section describes the dataset employed and parametric details of the algorithm used as follows Datasets. We employed the same training and independent test datasets developed for the prediction of Cα based angles (θ ) and dihedral angles (τ)16. Briefly, a non-redundant (25% cutoff), high resolution (< 2.0 Å) structures of 5789 proteins were obtained from the sequence culling server PISCES31 and fol- lowed by removing obsolete structures. We then randomly selected 4590 proteins as the training set (TR4590) and the remaining 1199 proteins as an independent test (TS1199). In addition, we downloaded the targets from critical assessment of structure prediction technique (CASP 11, 2014, http://www.pre- dictioncenter.org/casp11/index.cgi). After removing the proteins with inconsistent sequences and the proteins with > 30% sequence identities between each other and to the training and test sets (TR4590 and TS1199), we obtained a set of 72 proteins (CASP11) out of original 99 proteins. This set contains 17382 amino acid residues. A list of 72 proteins is provided in the Supplementary material. Deep neural-network learning. Here, we employed the same deep learning neural network as we have employed for prediction of Cα -based θ and τ angles prediction by SPIDER16. Briefly, the deep artificial Neural Network (ANN) consists of three hidden layers, each with 150 nodes. Input data was normalized to the range of 0 to 1. Weights for each layer were initialized in a greedy layer-wise manner, Scientific Reports \| 5:11476 \| DOI: 10.1038/srep11476 2 www.nature.com/scientificreports/ Figure 1. The general architecture of the parallel multi-step iterative algorithm. Black arrows indicate that position-specific scoring matrix (PSSM) and physical chemical properties (PP) are presented as input in every neural network predictor. There is no connection between each network. Figure 1. The general architecture of the parallel multi-step iterative algorithm. Black arrows indicate that position-specific scoring matrix (PSSM) and physical chemical properties (PP) are presented as input in every neural network predictor. There is no connection between each network. using stacked sparse auto-encoders which map the layer’s inputs back to themselves32 and refined using standard backward propagation. The learning rate for auto encoder stage was 0.05 and the number of epochs in auto encoder stage was 10. The learning rates for backward propagation were 1, 0.5, 0.2, and 0.05, respectively, with 30 epochs at each learning rate. In this study, we used the deep neural network MATLAB toolbox, implemented by Palm33. Methods Th Linear activation function was used for the hidden layers of auto encoder training whereas sigmoid activation function was employed at the stage of back propaga- tion. All these hyper parameters were obtained by a few initial studies of a single fold (90% for training and 10% for test), randomly selected from the training TR4590 dataset. Parallel multi-step iterative algorithm. Figure 1 shows the parallel, multi-step iterative algorithm for predicting secondary structure (SS), angles (backbone torsion angles, Cα -based angles) and ASA at the same time. In the first iteration, only seven representative physical chemical properties of amino acid residues34 and Position Specific Scoring Matrix (PSSM) from PSIBLAST35 were employed to predict SS, angles, and ASA, separately. The seven physicochemical properties (PP) of the amino acids employed are steric parameter (graph shape index), hydrophobicity, volume, polarizability, isoelectric point, helix probability, and sheet probability properties of the amino acids. PSSM was obtained by three iterations of searching against 90% non-redundant (NR90, ftp://toolkit.genzentrum.lmu.de/pub/HH-suite/databases/ nr90.tar.gz) protein data bank with a cut off value (so called E-value) set to 0.001. PSSM represents the substitution probability of a given amino acid based on its position in the protein sequence with all 20 amino acids.i In the second iteration, PSSM/PP plus predicted SS, angles, and ASA from the first iteration were employed to predict SS, angles, and ASA, separately. Additional iterations can be followed by using SS, angles, and ASA from the previous iteration in addition to PSSM and PP. We found three iterations are sufficient for achieving the best predictive power. Thus, each iteration has three separate predictors. Each predictor utilizes one stacked auto-encoder deep neural network as described above. Input. We employed a window size of 17 amino acids (8 amino acids at each side of the target amino acid). For the residues on terminal ends of a protein sequence, we simply repeat the residue type of the first (or last) residue to fill the window. This led to a total of 459 input features (17 × (20 PSSM + 7PP)) for a given amino acid residue in the first iteration. This window size was optimized by 10-fold cross validation. The dependence on window size is small. For example, the accuracy of secondary structure prediction for the first iteration is 80.4–80.5% for the window size of 13, 15, 17, 19, and 21. Output. Scientific Reports \| 5:11476 \| DOI: 10.1038/srep11476 Methods Th For output nodes, the SS predictor has three output nodes representing helix, strand, and coil, respectively; the ASA predictor has only one output node, and the angle predictor has eight output nodes representing sin(θ ), cos(θ ), sin(τ), cos(τ), sin(φ ), cos(φ ), sin(ψ ), and cos(ψ ), respectively. Sine and cosine were employed to remove the effect of angle periodicity. Predicted sine and cosine values are converted back to angles by using the equation α = tan−1[sin(α)/cos(α)]. In the second iteration, the number of inputs for each predictor is 663 (= 17 × (20 PSSM + 7 PP + 3 SS + 1 ASA + 8 Angles)). Only Scientific Reports \| 5:11476 \| DOI: 10.1038/srep11476 3 www.nature.com/scientificreports/ Figure 2. The accuracy of secondary structure (Q3), ASA (correlation coefficient), φ (Mean absolute error, MAE), ψ (MAE), θ (MAE) and τ (MAE) at four different iterations. Open and filled bars denote results from 10 fold cross validation and independent test, respectively. Figure 2 The accuracy of secondary structure (Q3) ASA (correlation coefficient) φ (Mean absolute Figure 2. The accuracy of secondary structure (Q3), ASA (correlation coefficient), φ (Mean absolute error, MAE), ψ (MAE), θ (MAE) and τ (MAE) at four different iterations. Open and filled bars denote results from 10 fold cross validation and independent test, respectively. sin(θ ), cos(θ ), sin(τ), cos(τ), sin(φ ), cos(φ ), sin(ψ ), and cos(ψ ) are utilised in the input for angles. The same number of inputs was employed for additional iterations. Ten-fold cross validation and independent test. The method was first examined using ten-fold cross validation where TR4590 was randomly divided into 10 folds. Nine folds were used in turn for training and the remaining one for test until all 10 folds were tested. In addition, we tested our method for the independent test sets TS1199 and CASP11 by using TR4590 as the training set. Performance measure. For secondary structure, we use the fraction of correctly predicted secondary structure elements for accuracy measurement (Q3)3. The accuracy of predicted angles was measured by a Mean Absolute Error (MAE), the average absolute difference between predicted and experimentally determined angles. The periodicity of an angle was taken care of by utilizing the smaller value of the absolute difference d A A i i i Pred Expt (= − ) and 360 − di for average. Methods Th For ASA, we report both MAE and the Pearson correlation coefficient between predicted and actual ASA. Resultsh The correlation coefficient between predicted and measured ASA is 0.751 for 10 fold cross validation and 0.756 for independent test. This is the correlation coefficient for un-normalized ASA. For normalized ASA (rASA), the correlation coefficient is slightly lower (0.731 for the independent test set). The mean absolute error for rASA is 0.145. The mean absolute errors of the angles for 10 fold cross validation (or independent test) are 19.2° (19.2°) for φ , 30.1° (29.9°) for ψ , 8.15° (8.03°) for θ , 32.4° (32.2°) for τ . Similar accuracy between 10 fold cross validation and independent test indicates the robustness of the method being developed. angles is the most significant, representing 5%–6%, relative improvement. At the third iteration, Q3 for the secondary structure is 81.6% for 10 fold cross validation and 81.8% for the independent test. The correlation coefficient between predicted and measured ASA is 0.751 for 10 fold cross validation and 0.756 for independent test. This is the correlation coefficient for un-normalized ASA. For normalized ASA (rASA), the correlation coefficient is slightly lower (0.731 for the independent test set). The mean absolute error for rASA is 0.145. The mean absolute errors of the angles for 10 fold cross validation (or independent test) are 19.2° (19.2°) for φ , 30.1° (29.9°) for ψ , 8.15° (8.03°) for θ , 32.4° (32.2°) for τ . Similar accuracy between 10 fold cross validation and independent test indicates the robustness of the method being developed. g p It is of interest to know if this improvement in angle MAE also translates into improvement in large angle errors. Reducing large angle errors is essential for sampling in the correct conformational space when used as restraints. Because both φ and ψ have two peaks in their distributions, they can be divided into two states associated with the two peaks. Here we define [0° to 150°] and the rest angle range [(150° to 180°) and (− 180° to 0°)] for two states in φ , and [− 100° to 60°] and the rest angle range [(− 180° to − 100°) and (60° to 180°)] for two states in ψ . We found that for the independent test set, the two-state accuracy for φ only increases marginally from 96.4%, 96.5% to 96.6% from the first to the third iteration. The two-state accuracy for ψ increases by a significant 1% from 85.3% , 86.4% to 86.8%. Resultsh The overall accuracy for all six structural properties (secondary structure, ASA, φ , ψ , θ , and τ) as a function of iterations is shown in Fig. 2. The improvement is clear at the second iteration and converged at the third iteration, regardless if it is 10 fold cross validation or independent test. Thus, we stopped the iteration at the third iteration. Three iterations led to about 1% improvement in Q3. In Table 1, we monitored the accuracy of each amino acid residue for secondary structure prediction. We found that for 17 of 20 amino acids, the accuracy improves in all three iterations. This confirms the robustness of improvement by iterations. p y In addition to improvement in secondary structure prediction, there is a 2% improvement in ASA correlation coefficient, 1°, 2°, 0.5° and 2° improvement in φ ,ψ , θ , and τ, respectively. Improvement in Scientific Reports \| 5:11476 \| DOI: 10.1038/srep11476 4 www.nature.com/scientificreports/ Secondary Structure Iteration Amino acids Abundance Frequency 1 2 3 4 A 21477 8.27 82.3 83.4 83.8 83.6 C 3557 1.37 74.4 75.3 76.3 76.6 D 15271 5.88 80.8 81.9 82.4 82.1 E 17413 6.71 81.5 83.0 83.5 83.3 F 10457 4.03 78.3 79.3 79.9 80.2 G 18723 7.21 80.6 81.8 82.1 82.0 H 5942 2.29 77.3 78.1 78.1 78.8 I 14577 5.61 82.2 83.4 84.0 83.8 K 15216 5.86 79.7 81.2 81.7 81.4 L 23835 9.18 81.7 83.3 83.6 83.4 M 5615 2.16 80.2 81.5 81.8 82.1 N 11306 4.35 79.8 80.8 80.8 80.9 P 11860 4.57 81.1 82.6 83.2 82.8 Q 9927 3.82 79.7 81.7 81.0 81.3 R 13307 5.12 79.6 81.0 81.2 81.5 S 15363 5.92 77.3 78.6 79.0 78.7 T 14445 5.56 77.8 78.9 79.0 79.1 V 18270 7.04 82.0 83.4 83.6 83.5 W 3828 1.47 76.7 78.3 79.4 78.8 Y 9273 3.57 76.8 78.4 79.0 79.0 Overall 259662 100.0 80.2 81.4 81.8 81.7 Table 1. The accuracy of predicted secondary structure for each amino acid residues for TS1199 for 4 iterations. Resultsh Secondary Structure Iteration Amino acids Abundance Frequency 1 2 3 4 A 21477 8.27 82.3 83.4 83.8 83.6 C 3557 1.37 74.4 75.3 76.3 76.6 D 15271 5.88 80.8 81.9 82.4 82.1 E 17413 6.71 81.5 83.0 83.5 83.3 F 10457 4.03 78.3 79.3 79.9 80.2 G 18723 7.21 80.6 81.8 82.1 82.0 H 5942 2.29 77.3 78.1 78.1 78.8 I 14577 5.61 82.2 83.4 84.0 83.8 K 15216 5.86 79.7 81.2 81.7 81.4 L 23835 9.18 81.7 83.3 83.6 83.4 M 5615 2.16 80.2 81.5 81.8 82.1 N 11306 4.35 79.8 80.8 80.8 80.9 P 11860 4.57 81.1 82.6 83.2 82.8 Q 9927 3.82 79.7 81.7 81.0 81.3 R 13307 5.12 79.6 81.0 81.2 81.5 S 15363 5.92 77.3 78.6 79.0 78.7 T 14445 5.56 77.8 78.9 79.0 79.1 V 18270 7.04 82.0 83.4 83.6 83.5 W 3828 1.47 76.7 78.3 79.4 78.8 Y 9273 3.57 76.8 78.4 79.0 79.0 Overall 259662 100.0 80.2 81.4 81.8 81.7 Table 1. The accuracy of predicted secondary structure for each amino acid residues for TS1199 for 4 iterations. Secondary Structure Iteration Amino acids Abundance Frequency 1 2 3 4 A 21477 8.27 82.3 83.4 83.8 83.6 C 3557 1.37 74.4 75.3 76.3 76.6 D 15271 5.88 80.8 81.9 82.4 82.1 E 17413 6.71 81.5 83.0 83.5 83.3 F 10457 4.03 78.3 79.3 79.9 80.2 G 18723 7.21 80.6 81.8 82.1 82.0 H 5942 2.29 77.3 78.1 78.1 78.8 I 14577 5.61 82.2 83.4 84.0 83.8 K 15216 5.86 79.7 81.2 81.7 81.4 L 23835 9.18 81.7 83.3 83.6 83.4 M 5615 2.16 80.2 81.5 81.8 82.1 N 11306 4.35 79.8 80.8 80.8 80.9 P 11860 4.57 81.1 82.6 83.2 82.8 Q 9927 3.82 79.7 81.7 81.0 81.3 R 13307 5.12 79.6 81.0 81.2 81.5 S 15363 5.92 77.3 78.6 79.0 78.7 T 14445 5.56 77.8 78.9 79.0 79.1 V 18270 7.04 82.0 83.4 83.6 83.5 W 3828 1.47 76.7 78.3 79.4 78.8 Y 9273 3.57 76.8 78.4 79.0 79.0 Overall 259662 100.0 80.2 81.4 81.8 81.7 Table 1 The accuracy of predicted secondary structu Table 1. The accuracy of predicted secondary structure for each amino acid residues for TS1199 for iterations. angles is the most significant, representing 5%–6%, relative improvement. At the third iteration, Q3 for the secondary structure is 81.6% for 10 fold cross validation and 81.8% for the independent test. Resultsh Method PSIPRED SPINE-X SCORPION SPIDER This Work SS (Q3) 79.7%(80.8%a) 81.0%(80.6%a) 82.0%(82.4%a) 81.8%(83.3%a) ASA (CC) – 0.74 – – 0.76 MAE: φ (°) – 20.2 – – 19.2 MAE: ψ (°) – 33.7 – – 29.9 MAE: θ (°) – – – 8.6 8.0 MAE: τ (°) – – – 33.6 32.2 Table 2. Accuracy comparison between our technique and several techniques for secondary structure, ASA and angle prediction for the independent test set (TS1199). a66 proteins of TS1199 that are not in the training set for SCORPION. Method PSIPRED SPINE-X SCORPION SPIDER This Work SS (Q3) 79.7%(80.8%a) 81.0%(80.6%a) 82.0%(82.4%a) 81.8%(83.3%a) ASA (CC) – 0.74 – – 0.76 MAE: φ (°) – 20.2 – – 19.2 MAE: ψ (°) – 33.7 – – 29.9 MAE: θ (°) – – – 8.6 8.0 MAE: τ (°) – – – 33.6 32.2 Table 2. Accuracy comparison between our technique and several techniques for secondary structure, ASA and angle prediction for the independent test set (TS1199). a66 proteins of TS1199 that are not in the training set for SCORPION. Table 2. Accuracy comparison between our technique and several techniques for secondary structure, ASA and angle prediction for the independent test set (TS1199). a66 proteins of TS1199 that are not in the training set for SCORPION. Table 2. Accuracy comparison between our technique and several techniques for secondary structure, ASA and angle prediction for the independent test set (TS1199). a66 proteins of TS1199 that are not in the training set for SCORPION. derived fragments has the largest improvement during iterations (from to 2.54 Å to 1.92 Å). Results for other sizes of fragments follow similar trend. This further confirms the power of iterative learning. derived fragments has the largest improvement during iterations (from to 2.54 Å to 1.92 Å). Results for other sizes of fragments follow similar trend. This further confirms the power of iterative learning. Our method is further applied to the most recent CASP targets (CASP11, 2014). It achieves 80.8% in secondary structure, 0.74 for correlation coefficient between measured and predicted ASA, 19.7° MAE for φ , 30.3° for ψ , 8.2° for θ , 32.6° for τ . The prediction accuracy for most structural properties is reduced somewhat from the independent test set to CASP 11 set. This type of reduction for CASP sets was observed previously4. Resultsh This significant increase confirms the usefulness of iterative learning. By comparison, SPINE-X36 was trained for two-state prediction and achieved two state accuracy of 96.4% for φ and 85.6% for ψ . p y φ ψ Once φ and ψ or θ and τ are known protein backbone structure can be constructed. Fragment structures of a length L are derived from predicted angles with a sliding window (1 to L, 2 to L + 1, 3 to L + 2, and etc.). For L = 15, a total of 229,681 fragments are constructed. For φ /ψ derived fragments, each fragment structure was built by standard bond lengths and angles and ω = 180°. For θ /τ derived fragments, each fragment structure was built by the standard Cα -Cα distance of 3.8 Å. The accuracy of a fragment structure can be measured by root-mean-squared distance (RMSD) from the corresponding native fragment. The accuracy of fragment structures either from φ and ψ (Fig. 3A) or from θ and τ (Fig. 3C) improves during iterations (from 3.37 to 3.09 Å for φ /ψ derived fragments and from 3.22 to 2.95 Å for θ /τ derived fragments. Perhaps, not surprisingly, the consistency between φ /ψ and θ /τ Scientific Reports \| 5:11476 \| DOI: 10.1038/srep11476 5 www.nature.com/scientificreports/ tificreports/ Figure 3. The improvement of fragment structures of 15 residues for the TS1199 dataset : (A) RMSD between the native fragments and the fragments generated from predicted φ and ψ for three iterations I1, I2, and I3 and the result from SPINE X (in grey bar). (B) RMSD between the native fragments and the fragments generated from predicted θ and τ for three iterations I1, I2, and I3. (C) The consistency between fragments from predicted φ and ψ and fragments from predicted θ and τ for three iterations I1, I2, and I3. Figure 3. The improvement of fragment structures of 15 residues for the TS1199 dataset : (A) RMSD between the native fragments and the fragments generated from predicted φ and ψ for three iterations I1, I2, and I3 and the result from SPINE X (in grey bar). (B) RMSD between the native fragments and the fragments generated from predicted θ and τ for three iterations I1, I2, and I3. (C) The consistency between fragments from predicted φ and ψ and fragments from predicted θ and τ for three iterations I1, I2, and I3. Resultsh Accuracy comparison between our technique and several techniques for secondary structur ASA and angle prediction for the independent CASP11 set. Table 3. Accuracy comparison between our technique and several techniques for secondary structure, ASA and angle prediction for the independent CASP11 set. ASA and angle prediction for the independent CASP11 set. Figure 4. (A) The accuracy of helical, sheet and coil residues predicted by PSIPRED, SPINE X, SCORPION and the present study for the CASP 11 dataset. (B) The misclassification errors between helix and coil, between sheet and coil and between helix and sheet for the four methods as labelled for the CASP11 dataset. Figure 4. (A) The accuracy of helical, sheet and coil residues predicted by PSIPRED, SPINE X, SCORPION and the present study for the CASP 11 dataset. (B) The misclassification errors between helix and coil, between sheet and coil and between helix and sheet for the four methods as labelled for the CASP11 dataset. In Fig. 4A we compare the accuracy of secondary structure prediction for helix, coil and sheet given by four methods for the CASP11 dataset (PSIPRED, SPINE X, SCORPION and present study). Our method provides the highest accuracy for sheet (76.4%) but lower accuracy in helical prediction (83.7%) than SPINE X (85.5%) and lower accuracy in coil prediction (80.8%) than PSIPRED (85.4%). PSIPRED is significantly more accurate in coil prediction because it over-predicts coil residues4.ifhi i Figure 4B further compares misclassification errors associated with different methods. This confirms that our method gives lower error in misclassification between helix and sheet. It gives a comparable error to SCORPION between sheet and coil and to SPINE X between helix and coil. It is of interest to know how the predicted values are compared to those of models in CASP 11. Methods compared are Zhang-server39, BAKER-ROSETTA40, FFAS41, myprotein-me (http://mypro- tein.me), nns42, 3D-Jigsaw43, RaptorX44, Quark45, TASSER46, and Fusion/MULTICOM47. Figures 5A,B shows that the MAE of predicted ψ and τ angles are 14% and 10% smaller than the lowest MAEs from BAKER-ROSETTA40 and Zhang Server39, respectively. Figure 5C further shows that predicted relative ASA values are also 12% better than those of model structures.hi The significant improvement in fragment structures revealed in Fig. 3 leads to an interesting ques- tion: can predicted angles be directly employed in building accurate protein structures? Resultsh This is in part due to a smaller number of targets (72 proteins) and in part because CASP targets were a carefully selected set for challenging structure prediction techniques. Tables 2 and 3 compare our method with several techniques for secondary structure (PSIPRED3.337, SCORPION38, SPINE-X4), ASA (SPINE-X4), backbone torsion angles (SPINE-X4) and backbone Cα angles and dihedral angles (SPIDER16) for TS1199 and CASP11 test datasets. We noted that TS1199 is not necessarily independent test set for other methods. In fact we found that the majority of TS1199 (all but 66 proteins) are contained in the training set for SCORPION. The accuracy for secondary structure predicted by our method is more accurate than that predicted by PSIPRED and SPINE-X and is compa- rable to SCORPION for the full TS1199 dataset. However, for the 66 proteins not trained by SCORPION, it achieves an accuracy of 82.4%, compared to 83.3% by our method. For ASA prediction, our technique continues to make an improvement over SPINE-X despite its high accuracy. The best improvement over previous methods is angle prediction. For example, there is almost 4° degree improvement (> 10% in relative improvement) over SPINE-X in ψ prediction. It is important to know the statistical significance of the difference among different methods. The p-values for the pair t-test in secondary structure of this work to SCORPION, PSIPRED, and SPINE X are 0.036, 0.00006, and 0.00009, respectively. That is, the improvement from this work over previous methods is statistically significant (< 0.05). 6 Scientific Reports \| 5:11476 \| DOI: 10.1038/srep11476 www.nature.com/scientificreports/ Method PSIPRED SPINE-X SCORPION SPIDER This Work SS (Q3) 78.8% 78.8% 79.9% 80.8% ASA (CC) – 0.72 – – 0.74 MAE: φ (°) – 20.7 – – 19.7 MAE: ψ (°) – 34.6 – – 30.3 MAE: θ (°) – – – 8.7 8.2 MAE: τ (°) – – – 34.1 32.6 Table 3. Accuracy comparison between our technique and several techniques for secondary structure, ASA and angle prediction for the independent CASP11 set. Method PSIPRED SPINE-X SCORPION SPIDER This Work SS (Q3) 78.8% 78.8% 79.9% 80.8% ASA (CC) – 0.72 – – 0.74 MAE: φ (°) – 20.7 – – 19.7 MAE: ψ (°) – 34.6 – – 30.3 MAE: θ (°) – – – 8.7 8.2 MAE: τ (°) – – – 34.1 32.6 Table 3. Scientific Reports \| 5:11476 \| DOI: 10.1038/srep11476 Resultsh The direct answer to this question is no because accumulation of errors in angles can lead to large deviation in three-dimensional structures. On the other hand, there is a small chance for cancellation of errors. The test dataset (1199 proteins) has 183924 40-mer fragments. The percentages of 40-mer fragments with a RMSD below 2.5Å by φ /ψ and θ /τ are 1.4% and 1.6%, respectively. In Fig. 6A, a 40-residue fragment of a three helical bundle constructed based on predicted φ /ψ angles (Residues 174 to 213 from PDB 1l3l chain A) is only 2.2Å RMSD from the native structure. Figure 6B shows an example of a mixed helix/strand fragment of 40 residues (Residues 77 to 116 from PDB 1jq5 chain A). The RMSD between Scientific Reports \| 5:11476 \| DOI: 10.1038/srep11476 7 www.nature.com/scientificreports/ Figure 5. (A) The mean absolute error (MAE) of predicted ψ for the CASP 11 dataset compared to best MAEs of ψ angles in the models from eight most accurate methods in CASP 11. (B) as in (A) but for the MAE of Cα based τ angles. (C) as in (A) but for the MAE of relative assessable surface area (rASA). Figure 5. (A) The mean absolute error (MAE) of predicted ψ for the CASP 11 dataset compared to best MAEs of ψ angles in the models from eight most accurate methods in CASP 11. (B) as in (A) but for the MAE of Cα based τ angles. (C) as in (A) but for the MAE of relative assessable surface area (rASA). predicted and native structure is 2.4 Å. The two constructed structures show that helical structures are more accurately reproduced than strands. What is most encouraging in Fig. 6A,B is well reproduced loop and turn regions that permitted accurate reproduction of fragments. predicted and native structure is 2.4 Å. The two constructed structures show that helical structures are more accurately reproduced than strands. What is most encouraging in Fig. 6A,B is well reproduced loop and turn regions that permitted accurate reproduction of fragments. Scientific Reports \| 5:11476 \| DOI: 10.1038/srep11476 Discussionh This paper developed an integrated sequence-based prediction of one-dimensional structural properties of proteins by iterative learning in a parallel scheme. The structural properties include local backbone structures represented by secondary structure, backbone torsion angle, and backbone Cα angles and dihedral angles. These three backbone representations are complementary to each other: backbone tor- sion angles are single residue properties, backbone Cα angles and dihedral angles involve three and four residues, respectively, and secondary structures involve three or more residues in sequence-position sep- aration. In addition, the method predicts a non-local property: solvent accessibility. We have shown that the input of these predicted structural properties can improve the accuracy of these structural properties iteratively (within three iterations).h y The method provides current state-of-the-art prediction accuracy for various structural properties. For secondary structure prediction, its accuracy reaches nearly 82% for the large test set of 1199 proteins. For solvent accessible surface area, the correlation coefficient between predicted and actual ASA values is 0.76. For angles, MAEs for φ , ψ , θ , and τ are 19.2, 29.9, 8.0 and 32.2 degrees, respectively. Application to a small but more challenging dataset of CASP 11 targets leads to only slightly lower accuracy. All these accuracies above are the best reported accuracies for test sets. Such an integrated collection of various predicted structural properties in one server makes it convenient for their use for other applications. p p p pp One interesting question is that whether or not improvement from iterations is due to addition of predicted secondary structures or other predicted structural information. Spencer et al.30 showed that 8 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 6. The structure (Red) constructed directly from φ/ψ angles compared to native structure (Green). (A) Residues 174 to 213 from PDB 1l3l chain A and (B) Residues 77 to 116 from PDB 1jq5 chain A. Figure 6. The structure (Red) constructed directly from φ/ψ angles compared to native structure (Green). (A) Residues 174 to 213 from PDB 1l3l chain A and (B) Residues 77 to 116 from PDB 1jq5 chain A. A. adding predicted secondary structures alone is sufficient to further improve secondary structure pre- diction. We performed independent tests by removing other non-secondary-structural features and achieved Q3 = 81.2% in the second iteration and Q3 = 81.2% in the third iteration, compared to 81.4% at the second iteration and 81.8% at the third iteration with non-secondary-structural features. Discussionh This indi- cates that adding predicted secondary structures alone contributes a large portion of the improvement whereas other features lead to additional improvement. p One obvious application is protein structure prediction. Previously, we have shown that predicted secondary structures are more accurate than the models predicted by various current state-of-the-art techniques4. Here we demonstrate that the same is true for backbone angles and solvent accessibility (Fig. 5). Indeed, employing predicted torsion angles as restraints doubled the success rate in ab initio structure prediction, compared to using predicted secondary structures36. 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Prediction of one-dimensional structural properties of proteins by integrated neural network. In: Protein Structure Prediction: Method and Algorithms ((ed^(eds Rangwala H., Karypis G.). Wiley (2010). diction: Method and Algorithms ((ed^(eds Rangwala H., Karypis 26. Hinton, G. E. Learning multiple layers of representation. Trends Cogn Sci 11, 428–434 (2007). ld h f d 27. Di Lena, P., Nagata, K. & Baldi, P. Deep architectures for protein contact map prediction. Author Contributions R.H., K.P., J.L., A.D., A.S. J.W., A.S., Y.Y. and Y.Z. designed the method; R.H., J.L., A.D. and Y.Y. developed and implemented methods and produced results; R.H., J.L., A.D., Y.Y. and Y.Z. wrote the manuscript; all authors reviewed the manuscript. Acknowledgementsh g This work was supported in part by National Health and Medical Research Council (1059775) of Australia and Australian Research Council’s Linkage Infrastructure, Equipment and Facilities funding scheme (project number LE150100161) to Y.Z., National Science Foundation of China 61271378 to J. W. and Y. Y., and the Microsoft Azure for Research Awarded to Y.Y. We also gratefully acknowledge the support of the Griffith University eResearch Services Team and the use of the High Performance Computing Scientific Reports \| 5:11476 \| DOI: 10.1038/srep11476 10 www.nature.com/scientificreports/ Cluster “Gowonda” to complete this research. This research/project has also been undertaken with the aid of the research cloud resources provided by the Queensland Cyber Infrastructure Foundation (QCIF). Cluster “Gowonda” to complete this research. This research/project has also been undertaken with the aid of the research cloud resources provided by the Queensland Cyber Infrastructure Foundation (QCIF). Scientific Reports \| 5:11476 \| DOI: 10.1038/srep11476 Additional Information Supplementary information accompanies this paper at http://www.nature.com/srepihi Competing financial interests: The authors declare no competing financial interests.f Competing financial interests: The authors declare no competing financial interests. How to cite this article: Heffernan, R. et al. Improving prediction of secondary structure, local backbone angles, and solvent accessible surface area of proteins by iterative deep learning. Sci. Rep. 5, 11476; doi: 10.1038/srep11476 (2015). How to cite this article: Heffernan, R. et al. Improving prediction of secondary structure, local backbone angles, and solvent accessible surface area of proteins by iterative deep learning. Sci. Rep. 5, 11476; doi: 10.1038/srep11476 (2015). 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https://openalex.org/W3162437384	https://www.research.ed.ac.uk/files/257304712/2046_3758.113.bjr_2021_0259.r1.pdf	English	null	The role of accelerated growth plate fusion in the absence of SOCS2 on osteoarthritis vulnerability	bioRxiv (Cold Spring Harbor Laboratory)	2,021	cc-by	7,568	General rights C i h f h General rights Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Edinburgh Research Explorer The role of accelerated growth plate fusion in the absence of SOCS2 on osteoarthritis vulnerability Edinburgh Research Explorer Citation for published version: Samvelyan, HJ, Huesa, C, Cui Lin, L, Farquharson, C & Staines, K 2022, 'The role of accelerated growth plate fusion in the absence of SOCS2 on osteoarthritis vulnerability', Bone & Joint Research, vol. 11, no. 3, pp. 162-170. https://doi.org/10.1302/2046-3758.113.BJR-2021-0259.R1 Results We observed a significant increase in mean number (wild-type (WT) DMM: 532 (SD 56); WT sham: 495 (SD 45); knockout (KO) DMM: 169 (SD 49); KO sham: 187 (SD 56); p < 0.001) and density (WT DMM: 2.2 (SD 0.9); WT sham: 1.2 (SD 0.5); KO DMM: 13.0 (SD 0.5); KO sham: 14.4 (SD 0.7)) of growth plate bridges in Socs2-/- in comparison with WT. Histological examination of WT and Socs2-/- knees revealed articular cartilage damage with DMM in com- parison to sham. Articular cartilage lesion severity scores (mean and maximum) were similar in WT and Socs2-/- mice with either DMM, or with ageing. Micro-CT analysis revealed signifi- cant decreases in SCB thickness, epiphyseal trabecular number, and thickness in the medial compartment of Socs2-/-, in comparison with WT (p < 0.001). DMM had no effect on the SCB thickness in comparison with sham in either genotype. Aims H. J. Samvelyan, C. Huesa, L. Cui, C. Farquharson, K. A. Staines H. J. Samvelyan, C. Huesa, L. Cui, C. Farquharson, K. A. Staines Osteoarthritis (OA) is the most prevalent systemic musculoskeletal disorder, character- ized by articular cartilage degeneration and subchondral bone (SCB) sclerosis. Here, we sought to examine the contribution of accelerated growth to OA development using a mu- rine model of excessive longitudinal growth. Suppressor of cytokine signalling 2 (SOCS2) is a negative regulator of growth hormone (GH) signalling, thus mice deficient in SOCS2 (Socs2-/-) display accelerated bone growth. From University of Brighton, Brighton, UK Conclusion Together, these data suggest that enhanced GH signalling through SOCS2 deletion acceler- ates growth plate fusion, however this has no effect on OA vulnerability in this model. Cite this article: Bone Joint Res 2022;11(3):162–170. Cite this article: Bone Joint Res 2022;11(3):162–170. Keywords: Osteoarthritis, SOCS2, Cartilage, Growth plate, Bone, Growth hormone Take down policy Take down policy The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and investigate your claim. Download date: 24. Oct. 2024 Follow us @BoneJointRes Methods We examined vulnerability of Socs2-/- mice to OA following surgical induction of disease (dest- abilization of the medial meniscus (DMM)), and with ageing, by histology and micro-CT. BJR The role of accelerated growth plate fusion in the absence of SOCS2 on osteoarthritis vulnerability H. J. Samvelyan, C. Huesa, L. Cui, C. Farquharson, K. A. Staines From University of Brighton, Brighton, UK Keywords: Osteoarthritis, SOCS2, Cartilage, Growth plate, Bone, Growth hormone Bone Joint Res 2022;11(3):162– 170. Introduction Osteoarthritis (OA) is the most prevalent systemic muscu- loskeletal disorder, characterized by degeneration of joint articular cartilage, osteophyte formation, subchon- dral bone plate thickening, synovial proliferation, and inflammation. OA has a multifactorial aetiology including ageing, trauma, obesity, and heredity. Further, a complex interplay of major molecules and signalling pathways play indispensable roles in OA development.1-5 Despite this, effective disease-modifying treatments are currently limited.i THE ROLE OF ACCELERATED GROWTH PLATE FUSION IN THE ABSENCE OF SOCS2 ON OSTEOARTHRITIS VULNERABILITY 163 Strengths and limitations has been shown as a primary intracellular suppressor of the growth hormone (GH) signalling pathway, thus mice deficient in SOCS2 (Socs2-/-) display an excessive growth phenotype.16 Here, we use this mouse model of accelerated bone growth to understand the association between aberrant growth dynamics and OA develop- ment. To achieve this, we examined the vulnerability of Socs2-/- mice to OA following surgical induction of disease (destabilization of the medial meniscus (DMM)), and with ageing, by histology and micro CT. This study reported, for the first time, accelerated growth plate fusion in Socs2 knockout mice in which an excessive growth phenotype is observed. This study reported, for the first time, accelerated growth plate fusion in Socs2 knockout mice in which an excessive growth phenotype is observed. g p yp A limitation of this study is the complexity of the GH/ insulin-like growth factor 1 (IGF-1) signalling pathway in OA, thus highlighting the need for a better under- standing of its role in disease pathology. A limitation of this study is the complexity of the GH/ insulin-like growth factor 1 (IGF-1) signalling pathway in OA, thus highlighting the need for a better under- standing of its role in disease pathology. Methods Animals. Socs2‐/‐ mice on a C57/BL6 genetic background were generated as previously described.17 For geno- typing, tail-biopsied DNA was analyzed by polymerase chain reaction (PCR) for Socs2 wild-type (WT) (Forward: TGTTTGACTGAGCTCGCGC, Reverse: CAACTTTAGTGT CTTGGATCT) or the neocassette (Socs2 −/−; Forward: ACCCTGCACACTCTCGTTTTG, Reverse: CCTCGACTAAAC ACATGTAAAGC). Mice were kept in polypropylene cages, with light/dark 12-hour cycles, at 21°C (± 2°C), and fed ad libitum with maintenance diet (Special Diet Services, UK). Ageing studies were completed in 12- to 13-month-old Socs2‐/‐ (n = 6) and C57/BL6 WT (n = 6) male mice (Charles River, UK). Analyses were conducted blindly to minimize the effects of subjective bias. All experimental protocols were approved by the local ethics committee, and the animals were maintained in accordance with UK Home Office guidelines for the care and use of laboratory animals. Animal studies were conducted in line with the ARRIVE guidelines. Endochondral ossification is an essential process for longitudinal bone growth. It requires hypertrophic differ- entiation of chondrocytes, characterized by secretion of type X collagen (COL10A1), matrix metalloproteinase-13 (MMP-13), and vascular endothelial growth factor (VEGF), followed by the subsequent degradation and conver- sion of the growth plate cartilage matrix into highly vascularized bone tissue.6,7 With sexual maturation, the human growth plate undergoes progressive narrowing as bony bridges form and span its width, establishing continuity between the cancellous bone of the epiphysis and metaphysis.8 These growth plate bridging events ultimately lead to complete growth plate closure and cessation of human growth.9 OA is widely accepted to involve the reversion of chondrocyte behaviour to an earlier developmental-like phenotype, which could drive the disease process. Indeed, re-expression of the type IIA procollagen, a spliced variant of the type II collagen gene (COL2A1) normally expressed in chondroprogenitor cells, in adult osteoarthritic articular chondrocytes indi- cates reversion of these cells to early developmental-like phenotype.10 Destabilization of the medial meniscus. Eight-week-old Socs2‐/‐ and C57/BL6 WT male mice (Charles River) were randomally allocated into 1) surgically induced DMM un- der isoflurane-induced anaesthesia (n = 6/genotype) or 2) sham operated (n = 6/genotype) groups. Following transection of the medial meniscotibial ligament to desta- bilize the medial meniscus, the left knee joint capsule and skin were closed and anaesthesia reversed. Sham‐operat- ed joints were used as controls. After eight weeks, knee joints were dissected, fixed in 4% paraformaldehyde for 24 hours at 4°C, and then stored in 70% ethanol. Micro-CT analysis. Key messages The reversion of chondrocyte behaviour to an earlier developmental-like phenotype in osteoarthritis (OA) has been defined, however the precise contribution of accel- erated growth to OA development remains unclear. We observed a significant increase in number and density of growth plate bridges in Socs2 knockout mice in compar- ison to wild-type (WT), thus indicating accelerated growth plate fusion in this model. Correspondence should be sent to Katherine Ann Staines; email: k.staines@brighton.ac.uk doi: 10.1302/2046-3758.113.BJR- 2021-0259.R1 Bone Joint Res 2022;11(3):162– 170. Correspondence should be sent to Katherine Ann Staines; email: k.staines@brighton.ac.uk doi: 10.1302/2046-3758.113.BJR- 2021-0259.R1 Bone Joint Res 2022;11(3):162– 170. Histological examination of Socs2 knockout and WT knee joints revealed similar artic- ular cartilage lesion severity and osteo- phyte formation scores, and therefore no effect of accelerated growth in this model on articular cartilage damage. Here, we used a murine model of exces- sive longitudinal growth (suppressor of cytokine signalling 2 (SOCS2) deletion – a suppressor of growth hormone signalling) and examined their vulnerability to surgical and ageing-related OA development. VOL. 11, NO. 3, MARCH 2022 162 THE ROLE OF ACCELERATED GROWTH PLATE FUSION IN THE ABSENCE OF SOCS2 ON OSTEOARTHRITIS VULNERABILITY VOL. 11, NO. 3, MARCH 2022 Methods Weights of wild type and Socs2-/- (knockout) mice during eight-week post-destabilization of the medial m Fig. 1 Socs2-deficient mice exhibit widened growth plates. Histological images of toluidine blue stained growth plates in a) wild type (WT) and Socs2-/- (knockout (KO)) destabilization of medial meniscus (DMM) and sham mice, and b) aged WT and KO mice (10×). Reduced staining in the KO growth plates suggests reduced proteoglycans in these animals. Quantification of c) proliferative zone, d) hypertrophic zone, and e) total growth plate width in WT and KO DMM and sham mice. f) Quantification of total growth plate width in aged WT and KO mice. Data are presented as mean and standard error of the mean. One-way analysis of variance was used for DMM/sham animals and an independent-samples t-test for aged animals. p < 0.05 *p < 0.001. Socs2-deficient mice exhibit widened growth plates. Histological images of toluidine blue stained growth plates in a) wild type (WT) and Socs2-/- (knockout (KO)) destabilization of medial meniscus (DMM) and sham mice, and b) aged WT and KO mice (10×). Reduced staining in the KO growth plates suggests reduced proteoglycans in these animals. Quantification of c) proliferative zone, d) hypertrophic zone, and e) total growth plate width in WT and KO DMM and sham mice. f) Quantification of total growth plate width in aged WT and KO mice. Data are presented as mean and standard error of the mean. One-way analysis of variance was used for DMM/sham animals and an independent-samples t-test for aged animals. p < 0.05 **p < 0.001. (SkyScan) to provide: subchondral bone plate thickness (SCB Th., mm); subchondral bone plate bone volume/tis- sue volume (SCB BV/TV, %); epiphyseal trabecular bone volume/tissue volume (Tb. BV/TV, %); trabecular number (Tb. N., mm-1); trabecular thickness (Tb. Th., mm); and trabecular separation (Tb. Sp., mm). and superimposed on the tibial joint surface (each bridge has a colour that represents the areal number density at the bridge location). (SkyScan) to provide: subchondral bone plate thickness (SCB Th., mm); subchondral bone plate bone volume/tis- sue volume (SCB BV/TV, %); epiphyseal trabecular bone volume/tissue volume (Tb. BV/TV, %); trabecular number (Tb. N., mm-1); trabecular thickness (Tb. Th., mm); and trabecular separation (Tb. Sp., mm). Histological analysis. Murine left knee joints were decal- cified in 10% ethylenediaminetetraacetic acid (EDTA) for approximately four weeks at 4°C, wax-embedded and 6 μm coronal sections cut. Methods Scans of the right knee joint were per- formed with an 1172 X-Ray microtomograph (SkyScan, Belgium) to evaluate the subchondral bone. High- resolution scans with voxel size of 5 µm were acquired (50 kV, 200µA, 0.5 mm aluminium filter, 0.6° rotation angle). The projection images were reconstructed us- ing NRecon software version 1.6.9.4 (SkyScan). Each dataset was rotated in DataViewer (SkyScan) to ensure similar orientation and alignment for analysis. Regions of interest (ROIs) were hand-drawn of the subchondral trabecular bone in the medial and lateral compartments of the femur and tibia. Subchondral bone ROIs were sub- sequently selected for each compartment. Analysis of subchondral bone plate thickness and the epiphyseal tra- becular bone was achieved using 3D algorithms in CTAn Consistent with this, Staines et al11 have previously shown the abnormal deployment of a transient chondro- cyte phenotype in the joints of a STR/ort mouse, a murine model for spontaneous OA.12 Staines et al11 further revealed accelerated long bone growth, a wider zone of growth plate proliferative chondrocytes, and widespread COL10A1 and MMP-13 expression beyond the expected hypertrophic zone distribution in these mice, which may underpin their OA onset. However, the precise contribu- tion of accelerated growth to OA development remains unclear. Suppressor of cytokine signalling 2 (SOCS2), one of the members of the suppressor of cytokine signalling family glycoproteins, is implicated in cancer and disorders of immune system and central nervous system.13-15 SOCS2 VOL. 11, NO. 3, MARCH 2022 H. J. SAMVELYAN, C. HUESA, L. CUI, C. FARQUHARSON, K. A. STAINES 164 Table I. Weights of wild type and Socs2-/- (knockout) mice during eight-week post-destabilization of the medial meniscus experimental timeline. Variable Mean weight, g (SD) Week 0 1 2 3 4 5 6 7 8 WT sham 23.2 (0.4) 22.2 (1.0) 24.0 (0.4) 25.6 (0.5) 26.4 (0.7) 27.0 (0.7) 27.6 (0.8) 28.2 (0.8) 29.0 (0.7) WT DMM 22.4 (0.3) 21.8 (0.3) 23.0 (0.3) 24.0 (0.4) 24.5 (0.3) 24.9 (0.3) 25.6 (0.4) 25.7 (0.3) 26.5 (0.3) KO sham 32.3 (1.2) 33.6 (0.9) 34.8 (1.1) 35.0 (1.0) 36.4 (1.1) 38.1 (1.3) 38.7 (0.9) 39.0 (0.9) 40.4 (0.9) KO DMM 31.3 (0.8) 33.0 (0.7) 33.7 (0.7) 34.9 (1.0) 35.8 (1.0) 39.8 (0.6) 38.3 (1.1) 38.9 (1.1) 40.3 (1.0) DMM, destabilization of the medial meniscus; KO, knockout; SD, standard deviation; WT, wild type. pe and Socs2-/- (knockout) mice during eight-week post-destabilization of the medial meniscus experimental timeline. Table I. Methods For assessment of OA severity, multiple sections (> 5/slide) from 120 μm intervals across the whole joint were stained with Toluidine blue (0.4% in 0.1 M acetate buffer, pH 4). The total width of the growth plate, as well as the proliferating and hypertrophic zones, were measured at ten different points along the length of the growth plate based on established cell morphology.19 This was conducted in 6 μm coronal sections from the middle region of the knee joint in a similar location of three individual animals per experimental group, using Growth plate bridging analysis. Growth plate bridging analysis was conducted using a 3D micro-CT quantifi- cation method as previously described.8,18 Briefly, micro- CT scans of the tibiae were segmented using a region- growing algorithm within the Avizo (V8.0, Thermo Fisher Scientific, USA) software. The central points of all bony bridges were identified (Supplementary Figure a) and projected on the tibial joint surface. The distribution of the areal number density of bridges (N, the number of bridg- es per 256 µm × 256 µm window) was then calculated BONE & JOINT RESEARCH THE ROLE OF ACCELERATED GROWTH PLATE FUSION IN THE ABSENCE OF SOCS2 ON OSTEOARTHRITIS VULNERABILITY 165 Fig. 2 Socs2-deficient mice exhibit accelerated growth plate fusion mechanisms. a) Location and areal densities of bridges across the growth plate projected on the medial (M) and lateral (L) tibial joint surface in wild type (WT) and Socs2-/- (knockout (KO)) destabilization of medial meniscus (DMM) and sham-operated joints. b) Number of growth plate (GP) bridges. c) Areal density of bridges defined as the number of bridges per 256 mm × 256 mm window. d) Location and areal densities of bridges across the growth plate projected on the medial (M) and lateral (L) tibial joint surface in aged WT and Socs2-/- (KO) mice. e) Number of GP bridges. f) Areal density (d) of bridges, defined as the number of bridges per 256 mm × 256 mm window. Data are presented as mean and standard error of the mean, and showing individual animals. Two-way analysis of variance with Bonferroni adjustments for multiple comparisons was used within each joint compartment. p < 0.05; p < 0.01; p < 0.001. Fig. 2 Socs2-deficient mice exhibit accelerated growth plate fusion mechanisms. Methods a) Location and areal densities of bridges across the growth plate projected on the medial (M) and lateral (L) tibial joint surface in wild type (WT) and Socs2-/- (knockout (KO)) destabilization of medial meniscus (DMM) and sham-operated joints. b) Number of growth plate (GP) bridges. c) Areal density of bridges defined as the number of bridges per 256 mm × 256 mm window. d) Location and areal densities of bridges across the growth plate projected on the medial (M) and lateral (L) tibial joint surface in aged WT and Socs2-/- (KO) mice. e) Number of GP bridges. f) Areal density (d) of bridges, defined as the number of bridges per 256 mm × 256 mm window. Data are presented as mean and standard error of the mean, and showing individual animals. Two-way analysis of variance with Bonferroni adjustments for multiple comparisons was used within each joint compartment. p < 0.05; p < 0.01; p < 0.001. a light microscope and ImageJ (National Institutes of Health, USA) software. Articular cartilage damage was assessed in the medial tibia using the well-established Osteoarthritis Research Society International (OARSI) grading scale, with scores averaged.20 Osteophytes were also scored where 0 = none; 1 = formation of cartilage- like tissue; 2 = increase in cartilaginous matrix; 3 = endo- chondral ossification.21 Scoring was conducted blindly by two observers (HJS and KAS). the mean (SEM). Normal distribution of data was assessed using the Shapiro-Wilk normality test. For the growth plate bridging and micro-CT analysis, one- or two-way analysis of variance (ANOVA) with Bonferroni adjustments for mul- tiple comparisons were used, and independent-samples t-test was used for ageing studies. For articular cartilage damage, the Kruskal-Wallis one-way ANOVA was used for DMM studies, and the Mann-Whitney U test for ageing studies. The significance was set at p < 0.05. Statistical analysis. All analyses were performed with GraphPad Prism software 6.0 f version (GraphPad, USA). The results were presented as the mean and standard error of VOL. 11, NO. 3, MARCH 2022 H. J. SAMVELYAN, C. HUESA, L. CUI, C. FARQUHARSON, K. A. STAINES H. J. SAMVELYAN, C. HUESA, L. CUI, C. FARQUHARSON, K. A. STAINES H. J. SAMVELYAN, C. HUESA, L. CUI, C. FARQUHARSON, K. A. STAINES 166 H. J. SAMVELYAN, C. HUESA, L. CUI, C. FARQUHARSON, K. A. STAINES 166 Fig. 3 Deletion of Socs2 does not prevent osteoarthritic articular cartilage lesions in a surgical model of osteoarthritis. Results two-way ANOVA) and density (Figures 2a and 2c) (WT DMM: 2.2 (SD 0.9); WT sham: 1.2 (SD 0.5); KO DMM: 13.0 (SD 0.5); KO sham: 14.4 (SD 0.7); p < 0.001, two- way ANOVA) in Socs2-/- sham and DMM tibiae (16 weeks of age), in comparison to WT sham and DMM tibiae . Further, we saw no significant differences between growth plate bridges in the medial and lateral condyles in WT animals, however in Socs2-/- there is a significant reduction in growth plate bridge number and density in the lateral condyle, regardless of OA development (p < 0.001, two-way ANOVA (Figures 2b and 2c)). No differ- ences in growth plate bridges were observed with OA development in either genotype. Concurrent with this, in aged mice (around one year), a significant increase in growth plate bridge number (Figures 2d and 2e) and density (Figures 2d and 2f) was observed in Socs2-/- mice in comparison to WT mice (WT: 585 (SD 70); KO: 1,659 (SD 91); densities – WT: 8.6 (SD 2.2); KO: 21.3 (SD 1.4); p < 0.001, two-way ANOVA). two-way ANOVA) and density (Figures 2a and 2c) (WT DMM: 2.2 (SD 0.9); WT sham: 1.2 (SD 0.5); KO DMM: 13.0 (SD 0.5); KO sham: 14.4 (SD 0.7); p < 0.001, two- way ANOVA) in Socs2-/- sham and DMM tibiae (16 weeks of age), in comparison to WT sham and DMM tibiae . Further, we saw no significant differences between growth plate bridges in the medial and lateral condyles in WT animals, however in Socs2-/- there is a significant reduction in growth plate bridge number and density in the lateral condyle, regardless of OA development (p < 0.001, two-way ANOVA (Figures 2b and 2c)). No differ- ences in growth plate bridges were observed with OA development in either genotype. Concurrent with this, in aged mice (around one year), a significant increase in growth plate bridge number (Figures 2d and 2e) and density (Figures 2d and 2f) was observed in Socs2-/- mice in comparison to WT mice (WT: 585 (SD 70); KO: 1,659 (SD 91); densities – WT: 8.6 (SD 2.2); KO: 21.3 (SD 1.4); p < 0.001, two-way ANOVA). Socs2-/- mice exhibit accelerated growth plate fusion. Methods a) Toluidine blue stained sections of the knee joint of wild type (WT) and Socs2-/- (knockout (KO)) mice showing development of articular cartilage lesions in the medial tibia (10×). b) Osteophyte severity score, c) mean articular cartilage damage Osteoarthritis Research Society International (OARSI) score,20 and d) maximum articular cartilage damage OARSI score in WT sham (n = 3), WT destabilization of the medial meniscus (DMM) (n = 6), KO sham (n = 6), and KO DMM (n = 6). Scale bar = 0.05 mm. Data are presented as mean and standard error of the mean, showing individual animals; Kruskal-Wallis one-way analysis of variance was used. Fi 3 Deletion of Socs2 does not prevent osteoarthritic articular cartilage lesions in a surgical model of osteoarthritis. a) Toluidine blue stained sections of the knee joint of wild type (WT) and Socs2-/- (knockout (KO)) mice showing development of articular cartilage lesions in the medial tibia (10×). b) Osteophyte severity score, c) mean articular cartilage damage Osteoarthritis Research Society International (OARSI) score,20 and d) maximum articular cartilage damage OARSI score in WT sham (n = 3), WT destabilization of the medial meniscus (DMM) (n = 6), KO sham (n = 6), and KO DMM (n = 6). Scale bar = 0.05 mm. Data are presented as mean and standard error of the mean, showing individual animals; Kruskal-Wallis one-way analysis of variance was used. Results Micro-CT analysis of the medial and lateral tibial a) subchondral bone plate (SCB) thickness (mm), b) SCB bone volume/ tissue volume (BV/TV; %), c) epiphyseal trabecular BV/TV (%), d) epiphyseal trabecular number (mm-1), e) epiphyseal trabecular thickness (mm), and f) epiphyseal trabecular separation (mm) in wild type (WT) and Socs2-/- (knockout (KO)) mice with DMM or sham surgery (n = 6 per group). Data are presented as mean and standard error of the mean and show individual animal data. Two-way analysis of variance with Bonferroni adjustments was used for multiple comparisons. p < 0.05; **p < 0.001. Fig. 5 Deletion of Socs2 does not prevent osteoarthritic articular cartilage lesions from ageing. a) Toluidine blue stained sections of the knee joint of 12- to 13-month-old WT and Socs2-/- (knockout (KO)) mice showing development of articular cartilage lesions in the medial tibia (10×). b) Mean articular cartilage damage Osteoarthritis Research Society International (OARSI) score across the knee joint and c) maximum articular cartilage damage OARSI score between wild type (WT) (n = 5) and KO (n = 6) mice with ageing, in the medial tibia of the knee joint. Scale bar = 0.05 mm. Data are presented as mean and standard error of the mean, and show individual animals; Mann-Whitney U test was used. Deletion of Socs2 does not prevent osteoarthritic articular cartilage lesions from ageing. a) Toluidine blue stained sections of the knee joint of 12- to 13-month-old WT and Socs2-/- (knockout (KO)) mice showing development of articular cartilage lesions in the medial tibia (10×). b) Mean articular cartilage damage Osteoarthritis Research Society International (OARSI) score across the knee joint and c) maximum articular cartilage damage OARSI score between wild type (WT) (n = 5) and KO (n = 6) mice with ageing, in the medial tibia of the knee joint. Scale bar = 0.05 mm. Data are presented as mean and standard error of the mean, and show individual animals; Mann-Whitney U test was used. compartment of Socs2-/- knee joints, in comparison to WT knee joints (Figure 4a; SCB Th. WT: 0.15 mm (SD 0.003); KO: 0.11 mm (SD 0.003); p < 0.001, two-way ANOVA). DMM had no effect on the subchondral bone plate thick- ness in comparison to sham in either WT or Socs2-/- knee joints (Figure 4a). Similarly, no differences were observed in the subchondral bone % BV/TV between genotypes or OA interventions (Figure 4b). Results In ac- cordance with the known effects of increased GH signal- ling on the skeleton, we observed a significant increase in Socs2-/- body weight in comparison to WT controls in age- ing (Socs2-/- 51.9 g (SD 1.4); WT 32.0 g (SD 0.5); p = 0.000; independent-samples t-test), and throughout the eight- week DMM experiment (Table I; p < 0.001; independent- samples repeated measures t-test). We also observed wid- ened growth plates in our Socs2-/- mice, in comparison to WT in the DMM experiment, specifically in the prolifera- tive zone of chondrocytes, as in accordance with previous studies at a younger age (Figures 1a and 1b).19 However, no differences were observed in aged animals (Figures 1b and 1f). We next sought to examine growth plate fusion in these mice. We observed a significant increase in the number of growth plate bridges in Socs2-/- mice in comparison to WT mice at both ages examined (Figure 2 and Supplemen- tary Figure a). Specifically, we saw a significant increase in growth plate bridge number (Figure 2) (WT DMM: 532 (SD 56); WT sham: 495 (SD 45); knockout (KO) DMM: 169 (SD 49); KO sham: 187 (SD 56); p < 0.001, Socs2 deletion does not exacerbate the development of OA in a DMM model. Assessment of cartilage damage in the medial tibia of WT mice revealed an increased artic- ular cartilage OARSI score with DMM in comparison to BONE & JOINT RESEARCH THE ROLE OF ACCELERATED GROWTH PLATE FUSION IN THE ABSENCE OF SOCS2 ON OSTEOARTHRITIS VULNERABILITY 167 Fig. 4 Socs2-deficient mice exhibit decreased subchondral bone plate and trabecular number, irrespective of destabilization of the medial meniscus (DMM) surgically induced osteoarthritis. Micro-CT analysis of the medial and lateral tibial a) subchondral bone plate (SCB) thickness (mm), b) SCB bone volume/ tissue volume (BV/TV; %), c) epiphyseal trabecular BV/TV (%), d) epiphyseal trabecular number (mm-1), e) epiphyseal trabecular thickness (mm), and f) epiphyseal trabecular separation (mm) in wild type (WT) and Socs2-/- (knockout (KO)) mice with DMM or sham surgery (n = 6 per group). Data are presented as mean and standard error of the mean and show individual animal data. Two-way analysis of variance with Bonferroni adjustments was used for multiple comparisons. p < 0.05; **p < 0.001. Fig 4 Socs2-deficient mice exhibit decreased subchondral bone plate and trabecular number, irrespective of destabilization of the medial meniscus (DMM) surgically induced osteoarthritis. Results Micro-CT analysis of the ep- iphyseal trabecular bone also revealed no effect of DMM on % BV/TV (Figure 3c), however we observed decrease in trabecular number (Figure 4d; Tb. N. WT: 12.3 mm-1 sham (p < 0.050, Kruskal-Wallis one-way ANOVA; Figures 3a, 3c, and 3d). However, no significant differences in the articular cartilage mean and maximum OARSI sever- ity scores were observed between WT and Socs2-/- mice in the medial tibia (no significant difference (Figures 3c and 3d)). Similar results were observed in the lateral tib- ia (data not shown). Similarly, while osteophytes were observed in both WT and Socs2-/- DMM mice, there was no difference between genotypes (Figure 3b). Micro-CT analysis of the subchondral bone plate revealed a sig- nificantly thinner subchondral bone plate in the medial VOL. 11, NO. 3, MARCH 2022 H. J. SAMVELYAN, C. HUESA, L. CUI, C. FARQUHARSON, K. A. STAINES 168 Fig. 6 Aged Socs2-deficient mice exhibit decreased subchondral bone plate thickness. Micro-CT analysis of the medial and lateral tibial a) subchondral bone plate (SCB) thickness (mm), b) SCB bone volume/tissue volume (BV/TV; %), c) epiphyseal trabecular BV/TV (%), d) epiphyseal trabecular number (mm-1), e) epiphyseal trabecular thickness (mm), and f) epiphyseal trabecular separation (mm) in aged wild type (WT) and Socs2-/- (knockout (KO)) mice (n = 6 per group). Data are presented as mean and standard error of the mean, and show individual animals. Two-way analysis of variance with Bonferroni adjustments for multiple comparisons was used. p < 0.05; **p < 0.001. Fi 6 Aged Socs2-deficient mice exhibit decreased subchondral bone plate thickness. Micro-CT analysis of the medial and lateral tibial a) subchondral bone plate (SCB) thickness (mm), b) SCB bone volume/tissue volume (BV/TV; %), c) epiphyseal trabecular BV/TV (%), d) epiphyseal trabecular number (mm-1), e) epiphyseal trabecular thickness (mm), and f) epiphyseal trabecular separation (mm) in aged wild type (WT) and Socs2-/- (knockout (KO)) mice (n = 6 per group). Data are presented as mean and standard error of the mean, and show individual animals. Two-way analysis of variance with Bonferroni adjustments for multiple comparisons was used. p < 0.05; ***p < 0.001. (SD 0.2); KO: 10.6 mm-1 (SD 0.2); p < 0.001, two-way ANOVA) and increase in trabecular thickness (Figure 4e; Tb. Th. WT: 0.06 mm (SD 0.001); KO: 0.07 mm (SD 0.002); p = 0.007, two-way ANOVA) in Socs2-/- knee joints in comparison to WT knee joints. Results DMM had no effect on the epiphyseal bone parameters in comparison to sham, in either WT or Socs2-/- knee joints (Figures 4c to 4f). observed in trabecular number (Figure 6d), trabecular thickness (Figure 6e), or trabecular separation (Figure 6f) between aged WT and Socs2-/- mice. Discussion This may be due to the normal serum levels of GH/IGF-1 observed in Socs2-/- mice,27 and suggests that compen- satory cellular mechanisms exist in the Socs2-/- which may involve other SOCS members. Indeed, eight SOCS proteins have been identified, and work by de Andrés et al32 suggests that SOCS1 and SOCS3 are likely to play such a compensatory role. Further, the GH/IGF-1 status of the STR/Ort mouse, which exhibits a similar overgrowth phenotype to the Socs2-/- mice but with spontaneous OA development, is unknown, and this may provide further insights into this mechanism. Similarly, we know that the GH signalling pathway is extremely complex, thus high- lighting the need to better understand GH/IGF-1 signal- ling in the aetiology of OA. Specifically, they revealed that STR/Ort mice show an over- growth phenotype with enriched growth plate bridging, which was associated with articular cartilage lesions at 18 to 20 weeks of age.11 This is similar to the growth pheno- type observed in the Socs2-/- described herein. Further, previous work on the MRC National Survey of Health and Development revealed modest associations between greater gains in height in childhood and decreased risk of knee OA at 53 years.23 Further, canine studies have shown that femoral lengthening by 30% leads to knee articular cartilage damage, which is protected by apparatus exten- sion with a hinged fixation system to the tibia.24 Together, this suggests that an accelerated growth rate may play a role in the development of OA, although what that role is has yet to be fully defined. y yi SOCS2 is a negative regulator of GH signalling, via inhibition of the Janus kinase/signal transducers and acti- vators of transcription (JAK/STAT) pathway.25 Thus, mice deficient in Socs2-/- display an excessive growth pheno- type.16 Characterization of their growth phenotype has revealed increased bone growth rates, growth plate widths, and chondrocyte proliferation in Socs2-/- six‐week‐ old mice compared to age-matched WT mice, suggestive of accelerated growth.26 Socs2-/- mice have normal serum levels of GH and insulin-like growth factor-1 (IGF-1), and their longitudinal overgrowth phenotype is due to local effects of the GH/IGF-1 axis on the growth plate.27 Consistent with this, we revealed increased numbers and densities of growth plate bridges in Socs2-/- mice, in comparison to WT mice. Growth plate bridges form in coordination with chondrocytes of the growth plate, exhausting their proliferative potential and undergoing senescence. Supplementary materiali Supp ‍ ‍ ‍Figure displaying identification of bony bridges crossing the epiphyseal growth plate. An ARRIVE checklist is also included to show that the ARRIVE guidelines were adhered to in this study. Using finite element modelling, work by Madi et al18 and Staines et al8 has previously shown growth plate bridging to increase stress dissipation in the subchondral bone region of the joint. It is therefore surprising that the increased growth plate bridging observed in our Socs2-/- mice here had reduced subchondral bone plate and trabecular parameters. Previous studies have examined the Socs2-/- bone phenotype with contradictory results. Our previous work has shown that Socs2-/- mice have increased bone mass, trabecular number, and trabecular thickness.17,27 However, others have shown the absence of SOCS2 to induce losses in cortical and trabecular bone mineral density.29 These results are not consistent with the expected augmented GH/IGF-1 axis, but are consistent with our findings here and highlight the complexity of this pathway and the need for further studies to elucidate the precise role of SOCS2 signalling in bone homeostasis. I d GH b t d d IGF 1 t ti Discussion Here we sought to examine whether altered growth plate dynamics underpin OA through examination of OA vulner- ability in a murine model of accelerated growth (Socs2-/-). We describe accelerated growth plate fusion in these mice, consistent with their known overgrowth pheno- type. However, we found no effect of surgical intervention or ageing on the articular cartilage or subchondral bone phenotype in these mice. This suggests that in this murine model, aberrant growth dynamics are not associated with vulnerability to OA development. Socs2 deletion has no effect on joint ageing. To examine the effects of Socs2 deletion in aged joints, histological ex- amination revealed no differences in the articular cartilage lesion mean and maximum severity scores between aged WT and Socs2-/- mice in any of the joint compartments (Figures 5a to 5c). Similar to our previous analysis of DMM- treated WT and Socs2-/- mice (Figure 4), micro-CT analysis of the subchondral bone revealed thinner subchondral bone plate in the medial compartment of Socs2-/- knee joints, in comparison to WT knee joints (Figure 6a; SCB Th. WT: 0.2 mm (SD 0.005); KO: 0.1 mm (SD 0.004); p < 0.001, two-way ANOVA). No differences were observed in the subchondral bone % BV/TV between genotypes (Figure 6b), however the epiphyseal trabecular BV/TV was decreased in Socs2-/- knee joints (Figure 6c; Tb. BV/ TV WT: 73.5% (SD 1.5%); KO: 64.6% (SD 2.2%); p = 0.001, two-way ANOVA). No significant differences were It is well established that in OA, chondrocytes in the articular cartilage adopt a more transient phenotype, similar to that seen in the growth plate.22 This raises the question as to whether a greater understanding of the discordant chondrocyte phenotype may inform on mech- anisms underpinning OA, and strategies for treatment. Staines et al11 have shown that in a spontaneous model of OA (STR/Ort mouse), there is an association between aberrant growth plate dynamics and OA development. BONE & JOINT RESEARCH THE ROLE OF ACCELERATED GROWTH PLATE FUSION IN THE ABSENCE OF SOCS2 ON OSTEOARTHRITIS VULNERABILITY 169 of osteoarthritic articular cartilage lesions is observed.31 Further, it has previously been shown that Socs2 messenger RNA (mRNA) levels are decreased in chondro- cytes from osteoarthritic femoral heads.32 Together, these data suggest a role for the GH/IGF-1/SOCS2 pathway in the pathology of OA. However, our results indicate that there is no effect of SOCS2 deficiency on OA vulnerability. Twitter Follow H. J. Samvelyan @JSamvelyan Follow C. Huesa @CHuesa Follow K. A. Staines @Dr_KatherineS Discussion They are also known to form upon growth plate injury, thought to be through an intramembranous ossification mechanism.28 However, whether growth plate fusion occurs prior to or after the cessation of growth is of significant controversy in the field and has been some- what overlooked.9 g gy In summary, our data show that deletion of SOCS2 leads to accelerated growth plate fusion, but this had no effect on OA vulnerability in a surgical model of murine OA. Future studies will determine whether this lack of vulnerability is specific to this model of accelerated longi- tudinal growth, or whether this is characteristic of OA in general. Author information: H. J. Samvelyan, MS, MRes, PhD, Lecturer, School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton, UK; Centre for Stress and Age-Related Dis- ease, University of Brighton, Brighton, UK; The Faculty of Health, Education, Medicine and Social Care, School of Medicine, Anglia Ruskin University, Chelmsford, UK.l 15. Cramer A, de Lima Oliveira BC, Leite PG, et al. Role of SOCS2 in the Regulation of Immune Response and Development of the Experimental Autoimmune Encephalomyelitis. Mediators Inflamm. 2019;2019:1872593. , , g y, , C. Huesa, BSc, PhD, Research Fellow, Institute of Infection, Immunity & Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK. L C i PhD P td t l R h A i t C. Huesa, BSc, PhD, Research Fellow, Institute of Infection, Immunity & Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK. l 16. Metcalf D, Greenhalgh CJ, Viney E, et al. Gigantism in mice lacking suppressor of cytokine signalling-2. Nature. 2000;405(6790):1069–1073. L. Cui, PhD, Postdoctoral Research Associate L. Cui, PhD, Postdoctoral Research Associate C. Farquharson, PhD, Professor of Skeletal Biology The Roslin Institute The University of Edinburgh Ed C. Farquharson, PhD, Professor of Skeletal Biology C. Farquharson, PhD, Professor of Skeletal Biology The Roslin Institute, The University of Edinburgh, Ed The Roslin Institute, The University of Edinburgh, Edinburgh, UK. 17. Dobie R, MacRae VE, Pass C, Milne EM, Ahmed SF, Farquharson C. Suppressor of cytokine signaling 2 (Socs2) deletion protects bone health of mice with DSS- induced inflammatory bowel disease. Dis Model Mech. 2018;11(1):dmm028456. K. A. Staines, BSc, PhD, Senior Lecturer, School of Pharmacy and Biomolecular S i U i it f B i ht B i ht UK C t f St d A R l t d K. A. Staines, BSc, PhD, Senior Lecturer, School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton, UK; Centre for Stress and Age-Related Disease, University of Brighton, Brighton, UK. K. A. Staines, BSc, PhD, Senior Lecturer, School of Pharmacy a K. A. Staines, BSc, PhD, Senior Lecturer, School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton, UK; Centre for Stress and Age-Related Disease, University of Brighton, Brighton, UK. , , , , y Sciences, University of Brighton, Brighton, UK; Centre for Stress and Age-Related Disease, University of Brighton, Brighton, UK. 18. H. J. SAMVELYAN, C. HUESA, L. CUI, C. FARQUHARSON, K. A. STAINES 9. Parfitt AM. Misconceptions (1): epiphyseal fusion causes cessation of growth. Bone. 2002;30(2):337–339. 29. Lorentzon M, Greenhalgh CJ, Mohan S, Alexander WS, Ohlsson C. Reduced bone mineral density in SOCS-2-deficient mice. Pediatr Res. 2005;57(2):223–226. 10. 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https://openalex.org/W4285785395	https://scholarworks.smith.edu/cgi/viewcontent.cgi?article=1116&context=ast_facpubs	English	null	Asymmetries in adaptive optics point spread functions	arXiv (Cornell University)	2,019	cc-by	14,567	Astronomy: Faculty Publications Astronomy This Article has been accepted for inclusion in Astronomy: Faculty Publications by an authorized administrator of Smith ScholarWorks. For more information, please contact scholarworks@smith.edu Smith ScholarW Smith ScholarW Astronomy: Faculty Publications Astro 10-1-2019 Asymmetries in Adaptive Optics Point Spread Functions Asymmetries in Adaptive Optics Point Spread Functions Alexander Madurowicz Kavli Institute for Particle Astrophysics and Cosmology Bruce Macintosh Kavli Institute for Particle Astrophysics and Cosmology Jeffrey Chilcote University of Notre Dame Marshall Perrin Space Telescope Science Institute Lisa Poyneer Lawrence Livermore National Laboratory See next page for additional authors Follow this and additional works at: https://scholarworks.smith.edu/ast_facpubs Part of the Astrophysics and Astronomy Commons Recommended Citation Recommended Citation Madurowicz, Alexander; Macintosh, Bruce; Chilcote, Jeffrey; Perrin, Marshall; Poyneer, Lisa; Pueyo, Laurent; Ruffio, Jean Baptiste; Bailey, Vanessa P.; Barman, Travis; Bulger, Joanna; Cotten, Tara; De R Robert J.; Doyon, Rene; Duchêne, Gaspard; Esposito, Thomas M.; Fitzgerald, Michael P.; Follette, Kat B.; Gerard, Benjamin L.; Goodsell, Stephen J.; Graham, James R.; Greenbaum, Alexandra Z.; Hibon, Pascale; Hung, Li Wei; Ingraham, Patrick; Kalas, Paul; Konopacky, Quinn; Maire, Jérôme; Marchis, Fr Marley, Mark S.; Marois, Christian; Metchev, Stanimir; Millar-Blanchaer, Maxwell A.; and Ward-Duong Kimberly, "Asymmetries in Adaptive Optics Point Spread Functions" (2019). Astronomy: Faculty Publications, Smith College, Northampton, MA. htt // h l k ith d / t f b /117 Smith ScholarWorks Smith ScholarWorks Authors Authors Alexander Madurowicz, Bruce Macintosh, Jeffrey Chilcote, Marshall Perrin, Lisa Poyneer, Laurent Pueyo, Jean Baptiste Ruffio, Vanessa P. Bailey, Travis Barman, Joanna Bulger, Tara Cotten, Robert J. De Rosa, Rene Doyon, Gaspard Duchêne, Thomas M. Esposito, Michael P. Fitzgerald, Katherine B. Follette, Benjamin L. Gerard, Stephen J. Goodsell, James R. Graham, Alexandra Z. Greenbaum, Pascale Hibon, Li Wei Hung, Patrick Ingraham, Paul Kalas, Quinn Konopacky, Jérôme Maire, Franck Marchis, Mark S. Marley, Christian Marois, Stanimir Metchev, Maxwell A. Millar-Blanchaer, and Kimberly Ward-Duong Recommended Citation Recommended Citation Madurowicz, Alexander; Macintosh, Bruce; Chilcote, Jeffrey; Perrin, Marshall; Poyneer, Lisa; Pueyo, Laurent; Ruffio, Jean Baptiste; Bailey, Vanessa P.; Barman, Travis; Bulger, Joanna; Cotten, Tara; De Rosa, Robert J.; Doyon, Rene; Duchêne, Gaspard; Esposito, Thomas M.; Fitzgerald, Michael P.; Follette, Katherine B.; Gerard, Benjamin L.; Goodsell, Stephen J.; Graham, James R.; Greenbaum, Alexandra Z.; Hibon, Pascale; Hung, Li Wei; Ingraham, Patrick; Kalas, Paul; Konopacky, Quinn; Maire, Jérôme; Marchis, Franck; Marley, Mark S.; Marois, Christian; Metchev, Stanimir; Millar-Blanchaer, Maxwell A.; and Ward-Duong, Kimberly, "Asymmetries in Adaptive Optics Point Spread Functions" (2019). Astronomy: Faculty Publications, Smith College, Northampton, MA. https://scholarworks.smith.edu/ast_facpubs/117 This Article has been accepted for inclusion in Astronomy: Faculty Publications by an authorized administrator of Smith ScholarWorks. For more information, please contact scholarworks@smith.edu This article is available at Smith ScholarWorks: https://scholarworks.smith.edu/ast_facpubs/117 Katherine B. Follette Benjamin L. Gerard Stephen J. Goodsell James R. Graham Alexandra Z. Greenbaum Pascale Hibon Li-Wei Hung Patrick Ingraham Paul Kalas Quinn Konopacky Jérôme Maire Franck Marchis Mark S. Marley Christian Marois Stanimir Metchev Maxwell A. Millar-Blanchaer Eric L. Nielsen Rebecca Oppenheimer David Palmer Jennifer Patience Abhijith Rajan Julien Rameau Fredrik T. Rantakyrö Dmitry Savransky Anand Sivaramakrishnan Inseok Song Remi Soummer Melissa Tallis Sandrine Thomas Jason J. Wang Kimberly Ward-Duong Schuyler Wolff Alexander Madurowicz, Bruce Macintosh, Jeffrey Chilcote, Marshall Perrin, Lisa Poyneer, Laurent Pueyo, Jean-Baptiste Ruffio, Vanessa P. Bailey, Travis Barman, Joanna Bulger, Tara Cotten, Robert J. De Rosa, Rene Doyon, Gaspard Duchêne, Thomas M. Esposito, Micha P. Fitzgerald, Katherine B. Follette, Benjamin L. Gerard, Stephen J. Goodsell, James R. Graha Alexandra Z. Greenbaum, Pascale Hibon, Li-Wei Hung, Patrick Ingraham, Paul Kalas, Quinn Konopacky, Jérôme Maire, Franck Marchis, Mark S. Marley, Christian Marois, Stanimir Metchev, Maxwell A. Millar-Blanchaer, Eric L. Nielsen, Rebecca Oppenheimer, David Palmer, Jennifer Patience, Abhijith Rajan, Julien Rameau, Fredrik T. Rantakyrö, Dmitry Savransky, Anand Sivaramakrishnan, Inseok Song, Remi Soummer, Melissa Tallis, Sandrine Thomas, Jason J. Wang, Kimberly Ward-Duong, Schuyler Wolff, “Asymmetries in adaptive optics point spread functions,” J. Astron. Telesc. Instrum. Syst. 5(4), 049003 (2019), doi: 10.1117/1.JATIS.5.4.049003. Authors Authors Authors Authors Alexander Madurowicz, Bruce Macintosh, Jeffrey Chilcote, Marshall Perrin, Lisa Poyneer, Laurent Pueyo, Jean Baptiste Ruffio, Vanessa P. Bailey, Travis Barman, Joanna Bulger, Tara Cotten, Robert J. De Rosa, Rene Doyon, Gaspard Duchêne, Thomas M. Esposito, Michael P. Fitzgerald, Katherine B. Follette, Benjamin L. Gerard, Stephen J. Goodsell, James R. Graham, Alexandra Z. Greenbaum, Pascale Hibon, Li Wei Hung, Patrick Ingraham, Paul Kalas, Quinn Konopacky, Jérôme Maire, Franck Marchis, Mark S. Marley, Christian Marois, Stanimir Metchev, Maxwell A. Millar-Blanchaer, and Kimberly Ward-Duong This article is available at Smith ScholarWorks: https://scholarworks.smith.edu/ast_facpubs/117 Asymmetries in adaptive optics point spread functions Wang,j Kimberly Ward-Duong,y and Schuyler Wolffab aStanford University, Kavli Institute for Particle Astrophysics and Cosmology, Stanford, California, United States bUniversity of Notre Dame Department of Physics Notre Dame Indiana United States p p y dLawrence Livermore National Laboratory, Livermore, California, United States y eJet Propulsion Laboratory, California Institute of Technology, Pasadena, California, United States f p hUniversity of Georgia, Department of Physics and Astronomy, Athens, Georgia, United States i versity of California, Berkeley, Department of Astronomy, Berkeley, California, United States kUniversity of California, Los Angeles, Department of Physics and Astronomy, Los Angeles, Califo lAmherst College Physics and Astronomy Department Amherst Massachusetts United States kUniversity of California, Los Angeles, Department of Physics and Astronomy, Los Angeles, California, United States l mUniversity of Victoria, Victoria, British Columbia, Canada nGemini Observatory, Hilo, Hawaii, United States nGemini Observatory, Hilo, Hawaii, United States y, , , oUniversity of Michigan, Department of Astronomy, Ann Arbor, Michigan, United States pEuropean Southern Observatory, Vitacura, Santiago, Chile y g qNatural Sounds and Night Skies Division, Fort Collins, Colorado, United States g y p y p , , , sUniversity of California, San Diego, Center for Astrophysics and Space Science, La Jolla, California, United States S C S C C S University of California, San Diego, Center for Astrophysics and Space Science, La Jolla, California, United State tSETI Institute, Carl Sagan Center, Mountain View, California, United States y g y tSETI Institute, Carl Sagan Center, Mountain View, California, United States vNational Research Council of Canada Herzberg, Victoria, British Columbia, Canada wThe University of Western Ontario, Department of Physics and Astronomy, Center for Planetary Science and Exploration, London, Ontario, Canada wThe University of Western Ontario, Department of Physics and Astronomy, Center for Planetary Science and Exploration, London, O t i C d wThe University of Western Ontario, Department of Physics and Astronomy, Center for Planetary Science a Ontario, Canada y p y y y Ontario, Canada xAmerican Museum of Natural History, Department of Astrophysics, New York, New York, United States yArizona State University, School of Earth and Space Exploration, Tempe, Arizona, United States zGemini Observatory, La Serena, Chile xAmerican Museum of Natural History, Department of Astrophysics, New York, New York, United States xAmerican Museum of Natural History, Department of Astrophysics, New York, New York, United States yArizona State University, School of Earth and Space Exploration, Tempe, Arizona, United States zGemini Observatory, La Serena, Chile xAmerican Museum of Natural History, Department of Astrophysics, New York, New York, United States yArizona State University School of Earth and Space Exploration Tempe Arizona United States y, p p y , , , yArizona State University, School of Earth and Space Exploration, Tempe, Arizona, United States zGemini Observatory, La Serena, Chile y aaCornell University, Sibley School of Mechanical and Aerospace Engineering, Ithaca, New York, United States abLeiden University Leiden Observatory Leiden The Netherlands aaCornell University, Sibley School of Mechanical and Aerospace Engineering, Ithaca, New York, United States abLeiden University, Leiden Observatory, Leiden, The Netherlands abLeiden University, Leiden Observatory, Leiden, The Netherlands Abstract. Asymmetries in adaptive optics point spread functions Alexander Madurowicz,a,* Bruce Macintosh,a Jeffrey Chilcote,b Marshall Perrin,c Lisa Poyneer,d Laurent Pueyo,c Jean-Baptiste Ruffio,a Vanessa P. Bailey,e Travis Barman,f Joanna Bulger,g Tara Cotten,h Robert J. De Rosa,a Rene Doyon,i Gaspard Duchêne,j Thomas M. Esposito,j Michael P. Fitzgerald,k Katherine B. Follette,l Benjamin L. Gerard,m Stephen J. Goodsell,n James R. Graham,j Alexandra Z. Greenbaum,o Pascale Hibon,p Li-Wei Hung,q Patrick Ingraham,r Paul Kalas,j Quinn Konopacky,s Jérôme Maire,s Franck Marchis,t Mark S. Marley,u Christian Marois,v Stanimir Metchev,w Maxwell A. Millar-Blanchaer,e Eric L. Nielsen,a Rebecca Oppenheimer,x David Palmer,d Jennifer Patience,y Abhijith Rajan,c Julien Rameau,i Fredrik T. Rantakyrö,z Dmitry Savransky,aa Anand Sivaramakrishnan,c Inseok Song,h Remi Soummer,c Melissa Tallis,a Sandrine Thomas,r Jason J. Downloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 06 Jul 2023 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use Asymmetries in adaptive optics point spread functions An explanation for the origin of asymmetry along the preferential axis of the point spread function (PSF) of an AO system is developed. When phase errors from high-altitude turbulence scintillate due to Fresnel propagation, wavefront amplitude errors may be spatially offset from residual phase errors. These correlated errors appear as asymmetry in the image plane under the Fraunhofer condition. In an analytic model with an open-loop AO system, the strength of the asymmetry is calculated for a single mode of phase aberration, which generalizes to two dimensions under a Fourier decomposition of the complex illumination. Other param- eters included are the spatial offset of the AO correction, which is the wind velocity in the frozen flow regime multiplied by the effective AO time delay and propagation distance or altitude of the turbulent layer. In this model, the asymmetry is strongest when the wind is slow and nearest to the coronagraphic mask when the turbulent layer is far away, such as when the telescope is pointing low toward the horizon. A great emphasis is made about the fact that the brighter asymmetric lobe of the PSF points in the opposite direction as the wind, which is con- sistent analytically with the clarification that the image plane electric field distribution is actually the inverse Fourier transform of the aperture plane. Validation of this understanding is made with observations taken from the Gemini Planet Imager, as well as being reproducible in end-to-end AO simulations. © The Authors. Published by SPIE under a Creative Commons Attribution 4.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI. [DOI: 10.1117/1.JATIS.5.4.049003] Keywords: adaptive optics; point-spread functions; scintillation; Fresnel propagation; turbulence. Keywords: adaptive optics; point-spread functions; scintillation; Fresnel propagation; turbulence. Paper 19027 received Mar. 20, 2019; accepted for publication Sep. 23, 2019; published online Oct. 25, 2019. y p p ; p p ; ; p p g ; Paper 19027 received Mar. 20, 2019; accepted for publication Sep. 23, 2019; published online Oct. 25, 2019. Paper 19027 received Mar. 20, 2019; accepted for publication Sep. 23, 2019; published online Oct. 25, 2019. *Address all correspondence to Alexander Madurowicz, E-mail: amaduro@ stanford.edu Oct–Dec 2019 • Vol. 5(4) Journal of Astronomical Telescopes, Instruments, and Systems 049003-1 Downloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 06 Jul 2023 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use Downloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 06 Jul 2023 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use Journal of Astronomical Telescopes, Instruments, and Systems 5(4), 049003 (Oct–Dec 2019) 1 Introduction The advancement of adaptive optics (AO) as a technology has enabled significant progress in astrophysics. Notably, the Gemini Planet Imager (GPI) is one such instrument,1 where fast and precise correction is pivotal to optimize instrument perfor- mance. The results so far have been spectacular. With detections of multiple planetary mass companions around various stars, as well as strong nondetection limits around many more, one can constrain planetary population distributions.2 Astrometric mea- surements of multibody systems probe dynamical constraints on planetary masses and system lifetimes3 and provide a spectacu- lar view of Kepler’s laws in action. Spectral measurements of individual giant planets constrain evolutionary and atmospheric models4 of these objects, paving the way toward characterization of extrasolar terrestrial planets. EQ-TARGET;temp:intralink-;e001;326;730Uðx; y; zÞ ¼ ZZ þ∞ −∞ ˜Uðkx; ky; zÞeiðkxxþkyyÞdkx dky: (1) (1) Direct application of the Helmholtz equation: EQ-TARGET;temp:intralink-;e002;326;662ð∇2 þ k2ÞUðx; y; zÞ ¼ 0 (2) (2) to this decomposition will allow us to derive a formula for the propagation through free space of an arbitrary illumination. For a wave propagating with wave vector ~k ¼ kx^x þ ky^y þ kz^z, implying k2 ¼ j~kj2 ¼ k2x þ k2y þ k2z, we find that propagation along the z axis is constrained by the second-order ordinary dif- ferential equation: For this generation of instruments and the next, understand- ing the point spread function (PSF) of AO instruments on giant telescopes will be important for the development of algorithms optimized in the search for planets.5,6 The analysis in this paper expands on our previous work,7 which demonstrated the origin of azimuthal asymmetry in the PSF as a consequence of the time lag error, to explore asymmetry along the preferential axis intro- duced by scintillation. This effect has been demonstrated previ- ously by Cantalloube et al.8 We will expand on their discussion using a more general method of analyzing the structure of the AO-corrected PSF analytically, as well as validating our conclu- sions with observations and atmospheric datasets. More specifi- cally, our formalism demonstrates that the asymmetry grows linearly only for small spatial frequencies, and at higher spatial frequencies becomes nonlinear. We include solutions for the zeros of the log of the asymmetry metric, which are image loca- tions with an observable return to symmetry. Journal of Astronomical Telescopes, Instruments, and Systems 049003-1 Downloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 06 Jul 2023 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use Madurowicz et al.: Asymmetries in adaptive optics point spread functions Madurowicz et al.: Asymmetries in adaptive optics point spread functions two-dimensional inverse Fourier transform in the plane ðx; yÞ at some constant z: Downloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 06 Jul 2023 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use 1 Introduction EQ-TARGET;temp:intralink-;e005;326;358Hðkx; ky; zÞ ¼ exp i ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 2π λ 2 −ðk2x þ k2yÞ s z : (5) (5) Making the substitution kx;y ¼ 2πfx;y to represent the true linear wavenumber as in Goodman9 recovers this expression for the free-space propagation transfer function: EQ-TARGET;temp:intralink-;e006;326;274Hðfx; fy; zÞ ¼ exp i 2π λ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −λ2ðf2x þ f2yÞ q z : (6) (6) This allows us to consider the propagation of light through free space as a linear spatial filter applied to each Fourier mode of the complex illumination independently. This allows us to consider the propagation of light through free space as a linear spatial filter applied to each Fourier mode of the complex illumination independently. 2.2 Single-Mode Analysis Now that we have described how free-space propagation affects the complex illumination of the electric field in the Fresnel regime, we will explore an analytic derivation of how this results in an asymmetric PSF for a single mode of phase aberration in one dimension. Journal of Astronomical Telescopes, Instruments, and Systems 049003-2 1 Introduction EQ-TARGET;temp:intralink-;e003;326;557 d2 dz2 ˜UðzÞ þ k2z ˜UðzÞ ¼ 0; (3) d2 dz2 ˜UðzÞ þ k2z ˜UðzÞ ¼ 0; (3) (3) where we have implicitly included the dependence of ˜U on the particular mode ðkx; kyÞ. This differential equation permits solutions of the form: EQ-TARGET;temp:intralink-;e004;326;479 ˜UðzÞ ¼ ˜Uðz ¼ 0Þeikzz; (4) (4) where the represents a wave traveling in the positive or neg- ative z direction. For a mode with kx, ky, and the magnitude of the wave vector constrained by jkj ¼ 2π∕λ, where λ is the wave- length of the propagating light (assumed monochromatic), this means we can find the angular spectrum at some later plane z from the angular spectrum at the origin z ¼ 0 by simply multi- plying by the free-space propagation transfer function HðzÞ, which takes on the form: The analysis in this paper is presented as a trident—theory, simulations, and observations. The first section derives the method of angular spectrum Fresnel propagation from the time- independent wave equation. This technique allows us to analyti- cally calculate the PSF formed from a single mode of phase aberration that is both scintillated and time-lag corrected. The PSF for this single mode is computed to second order in a Taylor expansion, which is well matched when compared to a numerical solution involving the discrete Fourier transform. In the second section, these methods are extended broadly to waves propagating through an atmospheric model with Kolmogorov turbulence in the frozen flow regime, which repro- duces the behavior in a moderately accurate simulation of an entire telescope employing AO. The third section demonstrates the effects from our analytic model are observable in real data taken from the Gemini Planet Imager. We then explore correla- tions in the observations when combined with a meteorological dataset containing the real wind velocities and directions in the atmosphere during the observations. Finally, this paper con- cludes with a brief discussion about the importance of this effect in the context of improving AO systems performance from design to postprocessing. 2.1 Angular Spectrum Fresnel Propagation To evaluate the effects of Fresnel propagation and scintillation, we derive the method of the angular spectrum. For an arbitrary complex illumination U of the electric field, we can consider its decomposition into its angular spectrum ˜U given by the When the inverse of the spatial frequency of the mode is much longer than the wavelength of the propagating light (or λ2f2 ≪1), a binomial expansion of the free-space propagation transfer function: Journal of Astronomical Telescopes, Instruments, and Systems 049003-2 Oct–Dec 2019 • Vol. 5(4) Madurowicz et al.: Asymmetries in adaptive optics point spread functions Madurowicz et al.: Asymmetries in adaptive optics point spread functions EQ-TARGET;temp:intralink-;e007;63;752HðzÞ ≈e−iπλf2z (7) EQ-TARGET;temp:intralink-;e014;326;752 ϕAO ¼ α cosð2πz∕zTÞ ﬃﬃﬃ 2 p ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −cosð2πvt∕pÞ p × sin 2πx∕p þ arctan −sinð2πvt∕pÞ cosð2πvt∕pÞ −1 : (14) EQ-TARGET;temp:intralink-;e007;63;752HðzÞ ≈e−iπλf2z (7) (14) allows us to perform the analysis up to a constant phase term. In the example of a single mode of sinusoidal phase aberration only (no amplitude errors), with period p ¼ 1∕f, and a phase variation amplitude α, the complex illumination can be written U ¼ Aeiϕ with To simplify this expression, we will use the substitutions: EQ-TARGET;temp:intralink-;e015;326;687A ¼ α sinð2πz∕zTÞ; (15) EQ-TARGET;temp:intralink-;e016;326;645P ¼ α cosð2πz∕zTÞ ﬃﬃﬃ 2 p ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −cosð2πvt∕pÞ p ; (16) EQ-TARGET;temp:intralink-;e017;326;622Δ ¼ −p 2π arctan −sinð2πvt∕pÞ cosð2πvt∕pÞ −1 ; (17) (15) EQ-TARGET;temp:intralink-;e008;63;673A ¼ 1; (8) EQ-TARGET;temp:intralink-;e009;63;631ϕ ¼ α sinð2πx∕pÞ: (9) EQ-TARGET;temp:intralink-;e008;63;673A ¼ 1; (8) (16) (9) (17) EQ-TARGET;temp:intralink-;e009;63;631ϕ ¼ α sinð2πx∕pÞ: EQ-TARGET;temp:intralink-;e009;63;631ϕ ¼ α sinð2πx∕pÞ: It can be shown10 that for small phase errors (α ≪1) the illu- mination vector at propagation distance z takes the form UðzÞ ¼ AðzÞeiϕðzÞ, where the phase and amplitude have appro- priately been “mixed” due to the scintillation effects: where A represents the term that modulates the amplitude, P is the term that modulates the phase, and Δ is a term that represents a phase shift between the amplitude term and the phase term. 2.1 Angular Spectrum Fresnel Propagation With these substitutions, the expressions for our AO-corrected amplitude and phase become EQ-TARGET;temp:intralink-;e010;63;576AðzÞ ¼ 1 þ α sinð2πz∕zTÞ sinð2πx∕pÞ; (10) EQ-TARGET;temp:intralink-;e011;63;534ϕðzÞ ¼ α cosð2πz∕zTÞ sinð2πx∕pÞ: (11) EQ-TARGET;temp:intralink-;e010;63;576AðzÞ ¼ 1 þ α sinð2πz∕zTÞ sinð2πx∕pÞ; (10) (10) EQ-TARGET;temp:intralink-;e018;326;543AAO ¼ 1 þ A sin½2πðx −vtÞ∕p; (18) EQ-TARGET;temp:intralink-;e019;326;501ϕAO ¼ P sin½2πðx −ΔÞ∕p: (19) (18) EQ-TARGET;temp:intralink-;e011;63;534ϕðzÞ ¼ α cosð2πz∕zTÞ sinð2πx∕pÞ: (11) (11) (19) Here zT is the Talbot length: An example of a single mode of AO-corrected illumination and its corresponding electric field distribution in the image plane is demonstrated in Fig. 1. For this figure, the principal observation is how the delayed AO correction and scintillation produce residual errors which are spatially offset, and how the corresponding speckles in the image plane are asymmetric. EQ-TARGET;temp:intralink-;e012;63;513zT ¼ λ 1 − ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −λ2f2 p ≈2 λf2 : (12) (12) With the assumption that an ideal AO system corrects phase aberrations after a short delay t due to the servo-lag error, our AO-corrected phase will be the difference between two propa- gated modes, one shifted along the direction of the wind with the coordinate transform x →x −vt, if the wind velocity v points along the positive x axis, and the other the initially measured phase: 2.3 Open-Loop Model Validation To validate the assumption that an open-loop AO model can accurately reproduce the behavior of a true closed-loop AO sys- tem, the frequency responses of both open-loop and closed-loop AO systems were modeled. Building on the analysis done for GPI’s AO system (see Sec. 4.D of Ref. 1 for a detailed treat- ment), the standard AO component models and control param- eters were used to generate error transfer functions (ETFs). EQ-TARGET;temp:intralink-;e013;63;390ϕAO ¼ ϕzðx −vtÞ −ϕzðxÞ; (13) (13) Fig. 1 An example of a single mode of AO-corrected complex illumination and corresponding asymmet- ric speckles in the PSF. Here α ¼ :01 rad, z ¼ 10 km, p ¼ 1 m, v ¼ 10 m∕s, and t ¼ 3.2 ms. The AO- corrected complex illumination shows small aberrations in both amplitude and phase from the Talbot mixing, with a spatial phase offset due to the delayed correction from the servo lag. The corresponding asymmetry is highlighted with red dots in the image plane, which is really the inverse Fourier transform, taken in the Fraunhofer diffraction limit. The large central speckle is due to the constant amplitude, on the left subtracted off from the displayed amplitude so that the small errors are more visible. Fig. 1 An example of a single mode of AO-corrected complex illumination and corresponding asymmet- ric speckles in the PSF. Here α ¼ :01 rad, z ¼ 10 km, p ¼ 1 m, v ¼ 10 m∕s, and t ¼ 3.2 ms. The AO- corrected complex illumination shows small aberrations in both amplitude and phase from the Talbot mixing, with a spatial phase offset due to the delayed correction from the servo lag. The corresponding asymmetry is highlighted with red dots in the image plane, which is really the inverse Fourier transform, taken in the Fraunhofer diffraction limit. The large central speckle is due to the constant amplitude, on the left subtracted off from the displayed amplitude so that the small errors are more visible. Oct–Dec 2019 • Vol. 5(4) Oct–Dec 2019 • Vol. 5(4) 2.5 Taylor Expansion of the Single-Mode PSF Although it may be possible to calculate the AO-corrected elec- tric field distribution in the image plane by taking the inverse Fourier transform of the AO-corrected aperture illumination by expanding it as an infinite series of Bessel functions, it is not clear that this expression can be easily squared to get the intensity distribution due to an infinite amount of cross terms. Instead we will follow the conventions of Sivaramakrishnan et al.12 and Perrin et al.13 to arrive at the intensity. However, both Sivaramakrishan et al. and Perrin et al. assume that the image plane PSF is the Fourier transform of the aperture plane and not the inverse. For all of their results, this fact does not matter but for ours we must be cautious, and remember to apply the appro- priate coordinate transform to recover the proper result. For small ϕ, the intensity distribution or PSF ¼ jF½uj2 can be expanded in a Taylor series: where τ is the delay time. The open-loop model does not agree at higher temporal frequencies, most obviously when temporal fre- quency is the inverse of τ. This is when an ideal open-loop AO system coincidentally achieves perfect correction of the translat- ing Fourier mode, which is not physically realizable. This region is beyond our wind speeds of interest, so the open-loop approxi- mation is suitable for our investigation. Journal of Astronomical Telescopes, Instruments, and Systems 049003-3 Downloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 06 Jul 2023 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use Madurowicz et al.: Asymmetries in adaptive optics point spread functions Madurowicz et al.: Asymmetries in adaptive optics point spread functions Fig. 2 Bode plot for comparing the transfer function of various open- and closed-loop AO models. By adding an integrating factor to the open-loop model, it becomes possible to recreate the performance of a closed loop system on static errors, although these are outside of the temporal frequency domain we care about, which is approxi- mately from 2 to 200 Hz. In this range, the standard open-loop model performs roughly as well as the integrator in mimicking the closed loop model, although the behavior diverges when the inverse of the tem- poral frequency becomes equal to the time delay. EQ-TARGET;temp:intralink-;e021;326;752Eðkx; kyÞ ¼ ZZ þ∞ −∞ Aðx; yÞ exp½iðkxx þ kyyÞdx dy: (21) (21) However, Goodman9 also claimed that the relationship between the two is the Fourier transform, yet they obtain the expression: EQ-TARGET;temp:intralink-;e022;326;671 Uðx; yÞ ¼ eikzeik∕2zðx2þy2Þ iλz ZZ ∞ −∞ Uðξ; ηÞ × exp −i 2π λz ðxξ þ yηÞ dξ dη: (22) (22) Comparing the two different expressions, it is not immediately obvious that they are nearly identical. However, after identifying the electric field E and U, identifying the geometric relationship between the coordinates in the aperture ðx; yÞ →ðξ; ηÞ and in the image plane ðkx; kyÞ →ð2πx λz ; 2πy λz Þ, and ignoring the phase prefactors (which do not affect the final intensity), the two answers are comparable with the exception of the sign in the exponent. Fig. 2 Bode plot for comparing the transfer function of various open- and closed-loop AO models. By adding an integrating factor to the open-loop model, it becomes possible to recreate the performance of a closed loop system on static errors, although these are outside of the temporal frequency domain we care about, which is approxi- mately from 2 to 200 Hz. In this range, the standard open-loop model performs roughly as well as the integrator in mimicking the closed loop model, although the behavior diverges when the inverse of the tem- poral frequency becomes equal to the time delay. Journal of Astronomical Telescopes, Instruments, and Systems 049003-3 Since the choice of defining which Fourier transform is the forward and which is the inverse is arbitrary, both authors choose opposite sign conventions to arrive at the same conclu- sion that the relationship is the forward transform. However, the physical relationship between the two planes should not have this sign ambiguity. This difference is traceable to an assumption at the beginning of the derivations, where the choice of the direc- tion of phasor rotation in a spherically converging or diverging wave eiðkr−ωtÞ∕r is used as a Green’s function to solve the Huygens–Fresnel diffraction integral. The GPI AO ETF is shown in red in Fig. 2. The system has a read-compute delay of 1.4 ms and a maximum controller gain of 0.3. An equivalent open-loop model, black curve in this figure, was fit by adjusting the effective delay such the measurement is applied 3.2 ms after it was taken. As shown in this figure, these two models agree very well in terms of both magnitude and phase response in the range of 2 to 200 Hz. Given a maximum spatial frequency in the AO-corrected dark hole of 2.78 m−1, these valid temporal frequencies in our model correspond to wind velocities in the range 8 to 72 m∕s. These velocities encompass the range of possible wind velocities we might see naturally occurring in the jet stream, which typically ranges from 10 to 60 m∕s. The open-loop model disagrees at the lowest temporal frequencies, which corresponds to static errors in the system. This discrepancy can be addressed by slightly scaling the phase measurement, e.g., In practice, the difference between the forward and inverse transforms is essentially a coordinate transform from x →−x and y →−y, and so the orientation of the PSF is flipped along both axes. For most cases, where the PSF is symmetric, this does not matter. However, for our purposes, properly orienting the PSF is critical and so taking note of this fact is important. Later, we will show that in order to remain consistent with obser- vations, the Fraunhofer diffraction limit should use the inverse transform, with a positive sign in the exponent, which results in the stronger asymmetric lobe of the PSF on the opposite side as the wind. (20) EQ-TARGET;temp:intralink-;e020;63;263ϕAOðtÞ ¼ ϕðtÞ −0.997ϕðt −τÞ; (20) Downloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 06 Jul 2023 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use 2.4 Fraunhofer Diffraction Limit According to Hecht,11 the relationship between the aperture and image planes taken in the far-field limit or the Fraunhofer dif- fraction limit is simply the Fourier transform, and their deriva- tion results in the expression: Oct–Dec 2019 • Vol. 5(4) Journal of Astronomical Telescopes, Instruments, and Systems 049003-4 Journal of Astronomical Telescopes, Instruments, and Systems 049003-4 Downloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 06 Jul 2023 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use Madurowicz et al.: Asymmetries in adaptive optics point spread functions Madurowicz et al.: Asymmetries in adaptive optics point spread functions EQ-TARGET;temp:intralink-;e023;63;752PSF ≈PSF0 þ PSF1 þ PSF2;halo þ PSF2;strehl; (23) whose first few terms are EQ-TARGET;temp:intralink-;e024;63;708PSF0 ¼ aa; (24) EQ-TARGET;temp:intralink-;e025;63;676PSF1 ¼ 2 Im½aða⋆ΦÞ; (25) EQ-TARGET;temp:intralink-;e026;63;655PSF2;halo ¼ ða⋆ΦÞða⋆ΦÞ; (26) EQ-TARGET;temp:intralink-;e036;326;741 PSF2ðf ¼ −1∕pÞ ¼ PSF2ðf ¼ 1∕pÞ ¼ P2 4 −A2P2 32 4 þ 2 cos 4π p ðvt −ΔÞ : (36) Our metric for the ratio of the right to left speckle asymmetry is the Taylor expansion sum of the PSF terms evaluated at these EQ-TARGET;temp:intralink-;e023;63;752PSF ≈PSF0 þ PSF1 þ PSF2;halo þ PSF2;strehl; (23) whose first few terms are EQ-TARGET;temp:intralink-;e024;63;708PSF0 ¼ aa; (24) PSF1 ¼ 2 Im½aða⋆ΦÞ (25) EQ-TARGET;temp:intralink-;e036;326;741 PSF2ðf ¼ −1∕pÞ ¼ PSF2ðf ¼ 1∕pÞ ¼ P2 4 −A2P2 32 4 þ 2 cos 4π p ðvt −ΔÞ : (36) whose first few terms are whose first few terms are EQ-TARGET;temp:intralink-;e024;63;708PSF0 ¼ aa; (24) EQ-TARGET;temp:intralink-;e025;63;676PSF1 ¼ 2 Im½aða⋆ΦÞ; (25) EQ-TARGET;temp:intralink-;e026;63;655PSF2;halo ¼ ða⋆ΦÞða⋆ΦÞ; (26) EQ-TARGET;temp:intralink-;e027;63;634PSF2;strehl ¼ −1 2½aða⋆Φ⋆ΦÞ þ aða⋆Φ⋆ΦÞ: (27) (36) EQ-TARGET;temp:intralink-;e024;63;708PSF0 ¼ aa; (24) EQ-TARGET;temp:intralink-;e025;63;676PSF1 ¼ 2 Im½aða⋆ΦÞ; (25) EQ-TARGET;temp:intralink-;e026;63;655PSF2;halo ¼ ða⋆ΦÞða⋆ΦÞ; (26) EQ-TARGET;temp:intralink-;e027;63;634PSF2;strehl ¼ −1 2½aða⋆Φ⋆ΦÞ þ aða⋆Φ⋆ΦÞ: (27) EQ-TARGET;temp:intralink-;e024;63;708PSF0 ¼ aa; Our metric for the ratio of the right to left speckle asymmetry is the Taylor expansion sum of the PSF terms evaluated at these appropriate locations: (27) EQ-TARGET;temp:intralink-;e037;326;657χ ¼ PSFðf ¼ 1∕pÞ PSFðf ¼ −1∕pÞ ; (37) (37) Here, the case change is used as shorthand for the Fourier transform, so a ¼ FðAÞ and Φ ¼ FðϕÞ. Additionally, ⋆is the convolution operation and is the complex conjugate. For our AO-corrected illumination, these can be calculated as follows: which is a function of propagation distance z, mode period p, and velocity times delay vt. A comparison of the asymmetry metric for both the numerical single-mode scintillation using the discrete Fourier transform and our analytic second-order Taylor expansion is given in Fig. 3. 2.4 Fraunhofer Diffraction Limit EQ-TARGET;temp:intralink-;e028;63;569aAO ¼ δðfÞ þ A 2i δðf −1∕pÞe−i2π p vt −A 2i δðf þ 1∕pÞei2π p vt; (28) EQ-TARGET;temp:intralink-;e029;63;503ΦAO ¼ P 2i δðf −1∕pÞe−i2π p Δ −P 2i δðf þ 1∕pÞei2π p Δ; (29) (28) Highlighting a few observations from this plot: first, we note that for wind layers at z ¼ 10 km, the first zero crossing is not until roughly 1 arcsec in the image, and so for most of the relevant observations, the strongest asymmetry will be on the side opposite to the direction of the wind, though for higher spatial frequencies corresponding to the edges of the PSF in the image plane, this will not always be true. Second, the strength of the asymmetry is greater for slower wind velocities in this region of interest. Third, it is worthwhile to note that our Taylor expan- sion generally gets the behavior of the asymmetry analytically correct, although differs from the numerical solution due to the absence of higher order terms in the expansion. (29) where δ is the Dirac delta distribution. It is worthwhile to note that here we implicitly are using the entire real line in the Fourier transform, which can be interpreted as using an infinitely large telescope, or as a telescope with an idealized perfect corona- graph. This leads to a PSF with terms: EQ-TARGET;temp:intralink-;e030;63;428PSF0 ¼ δðfÞ þ A2 4 ½δðf −1∕pÞ þ δðf þ 1∕pÞ; (30) EQ-TARGET;temp:intralink-;e031;63;374 PSF1 ¼ −AP 2 sin 2π p ðvt −ΔÞ δðf −1∕pÞ −δðf þ 1∕pÞ ; (31) EQ-TARGET;temp:intralink-;e032;63;334 2 (30) Fig. 3 The log of the speckle asymmetry ratio as a function of PSF location for parameters α ¼ :01 rad, t ¼ 3.2 ms, and z ¼ 10 km. The solid (or thin) lines represents the second-order Taylor expansion and the dashed (or thick) lines represent the numerical solution found using the discrete Fourier Transform. The x axis is a proxy for mode length, transformed in PSF location when imaged in H-band at λ ¼ 1.6 μm. The zeros corresponding to the particular mode lengths p for which the propagation distance z is an integer multiple of zT ∕4 are plotted in black stars (or squares). For these, PSF1 ¼ 0 because either A ∼sinð2πz∕zT Þ ¼ 0 or P ∼cosð2πz∕zT Þ ¼ 0, resulting in a symmetric PSF. 2.4 Fraunhofer Diffraction Limit Although there are additional zeros when the velocity times time delay is comparable to the mode length, visible in both the 30 and 50 m∕s case, though their analytic solution is more compli- cated [set 2π p ðΔ −vtÞ ¼ π∕2 þ nπ, n ∈Z]. (31) EQ-TARGET;temp:intralink-;e032;63;334 PSF2;halo ¼ P2 4 ½δðf þ 1∕pÞ þ δðf −1∕pÞ þ A2P2 16 2 1 þ cos 4π p ðvt −ΔÞ δðfÞ þ δðf þ 2∕pÞ þ δðf −2∕pÞ ; (32) EQ-TARGET;temp:intralink-;e033;63;243 PSF2;strehl ¼ −P2 2 δðfÞ −A2P2 32 4 þ 2 cos 4π p ðvt −ΔÞ × ½δðf −1∕pÞ þ δðf þ 1∕pÞ: (33) (33) Indeed, the even order terms are symmetric and the first odd order term PSF1 is responsible for the observed asymmetry. We will examine the ratio of the amplitude for the speckles on either side of the image, and to do so evaluate the PSF at the location appropriate f ¼ 1∕p: Fig. 3 The log of the speckle asymmetry ratio as a function of PSF Fig. 3 The log of the speckle asymmetry ratio as a function of PSF location for parameters α ¼ :01 rad, t ¼ 3.2 ms, and z ¼ 10 km. The solid (or thin) lines represents the second-order Taylor expansion and the dashed (or thick) lines represent the numerical solution found using the discrete Fourier Transform. The x axis is a proxy for mode length, transformed in PSF location when imaged in H-band at λ ¼ 1.6 μm. The zeros corresponding to the particular mode lengths p for which the propagation distance z is an integer multiple of zT ∕4 are plotted in black stars (or squares). For these, PSF1 ¼ 0 because either A ∼sinð2πz∕zT Þ ¼ 0 or P ∼cosð2πz∕zT Þ ¼ 0, resulting in a symmetric PSF. Although there are additional zeros when the velocity times time delay is comparable to the mode length, visible in both the 30 and 50 m∕s case, though their analytic solution is more compli- cated [set 2π p ðΔ −vtÞ ¼ π∕2 þ nπ, n ∈Z]. Fig. 3 The log of the speckle asymmetry ratio as a function of PSF location for parameters α ¼ :01 rad, t ¼ 3.2 ms, and z ¼ 10 km. 2.4 Fraunhofer Diffraction Limit The solid (or thin) lines represents the second-order Taylor expansion and the dashed (or thick) lines represent the numerical solution found using the discrete Fourier Transform. The x axis is a proxy for mode length, transformed in PSF location when imaged in H-band at λ ¼ 1.6 μm. The zeros corresponding to the particular mode lengths p for which the propagation distance z is an integer multiple of zT ∕4 are plotted in black stars (or squares). For these, PSF1 ¼ 0 because either A ∼sinð2πz∕zT Þ ¼ 0 or P ∼cosð2πz∕zT Þ ¼ 0, resulting in a symmetric PSF. Although there are additional zeros when the velocity times time delay is comparable to the mode length, visible in both the 30 and 50 m∕s case, though their analytic solution is more compli- cated [set 2π p ðΔ −vtÞ ¼ π∕2 þ nπ, n ∈Z]. EQ-TARGET;temp:intralink-;e034;63;147PSF0ðf ¼ −1∕pÞ ¼ PSF0ðf ¼ 1∕pÞ ¼ A2 4 ; (34) EQ-TARGET;temp:intralink-;e035;63;93 −PSF1ðf ¼ −1∕pÞ ¼ PSF1ðf ¼ 1∕pÞ ¼ −AP 2 sin 2π p ðvt −ΔÞ ; (35) (34) (35) Journal of Astronomical Telescopes, Instruments, and Systems 049003-5 Journal of Astronomical Telescopes, Instruments, and Systems 049003-5 From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 06 Jul 2023 : https://www.spiedigitallibrary.org/terms-of-use Journal of Astronomical Telescopes, Instruments, and Systems 049003-5 Oct–Dec 2019 • Vol. 5(4) Oct–Dec 2019 • Vol. 5(4) Downloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 06 Jul 2023 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use Madurowicz et al.: Asymmetries in adaptive optics point spread functions Madurowicz et al.: Asymmetries in adaptive optics point spread functions 3 Atmospheric Turbulence Scintillation Simulation However, when we run this simulation with many wind layers, each with unique velocities pulled from an instance g , g g y q p Fig. 4 For a single layer of atmospheric turbulence at z ¼ 10 km, the scintillation halo at low wind veloc- ities is highly apparent and still noticeable at rapid velocities. Each image has its own unique colorbar, so that the variation in the halo shape is visible, although from looking at the magnitude of the halo intensity, it is clear that slower wind velocities are corrected better in the metric of total scattered light. Fig. 5 For simulations with many wind layers, here L ¼ 18 is the number of layers used, the effect of the scintillation asymmetry is significantly less apparent, although at low wind velocities where the asym- metry is strongest, it is still visible. This is likely due to interference from the other wind layers causing the delicate spatial offset in the illumination to become washed out when combined with the finite sam- pling resolution of our simulation, and for real observations, the detector. Journal of Astronomical Telescopes, Instruments, and Systems 049003-6 Oct–Dec 2019 • Vol. 5(4) Fig. 4 For a single layer of atmospheric turbulence at z ¼ 10 km, the scintillation halo at low wind veloc- ities is highly apparent and still noticeable at rapid velocities. Each image has its own unique colorbar, so that the variation in the halo shape is visible, although from looking at the magnitude of the halo intensity, it is clear that slower wind velocities are corrected better in the metric of total scattered light. Fig. 4 For a single layer of atmospheric turbulence at z ¼ 10 km, the scintillation halo at low wind veloc- ities is highly apparent and still noticeable at rapid velocities. Each image has its own unique colorbar, so that the variation in the halo shape is visible, although from looking at the magnitude of the halo intensity, it is clear that slower wind velocities are corrected better in the metric of total scattered light. Fig. 5 For simulations with many wind layers, here L ¼ 18 is the number of layers used, the effect of the scintillation asymmetry is significantly less apparent, although at low wind velocities where the asym- metry is strongest, it is still visible. 3 Atmospheric Turbulence Scintillation Simulation This is likely due to interference from the other wind layers causing the delicate spatial offset in the illumination to become washed out when combined with the finite sam- pling resolution of our simulation, and for real observations, the detector. Fig. 5 For simulations with many wind layers, here L ¼ 18 is the number of layers used, the effect of the scintillation asymmetry is significantly less apparent, although at low wind velocities where the asym- metry is strongest, it is still visible. This is likely due to interference from the other wind layers causing the delicate spatial offset in the illumination to become washed out when combined with the finite sam- pling resolution of our simulation, and for real observations, the detector. Oct–Dec 2019 • Vol. 5(4) Oct–Dec 2019 • Vol. 5(4) 3 Atmospheric Turbulence Scintillation Simulation The jet stream is the strongest layer of turbulence which has enough relevant altitude to scintillate significantly [recall the strength of the scintillation amplitude errors grow like sinð2πz∕zTÞ]. The response of an ideal open-loop AO system to frozen flow Kolmogorov turbulence was developed in simulations using the method of the angular spectrum free-space propagation and Fourier decomposition. These simulations can place many layers of turbulence at arbitrary altitudes, and we explore the effects of single and multiple layers. Readers interested in the precise details of these simulations are referred to Sec. 6, which discusses our techniques at greater length. However, here, we will discuss the results of these simulations in the context of the previous analysis. ð Þ The wind in the single-layer example is propagating directly to the right, along the positive x axis. For the multilayer case, the wind velocity for the jet stream is generally pointing toward 2 o’clock (Fig. 5). As a consequence, we can see that the brighter asymmetric lobe of the PSF is on the opposite side of wind direction. To remain consistent with observations, it is necessary that the image plane is the inverse Fourier transform of the aperture plane in the Fraunhofer limit. Looking at just the single layer, it is apparent that the scintillation dominates for slower wind velocities. This scintillation halo would be close to the true PSF if the atmosphere was actually only a single layer of turbulence. Examining the edges of the PSF, one can identify the region where the asymmetry metric log χ ¼ 0 that was described analytically, noticeable here as dark bands. To start, the effect of scintillation in the halo of an AO PSF is most clearly seen for the case of a single layer of Kolmogorov turbulence, which is plotted in Fig. 4 for three characteristic wind velocities. The single-layer simulation uses a turbulent layer altitude of z ¼ 10 km, which roughly corresponds to the altitude of the jet stream. The layer altitude equals the propaga- tion distance of the light when the telescope is pointed at zenith; however, for low elevation pointing, the propagation distance and therefore strength of the scintillation, will grow larger. The halos for the single-layer case do not match real obser- vations well. Journal of Astronomical Telescopes, Instruments, and Systems 049003-6 Journal of Astronomical Telescopes, Instruments, and Systems 049003-6 Downloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 06 Jul 2023 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 06 Jul 2023 : https://www.spiedigitallibrary.org/terms-of-use Madurowicz et al.: Asymmetries in adaptive optics point spread functions axis. This allows us to fit for the preferential wind direction modulo 180 deg. These techniques follow exactly from our pre- vious work,7 yet in this work, we expand by defining the asym- metry metric χ between the two lobes of the PSF. χ is defined as the ratio of the summed intensities in the half-annulus perpendicular to the axis of wind propagation. This asymmetry metric is used to break the 180-deg symmetry of the wind axis to find the true asymmetric butterfly vector in the image plane. In addition, our metric χ is folded around 1 by inversion such that the half-annulus with greater intensity is always in the numer- ator, meaning χ always takes on values greater than 1, and larger values correspond to greater asymmetry. Note that this χ is not identical to the χ described earlier for the one-dimensional case, although both are intensity ratios, this one lives in two dimen- sions. A large sample of PSFs selected in this manner as dis- played in Fig. 6. of the NOAA GFS to mimic real observing conditions, the extreme scintillation halo washes out. This is likely an interfer- ence between multiple independent Kolmogorov layers masking the delicate spatial offset for a single mode needed to create the asymmetry demonstrated earlier. Although at a quick glance the PSF does not appear to vary with the scaled wind velocities cited in their titles, upon closer inspection the asymmetry remains, and is most obvious for the slowest wind velocity. For most observations, with jet stream velocities upward of 50 m∕s, there are no apparent deviations from the symmetry of the butterfly shaped halo that is often seen. But in the rare cases when the high-altitude winds are very slow, around 10 m∕s, the scintilla- tion becomes stronger and introduces a noticeable asymmetry in the PSF along the axis of the wind propagation. 4 Observational Correlations These asymmetric butterfly vectors are projected from the image plane onto the surface of the Earth, for comparison with meteorological wind data. Readers interested in the trigonomet- ric problem of relating the two are referenced to our techniques in Sec. 7. The directions and velocities of the wind for various layers in the Earth’s atmosphere are taken from the NOAA Global Forecast System,14 archives of which are available to the public. The distribution of wind directions for the jet stream and the distribution of directions for the asymmetric butterfly vector projected on the ground are displayed in Fig. 7 for a concise comparison. It is apparent that the jet stream predominately points East in accordance with its origin due to the rotation of the Earth. As a consequence, the resulting asymmetric butter- fly vector points entirely toward the West. This observation The previously discussed asymmetry for AO PSFs is observable in real data taken from observations using the Gemini Planet Imager. Over the course of a few years of observations, over 20,000 images were taken in H-band as a part of the GPI Exoplanet Survey. We exercise selection cuts to find a sample of PSFs exhibiting this characteristic asymmetry using the fol- lowing methods. The first metric is the fractional standard deviation (FSD) in an annulus 30 to 70 pixels wide centered around the star’s loca- tion. We first select images with FSD greater than one standard deviation above the mean. This selects for images where the rel- atively fast jet stream turbulence was dominant, producing PSFs with the characteristic butterfly shaped halo. Then we construct an angular profile of the image by integrating along the radial Fig. 6 A selection of images taken with the Gemini Planet Imager, ordered by decreasing asymmetry metric χ. Each image is the most strongly asymmetric image taken from each set of observations of a single target, such that there are no duplicates, which shows that this effect is often recurring and not limited to single cases. In addition, each image has been rotated and flipped such that north is up and east is to the left. The angular size of each GPI image is 2.8” × 2.8” arc sec. 4 Observational Correlations In each image, the direction of the strong asymmetry is plotted with a red arrow, and the corresponding direction in degrees azimuth projected onto the ground is printed in the corner of each image, alongside the ratio of the asymmetry metric χ. Glancing over the entire dataset, it is readily apparent that the strong asymmetry direction is predominately pointing into the west. Fig. 6 A selection of images taken with the Gemini Planet Imager, ordered by decreasing asymmetry metric χ. Each image is the most strongly asymmetric image taken from each set of observations of a single target, such that there are no duplicates, which shows that this effect is often recurring and not limited to single cases. In addition, each image has been rotated and flipped such that north is up and east is to the left. The angular size of each GPI image is 2.8” × 2.8” arc sec. In each image, the direction of the strong asymmetry is plotted with a red arrow, and the corresponding direction in degrees azimuth projected onto the ground is printed in the corner of each image, alongside the ratio of the asymmetry metric χ. Glancing over the entire dataset, it is readily apparent that the strong asymmetry direction is predominately pointing into the west. Oct–Dec 2019 • Vol. 5(4) Downloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 06 Jul 2023 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use Madurowicz et al.: Asymmetries in adaptive optics point spread functions The strong correlations are visible for the altitudes relevant to the jet stream in both the slope of the best fit line and the R coef- ficient. These correlations are much greater than the 2 to 4 sigma chances concerning the null hypothesis, generated from bootstrap sampling at random, shown as the shaded regions and solid lines for the slope and the R coefficient, respectively. Fig. 7 Angular histogram of the asymmetric butterfly vector’s projec- tion onto the ground for our sample in degrees from azimuth alongside the direction of the jet stream. The asymmetric butterfly nearly always points west while the jet stream is nearly always points east, which is only possible if the image plane electric field distribution is the inverse Fourier transform of the aperture plane. These distributions are not temporally matched but are rather the entire subset observations with strong asymmetry and the entire distribution of jet stream wind direc- tions over the course of the survey. For temporally matched correla- tions, see Figs. 8 and 11. smaller relative to the Talbot length zT in comparison to when the telescope is pointed lower toward the horizon. A lower elevation value corresponds to the telescope effectively pushing the layer for a given altitude to a further propagation distance, giving the light more time to scintillate. This pushes the relevant mode lengths for the asymmetry closer to the center of the image, see Fig. 12 for a remake of Fig. 3 with z ¼ 25 km. In addition, a low telescope elevation also has the effect of changing the apparent wind velocity over the aperture. A tele- scope pointing away from zenith can only observe relatively slower wind velocities than one pointing directly up as long as the winds are parallel to the surface of the Earth. These two effects work together to build the strong correlation between telescope elevation and asymmetry. confirms our prior analysis regarding the strength of the asym- metry being opposed to the direction of the wind. Madurowicz et al.: Asymmetries in adaptive optics point spread functions To further verify this correlation between the jet stream and the asymmetric butterfly, we take our sample of data where χ ≥1.2, which is where the asymmetry begins to be noticeable to the eye, and match the time of observation to time of the wind data for the approximate location of Cerro Pachon, Chile, where the Gemini South Telescope takes it observations from, and plot the resulting correlations for the various directions of the wind layers in Fig. 11. For most of the wind layers in the NOAA GFS, there is little to no correlation between the direction of the asym- metric butterfly and the direction of the wind, with the exception of the wind layers around 100 to 300 hPa, which roughly cor- responds to altitudes of 10 to 15 km, which is the approximate altitude of the jet stream. The strength of these correlations, as measured by the Pearson R coefficient, and the slopes of the best fit lines are plotted for a concise summary in Fig. 8. The rise of the correlations well beyond the limits imposed by a null hypothesis bootstrap show that these strong correlations are not spurious and a very real phenomenon. Downloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 06 Jul 2023 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use Madurowicz et al.: Asymmetries in adaptive optics point spread functions Madurowicz et al.: Asymmetries in adaptive optics point spread functions Fig. 7 Angular histogram of the asymmetric butterfly vector’s projec- tion onto the ground for our sample in degrees from azimuth alongside the direction of the jet stream. The asymmetric butterfly nearly always points west while the jet stream is nearly always points east, which is only possible if the image plane electric field distribution is the inverse Fourier transform of the aperture plane. These distributions are not temporally matched but are rather the entire subset observations with strong asymmetry and the entire distribution of jet stream wind direc- tions over the course of the survey. For temporally matched correla- tions, see Figs. 8 and 11. Fig. 8 Distilling the information from the scatter plots in Fig. 11 into their slopes and Pearson R coefficient as a function of altitude from the GFS. The model contains layers all the way to sea level despite the observatory being around 3 km up because of the uniform grid spacing. The strong correlations are visible for the altitudes relevant to the jet stream in both the slope of the best fit line and the R coef- ficient. These correlations are much greater than the 2 to 4 sigma chances concerning the null hypothesis, generated from bootstrap sampling at random, shown as the shaded regions and solid lines for the slope and the R coefficient, respectively. Fig. 8 Distilling the information from the scatter plots in Fig. 11 into their slopes and Pearson R coefficient as a function of altitude from the GFS. The model contains layers all the way to sea level despite the observatory being around 3 km up because of the uniform grid spacing. The strong correlations are visible for the altitudes relevant to the jet stream in both the slope of the best fit line and the R coef- ficient. These correlations are much greater than the 2 to 4 sigma chances concerning the null hypothesis, generated from bootstrap sampling at random, shown as the shaded regions and solid lines for the slope and the R coefficient, respectively. Fig. 8 Distilling the information from the scatter plots in Fig. 11 into their slopes and Pearson R coefficient as a function of altitude from the GFS. The model contains layers all the way to sea level despite the observatory being around 3 km up because of the uniform grid spacing. Journal of Astronomical Telescopes, Instruments, and Systems 049003-8 Oct–Dec 2019 • Vol. 5(4) Journal of Astronomical Telescopes, Instruments, and Systems 049003-7 Downloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 06 Jul 2023 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use Downloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 06 Jul 2023 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use Madurowicz et al.: Asymmetries in adaptive optics point spread functions However, since the scintillation halo intensity is driven by the effectiveness of the AO system, the halo appears fainter at longer wavelengths. Because the halo originates from a spectrum of turbulent modes which decide their final image plane location, the halo does not scale in image location with wavelength, unlike the static speckles best removed with SDI.27 In addition, the scintillation halo is fixed with respect to the direction of the high-altitude winds, and it does not track with the rotation of the instrumental errors or the parallactic rotation of the astrophysical signal in an observing strategy such as ADI.28 Scintillation errors are uncorrectable for an AO system oper- ating in single-DM phase-conjugation mode, even if it is infi- nitely fast (see Fig. 13). This in turn will set a performance floor for such systems, and the asymmetry detected here pro- vides a first measurement of the level in which those effects begin to dominate. One could address this with a system that corrects amplitude errors as well, using the Talbot or scintilla- tion mixing effect to one’s own advantage. Having two deform- able mirrors at two unique conjugate planes in the optical system enables some phase introduced by one DM to transform into amplitude, allowing one to correct amplitude errors from scintillation. This concept has been proposed for space-based coronagraphs17 to correct static amplitude errors, and laboratory testing is underway.18 Similar designs have been expressed for improvements in laser communications.19,20 But we are particu- larly interested in the future of high-contrast imaging, particu- larly in the era of ELTs. Such a system could also correct static amplitude errors in an ELT, such as reflectivity variations between segments which have been recoated at different times.21,22 Driving such a system would require knowledge of both the phase and amplitude of the science wavefront. Space- based coronagraphic instruments can achieve this using the sci- ence camera and making several measurements while modulat- ing the speckle field with the DM.23,24 Another similar focal plane wavefront sensing approach was recently proposed on ground-based telescopes,25 which could also correct for amplitude aberrations from scintillation. Madurowicz et al.: Asymmetries in adaptive optics point spread functions Madurowicz et al.: Asymmetries in adaptive optics point spread functions Fig. 9 Asymmetry strength χ when compared to the velocity of the jet stream and the telescope elevation for our sample. Both exhibit strong correlations which corroborate our analytic understanding of the origin of the asymmetry. Slower jet stream velocities directly cause stronger asymmetry, while decreased tele- scope elevation has two effects. One to decrease the apparent wind velocity and the other to push the turbulent layers father away, giving more distance to scintillate. Fig. 9 Asymmetry strength χ when compared to the velocity of the jet stream and the telescope elevation for our sample. Both exhibit strong correlations which corroborate our analytic understanding of the origin of the asymmetry. Slower jet stream velocities directly cause stronger asymmetry, while decreased tele- scope elevation has two effects. One to decrease the apparent wind velocity and the other to push the turbulent layers father away, giving more distance to scintillate. time lag error alone, without additionally compensating ampli- tude errors, the asymmetry will become larger as the effective wind velocity is decreased. This effect is worst when consider- ing the asymmetry in low-order modes, which correspond to small separations in the final images, the region where planets or protoplanetary disks are most likely to be found. Although it is still optimal to have as-fast-as-possible correction in the metric of total scattered light in the halo, as computers get faster and algorithms are optimized the asymmetry will begin to play a larger role relative to other errors in the final PSF formed in AO images, leading many to explore possible routes for correction. separate phase and amplitude. Other methods to estimate the amplitude errors could utilize fast interferometric focal-plane sensing,26 or something as simple as adding a high-speed direct pupil-imaging channel to a traditional AO wavefront sensor. The scintillation halo observed in AO PSFs is a challenge for effective postprocessing of datasets. Many algorithms are designed to subtract a static speckle field with respect to the detector plane whose origin is from optical phase errors from imperfections, misalignment, noncommon-path errors (NCPE), and other systematic sources. Since these speckles have a unique spectral dependence, moving to farther separations at longer wavelengths, instruments imaging with an integral field spectro- graph can measure that spectral dependence and remove those aberrations. Downloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 06 Jul 2023 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use Journal of Astronomical Telescopes, Instruments, and Systems 049003-9 5 Discussion High-contrast imaging systems, with large actuator counts, are often limited by time lag errors. In particular, when observing bright stars, they are the dominant source of scattered light within the “dark hole” region.15 Under mid-latitude Chilean conditions, the velocity of the jet stream is often the dominant source of these errors, even if its contribution to the total r0 is moderate. This implies that different observatory sites with slower winds may have a comparative advantage, as well as the merits in scheduling observations around poor atmospheric con- ditions. In our previous work,7 we demonstrate that the jet stream is highly correlated with these errors, in a very large sam- ple of observations. To further explore our observations of the asymmetry, we plot χ versus the velocity of the jet stream and the elevation of the telescope pointing for our subset of observations which have been temporally matched to the wind data in Fig. 9. Both plots have reasonable correlation strength with R coefficient around negative one half, and both of these correlations have an intuitive sense. Our analysis showed that the asymmetry should be strong for slower wind velocities, and this is indeed verified through the first plot. The second has contributions from two different effects. When the telescope is pointing toward zenith (elevation ¼ 90 deg), the turbulent wind layers in the jet stream are as close to the telescope as possible along the line of sight. As a consequence, the propagation distance z will be Scintillation has previously been identified as a performance limiting factor in high-contrast imaging,16 and here we have demonstrated the severity of this effect, visible in the form of PSF asymmetry. This has implications for both current and future AO systems, especially but not exclusively those designed for high-contrast imaging. Many analyses often assume that various sources of scattered light are uncorrelated16 for simplic- ity. In this paper, we show that correlations between scintillation amplitude errors and time lag phase errors exist and can domi- nate during ideal conditions. If one simply tries to minimize the Journal of Astronomical Telescopes, Instruments, and Systems 049003-8 Oct–Dec 2019 • Vol. 5(4) Oct–Dec 2019 • Vol. 5(4) Madurowicz et al.: Asymmetries in adaptive optics point spread functions Conventional Shack– Hartmann wavefront sensors measure some intensity informa- tion but this is complicated by spots in each subaperture moving out the active pixels of the detector, so it is impractical to Various different postprocessing algorithms29,30 often use a high-pass filter (HPF) to attempt to eliminate the diffuse back- ground halo, and this is effective for regions of the image at large separations. However, near the coronagraphic mask, the scintil- lation can have very sharp features, demonstrated analytically as regions where log χ ¼ 0, and observable in simulations as dark regions perpendicular to the axis of the wind direction. When an HPF that preserves the features of a planet is applied to this halo, residuals which vary on spatial scales comparable to the planet are not removed. Often, a quadrupolar residual artifact near the coronagraphic mask if left which is large compared to the speckle residuals in the smooth halo at large separations. These residuals contribute significant noise to planetary detec- tion attempts at the nearest separations, where the likelihood of detection is highest from their population distributions.2 In addi- tion, imaging extended objects such as debris disks cannot uti- lize an HPF in postprocessing, implying a limit to sensitivity even at wide separations for diffuse unpolarized structure. Journal of Astronomical Telescopes, Instruments, and Systems 049003-9 Oct–Dec 2019 • Vol. 5(4) Madurowicz et al.: Asymmetries in adaptive optics point spread functions Madurowicz et al.: Asymmetries in adaptive optics point spread functions Various methods of subtracting the scintillation halo have been suggested. One could take an empirical approach, using a PCA style analysis to model the shape of the halo over an averaged population of observations. Acknowledging this effect is unique and must be treated independently with this sort of approach can be effective at improving the final SNR in your detection algorithm.2 Another approach may be to model the PSF end-to-end with complete simulations of the instrument and atmosphere, although this approach is significantly limited by the extent to which your simulated instrument can account for all real sources of error. Not only do errors arise from instrumen- tal effects like DM fitting and NCPE, but also the non- Kolmogorov deviations in the turbulent spectrum from environ- mental effects,31 as well as the finite temporal resolution of available atmospheric information. Another path may attempt to estimate the PSF using a reconstruction from measured AO telemetry. 6 Appendix A: Propagation through the Atmosphere EQ-TARGET;temp:intralink-;e043;326;435ρðzÞ ¼ ρ0e −Mgz RT ; (43) (43) Tartarski32 has shown that the fluctuations in the optical index of refraction in three dimensions for a Kolmogorov turbulence spectrum follow the form: where ρ0 is the atmospheric pressure at sea level. An atmospheric temperature profile as a function from altitude can be determined empirically, or the values given in the GFS can be used, but small fluctuations in T hardly affect the end value of the index of refraction, compared to the pressure, which dominates. EQ-TARGET;temp:intralink-;e038;63;398ΦNðκ; zÞ ¼ 0.033C2 NðzÞκ−11∕3; (38) (38) where C2 N is the index of refraction structure constant and κ ¼ 2π∕l is the spatial wave vector for an eddy of size l. Here, we use the standard Kolmogorov power spectrum, which is fractally self-similar at all length scales, although it is in principle simple to extend this model to a Von-Karman spectrum by attenuating the power above and below the outer and inner scales. From the square root of the power spectrum, we can find the fluctuations from the inverse Fourier transform according to Johansson and Gavel33 with where C2 N is the index of refraction structure constant and κ ¼ 2π∕l is the spatial wave vector for an eddy of size l. Here, we use the standard Kolmogorov power spectrum, which is fractally self-similar at all length scales, although it is in principle simple to extend this model to a Von-Karman spectrum by attenuating the power above and below the outer and inner scales. From the square root of the power spectrum, we can find the fluctuations from the inverse Fourier transform according to Johansson and Gavel33 with For nonzenith observations, an additional term of sec ζ where ζ is the zenith angle should be included in the integral in Eq. (41) to account for additional atmospheric depth. When the accumulated phase on the aperture is very large, we can subtract off the average phase, which is equivalent to removing the piston term from a Zernike polynomial.35 In order to account for scintillation, each turbulent layer must be propagated according to the angular spectrum rule derived at the beginning of this paper. Madurowicz et al.: Asymmetries in adaptive optics point spread functions However, with current WFS measurements, esti- mating the amplitude error from scintillation is rather difficult, as current instruments are not designed to measure wavefront amplitude. It is likely that the optimal approach to handling these errors is at the instrument level itself, as discussed previ- ously, with a method to measure and correct the wavefront amplitude in real time. As high-contrast imaging strives for higher and higher performance levels, identification, measure- ment, estimation, and mitigation of scintillation errors will become increasingly important. exponent, and we have to pay careful attention the normalization factor used by a routine such as np.fft.fft2, which includes a nor- malization of 1 PQ on the inverse transform, but no normalization on the forward transform by default. The optical path length of a wavefront traversing a turbulent layer in the atmosphere from zenith can be found to first order by integrating the index of refraction over the thickness of the layer, and the accumulated phase is simply the wave vector of the ray k ¼ 2π∕λ times the optical path length: EQ-TARGET;temp:intralink-;e041;326;651ϕið~xÞ ¼ k Z ziþΔzi zi nð~x; zÞdz ¼ knð~x; zÞΔzi: (41) (41) Here ~x ¼ ðx; yÞ is the coordinate system in the aperture at z ¼ 0, Δzi is the range of altitudes relevant to the turbulent layer at altitude zi, and the baseline index of refraction of the atmosphere can be approximated34 with EQ-TARGET;temp:intralink-;e042;326;562N ≡ðn −1Þ106 ≈77.6 ρ T ; (42) (42) where ρ and T are the pressure (in millibars or equivalently hPa) and temperature (in Kelvin) of the atmosphere for a particular altitude. To obtain a model for the index of refraction as a func- tion of altitude, one can model the pressure as a decaying expo- nential with a scale height given by the local surface gravity g, the mean molecular mass M of the atmosphere, the ideal gas constant R, and an assumed isothermal uniform temperature of the surface T with Downloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 06 Jul 2023 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 06 Jul 2023 https://www.spiedigitallibrary.org/terms-of-use Downloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,- Terms of Use: https://www.spiedigitallibrary.org/terms-of-use 6 Appendix A: Propagation through the Atmosphere the previous description, we invoke the Taylor frozen-flow hypothesis, which requires that the timescale for turbulence is much greater than the time delay with which the AO system will respond. For our simulation, this means that the fluctuations in the field of view simply propagate by translations due to the wind velocity, which can be expressed by EQ-TARGET;temp:intralink-;e045;63;686δN½~x þ ~vðzÞτ; t0 þ τ ¼ δNð~x; t0Þ; (45) (45) where ~vðzÞ is the wind velocity at altitude z, which is assumed to lie only in the plane at altitude with no vertical component, t0 is a particular instant in time, and τ is the total time delay for the AO system to respond to a measurement from the wavefront sensor. We also assume a perfect noiseless wavefront sensor and deformable mirror with only a time lag or servo-lag error for an ideal open-loop AO simulation. The expression for the compen- sated phase in the aperture is a new complex illumination with the amplitude errors from the current timestep and the phase given by two subtracted phases, one from the current timestep and one from the previous, which is our AO correction: Fig. 10 Diagram of coordinates used to orient images on the sky demonstrating the relationship between the ground plane and the BT plane. where we have assumed the convention of the positive x axis pointing north, and the positive y axis pointing west. This con- veniently sets up the positive z axis to point toward zenith, as it should. azimuth is measured from north opening toward the east, and elevation is measured from the horizon upward. See Fig. 10 for an illustration. It is worth noting that the val- idity of this analogy, as well as is necessary to implement angu- lar differential imaging, a postprocessing technique for combining multiple exposures while the target star moves through the zenith that GPI operates in a fixed parallactic ori- entation, with the instrument derotator disabled, so that GPI is fixed with respect to the telescope orientation, which is uncommon. EQ-TARGET;temp:intralink-;e046;63;534ϕAO ¼ ϕð~x; t0Þ −ϕð~x; t0 −τÞ; (46) (46) where we implicitly have included the contributions from L tur- bulent layers at various altitudes z with a flat interpolation scheme for the structure constant. 6 Appendix A: Propagation through the Atmosphere From the compensated phase on the aperture, we can obtain the final image’s intensity dis- tribution with an inverse Fourier transform by assuming the tele- scope focus operates in a Fraunhofer diffraction limit, so that electric field distribution in the image plane is the inverse Fourier transform of the aperture function:11 With such conventions laid out, it becomes easy to identify the location of the image plane on the sky, as it must be perpendicular to the line of sight. Since there are infinitely many such planes, we will use the convention EQ-TARGET;temp:intralink-;e047;63;414Iðα; βÞ ¼ hjF −1ðAUAOÞj2i: (47) (47) EQ-TARGET;temp:intralink-;e050;326;399^a ¼ −sinðazÞ; −cosðazÞ; 0 ; (50) EQ-TARGET;temp:intralink-;e051;326;349^b ¼ h−sinðelÞ cosðazÞ; sinðelÞ sinðazÞ; cosðelÞi: (51) EQ-TARGET;temp:intralink-;e050;326;399^a ¼ −sinðazÞ; −cosðazÞ; 0 ; (50) (50) Here α and β are the coordinates in the image plane, UAO is our AO corrected complex illumination which includes ampli- tude errors from scintillation, A is the aperture function with the Blackman window apodization,36 parameterized radially from the center with r2 ¼ x2 þ y2: EQ-TARGET;temp:intralink-;e051;326;349^b ¼ h−sinðelÞ cosðazÞ; sinðelÞ sinðazÞ; cosðelÞi: (51) (51) So that one can think of ^a as pointing in the direction of increasing azimuth and ^b pointing toward increasing elevation. It is left to the reader to show that ^a · ^b ¼ 0, and that ^a × ^r ¼ ^b to verify the orthogonality of these unit vectors as a coordinate system. EQ-TARGET;temp:intralink-;e048;63;327 AðrÞ ¼ 1 −γ 2 −1 2 cos 2πðr −D∕2Þ D þ γ 2 cos 2πðr −D∕2Þ D ; (48) (48) With this elaborate set up, it becomes easy to convert vectors in the image plane into vectors in full three-dimensional space, and then project them onto the ground plane. Suppose we have a wind vector which appears in the image plane rotated ψ from ^a counterclockwise. Such a wind vector is with an aperture diameter of D ¼ 8 m, a falloff of γ ¼ :16, and the brackets denote time average over the whole length of the simulation. The Blackman apodization simulates a crude coronagraph and dampens the high-order airy rings, whose final intensity in the image plane can swamp the effect of the scin- tillation halo. EQ-TARGET;temp:intralink-;e052;326;219 ^w ¼ cosðψÞ^a þ sinðψÞ^b: (52) (52) 6 Appendix A: Propagation through the Atmosphere In order to make this simulation numerically tractable, the propagation through the turbulent phase screen is discretized into two steps, one where the phase is first accumulated according to the entire thickness of the layer at the start, and then second where the wave free-space propa- gates the entire distance of the layer. For an infinite number of layers, this assumption should recover the true propagation and indeed we are in a regime where the layer thickness is relatively small compared to the total propagation distance. A shorthand summary rule for the angular spectrum propagation is that the complex illumination at propagation distance z is related to the complex illumination at the origin with EQ-TARGET;temp:intralink-;e039;63;266 δNð~x; zÞ ¼ Re F −1h ξð~κ; zÞ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ ΦNðκ; zÞ p i ; (39) (39) where δN are the fluctuations of the index of refraction from unity in parts per million, ξ is a zero-mean unit-variance com- plex hermitian Gaussian noise process, and F −1 is the unnor- malized inverse discrete Fourier transform given by EQ-TARGET;temp:intralink-;e040;63;177ηab ¼ F −1ð˜ηpqÞ ¼ X P−1 p¼0 X Q−1 q¼0 ˜ηpq exp 2πi pa P þ qb Q ; (40) (40) EQ-TARGET;temp:intralink-;e044;326;126uðzÞ ¼ F −1fHðzÞF½ug; (44) (44) for a discrete array of size P × Q with P; Q ∈N. The discrete indices p; a ∈0;1; : : : ; P −1 and q; b ∈0;1; : : : ; Q −1 exist in Fourier and configuration space, respectively. The correspond- ing forward Fourier transform simply includes negation in the for a discrete array of size P × Q with P; Q ∈N. The discrete indices p; a ∈0;1; : : : ; P −1 and q; b ∈0;1; : : : ; Q −1 exist in Fourier and configuration space, respectively. The correspond- ing forward Fourier transform simply includes negation in the where H is the free-space propagation transfer function which is implicitly also a function of the particular modes k being propa- gated. With the complex illumination given at the aperture by tems 049003-10 Oct–Dec 2019 • Vol. 5(4) 0 Oct–Dec 2019 • Vol. 5(4) Journal of Astronomical Telescopes, Instruments, and Systems 049003-10 Madurowicz et al.: Asymmetries in adaptive optics point spread functions Fig. 10 Diagram of coordinates used to orient images on the sky demonstrating the relationship between the ground plane and the BT plane. 7 Appendix B: Relationship between the Sky and the Ground However, we would instead like to know ^wð^x; ^y; ^zÞ. By alge- braically substituting in our coordinate vectors ^a and ^b formulas in x, y, z space, we can arrive at an expression for the wind vec- tor in x, y, z space in terms of ψ, az, and el. This is The back of the telescope (BT) plane is the simplest way to imagine the relationship between an image on the sky and its orientation relative to the ground. Suppose you have a DSLR on a tripod, or a multimillion-dollar telescope with an Alt-Az tracking system. Either way, your imaging device is pointed at the celestial sphere along the line of sight vector: EQ-TARGET;temp:intralink-;e053;326;132 ^w ¼ −cosðψÞ sinðazÞ −sinðψÞ sinðelÞ cosðazÞ; −cosðψÞ cosðazÞ þ sinðψÞ sinðelÞ sinðazÞ; sinðψÞ cosðelÞ : (53) EQ-TARGET;temp:intralink-;e053;326;132 ^w ¼ −cosðψÞ sinðazÞ −sinðψÞ sinðelÞ cosðazÞ; −cosðψÞ cosðazÞ þ sinðψÞ sinðelÞ sinðazÞ; EQ-TARGET;temp:intralink-;e049;63;86^r ¼ cosðelÞ cosðazÞ; −cosðelÞ sinðazÞ; sinðelÞ ; (49) (53) (49) Journal of Astronomical Telescopes, Instruments, and Systems 049003-11 Journal of Astronomical Telescopes, Instruments, and Systems 049003-11 From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 06 Jul 2023 https://www.spiedigitallibrary.org/terms-of-use Journal of Astronomical Telescopes, Instruments, and Systems 049003-11 Oct–Dec 2019 • Vol. 5(4) Downloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 06 Jul 2023 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use Madurowicz et al.: Asymmetries in adaptive optics point spread functions Fig. 11 Correlations between the directions of the strong asymmetry of the image PSF and the wind direction for various wind layers in the NOAA GFS. Most wind layers do not exhibit significant correlation, with the exception of the layers around 100 to 250 hPa, which are the pressures corresponding the jet stream, at around 10 to 15 km of altitude. Madurowicz et al.: Asymmetries in adaptive optics point spread functions Fig. 11 Correlations between the directions of the strong asymmetry of the image PSF and the wind direction for various wind layers in the NOAA GFS. Most wind layers do not exhibit significant correlation, with the exception of the layers around 100 to 250 hPa, which are the pressures corresponding the jet stream, at around 10 to 15 km of altitude. With this done, we can easily project the vector onto the ground plane by simply removing the z component. If we need to find the direction of this wind vector as an azimuth, we can use the following trick: convenient to use a smart operator like arctan2 to get the quad- rant correct. However, images in the GPIES are not simply oriented as in the BT plane, but rather can be arbitrarily arranged due to the com- plexities of postprocessing. Fortunately for us, the orientation of each of the image has been previously calculated in celestial coor- dinates. These are represented as a CD matrix, which describe how x and y in pixels for the image correspond to right ascension and declination. Using the local sidereal time of the image during the exposure, it is possible to convert coordinates in right ascen- sion and declination to coordinates in azimuth and elevation, using EQ-TARGET;temp:intralink-;e054;63;138azimuth ¼ 360 deg −arctan 2 wy wx %360 deg; (54) (54) where wx, wy, are the x and y components of the wind vector, respectively, % is the modulo operator, and it is often Oct–Dec 2019 • Vol. 5(4) Journal of Astronomical Telescopes, Instruments, and Systems 049003-12 Downloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instrum Terms of Use: https://www.spiedigitallibrary.org/terms-of-use Acknowledgments This research was sponsored by grants from NSFAST-1411868, NASA NNX14AJ80G, NNX15AC89G, and NNX15AD95G. Research benefited from the Gemini Observatory, operated by AURA for NSF and the Gemini Consortium. Portions of this work were performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DEAC52-07NA27344. Special thanks are owed to Paul Williams, Alfredo Dubra, Julien Milli, Faustine Cantalloube, and Elena Masciadri for their helpful discussions. This research was sponsored by grants from NSFAST-1411868, NASA NNX14AJ80G, NNX15AC89G, and NNX15AD95G. Research benefited from the Gemini Observatory, operated by AURA for NSF and the Gemini Consortium. Portions of this work were performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DEAC52-07NA27344. Special thanks are owed to Paul Williams, Alfredo Dubra, Julien Milli, Faustine Cantalloube, and Elena Masciadri for their helpful discussions. Fig. 12 The log of the speckle asymmetry ratio for a single mode with a propagation distance of z ¼ 25 km. Such layers in the atmosphere viewed at zenith are very sparse due to the exponential decline in pressure of the Earth’s atmosphere, and so do not contribute signifi- cantly into observations done at zenith. For a jet stream layer at 15 km, a propagation distance of 25 km corresponds to telescope pointing elevation of 40 deg. When comparing this figure to Fig. 3, it becomes clear that modifying the propagation distance z effectively changes the particular mode lengths the asymmetry occurs at to larger mode lengths, pushing the asymmetry to smaller separations, and nearer to the coronagraphic mask. Madurowicz et al.: Asymmetries in adaptive optics point spread functions Madurowicz et al.: Asymmetries in adaptive optics point spread functions Fig. 12 The log of the speckle asymmetry ratio for a single mode with a propagation distance of z ¼ 25 km. Such layers in the atmosphere viewed at zenith are very sparse due to the exponential decline in pressure of the Earth’s atmosphere, and so do not contribute signifi- cantly into observations done at zenith. For a jet stream layer at 15 km, a propagation distance of 25 km corresponds to telescope pointing elevation of 40 deg. When comparing this figure to Fig. 3, it becomes clear that modifying the propagation distance z effectively changes the particular mode lengths the asymmetry occurs at to larger mode lengths, pushing the asymmetry to smaller separations, and nearer to the coronagraphic mask. where h ¼ θL −α is the hour angle, θL is the local sidereal time in radians, ϕ0 is the local latitude, α is right ascension, δ is declina- tion, and here we use the convention that azimuth starts at north and opens to the east. The modulo is there to handle overflow and the azimuth and elevation are the coordinates on the sky. Once these are calculated, we can orient images relative to the BT plane because ^a points toward increasing azimuth and ^b points toward increasing elevation. References 1. L. A. Poyneer et al., “Performance of the Gemini planet imager’s adaptive optics system,” Appl. Opt. 55(2), 323–340 (2016). 2. E. L. Nielsen et al., “The Gemini planet imager exoplanet survey: giant planet and brown dwarf demographics from 10–100 au,” Astron. J. 158(1), 13 (2019). Fig. 13 Horizontal slice through the center of the PSFs generated in the single-layer atmospheric simulation, overlaid on top of each other for direct comparison. Here it is more explicitly visible as the wind speed decreases how both the total intensity of the halo decreases, as expected for better corrections, but in addition, the asymmetry becomes stronger at lower wind velocities, as expected from our model. For an infinitely fast AO system, the effective wind velocity is v ¼ 0 m∕s, which demonstrates the residual uncorrected amplitude error. 3. J. J. Wang et al., “Dynamical constraints on the HR 8799 planets with GPI,” Astron. J. 156(5), 192 (2018). 4. A. Rajan et al., “Characterizing 51 ERI b from 1 to 5 μm: a partly cloudy exoplanet,” Astron. J. 154(1), 10 (2017). 5. J.-B. Ruffio et al., “Improving and assessing planet sensitivity of the GPI exoplanet survey with a forward model matched filter,” Astrophys. J. 842(1), 14 (2017). 6. F. Cantalloube et al., “Status of the MEDUSAE post-processing method to detect circumstellar objects in high-contrast multispectral images,” arXiv:1812.04312 (2018). 7. A. Madurowicz et al., “Characterization of Lemniscate atmospheric aberrations in Gemini planet imager data,” Proc. SPIE 10703, 107036E (2018). 8. F. Cantalloube et al., “Origin of the asymmetry of the wind driven halo observed in high-contrast images,” Astron. Astrophys. 620, L10 (2018). 9. J. W. Goodman, Introduction to Fourier Optics, McGraw-Hill Series in Electrical and Computer Engineering: Communications and Signal Processing, McGraw-Hill, New York (1996). Fig. 13 Horizontal slice through the center of the PSFs generated in the single-layer atmospheric simulation, overlaid on top of each other for direct comparison. Here it is more explicitly visible as the wind speed decreases how both the total intensity of the halo decreases, as expected for better corrections, but in addition, the asymmetry becomes stronger at lower wind velocities, as expected from our model. For an infinitely fast AO system, the effective wind velocity is v ¼ 0 m∕s, which demonstrates the residual uncorrected amplitude error. 10. P. Zhou and J. H. Burge, “Analysis of wavefront propagation using the Talbot effect,” Appl. Opt. 49(28), 5351–5359 (2010). 1. E. Journal of Astronomical Telescopes, Instruments, and Systems 049003-12 Downloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 06 Jul 2023 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use Madurowicz et al.: Asymmetries in adaptive optics point spread functions References Hecht, Optics, Addison-Wesley, Reading, Massachusetts 12. A. Sivaramakrishnan et al., “Speckle decorrelation and dynamic range in speckle noise-limited imaging,” Astrophys. J. 581(1), L59–L62 (2002). 13. M. D. Perrin et al., “The structure of high STREHL ratio point-spread functions,” Astrophys. J. 596, 702–712 (2003). 14. NOAA NCEP, “Global forecast system analysis dataset,” ftp://nomads .ncdc.noaa.gov/GFS/analysis_only/. 15. V. P. Bailey et al., “Status and performance of the Gemini planet imager adaptive optics system,” Proc. SPIE 9909, 99090V (2016). EQ-TARGET;temp:intralink-;e055;63;164 azimuth ¼ arctan2 cosðδÞsinðhÞ sinðϕ0ÞcosðδÞcosðhÞ−cosðϕ0ÞsinðδÞ %360; (55) EQ-TARGET;temp:intralink-;e056;63;83elevation ¼ arcsin½sinðϕ0Þ sinðδÞ þ cosðϕ0Þ cosðδÞ cosðhÞ; (56) EQ-TARGET;temp:intralink-;e055;63;164 azimuth azimuth ¼ arctan2 cosðδÞsinðhÞ sinðϕ0ÞcosðδÞcosðhÞ−cosðϕ0ÞsinðδÞ %360; (55) 16. O. Guyon, “Limits of adaptive optics for high-contrast imaging,” Astrophys. J. 629(1), 592–614 (2005). 17. C. de Jonge et al., “Effect of multiple deformable mirrors in broadband high-contrast coronagraphs,” Proc. SPIE 10703, 107035D (2018). (55) 18. B.-J. Seo et al., “Hybrid lyot coronagraph for WFIRST: high contrast testbed demonstration in flight-like low flux environment,” Proc. SPIE 10698, 106982P (2018). EQ-TARGET;temp:intralink-;e056;63;83elevation ¼ arcsin½sinðϕ0Þ sinðδÞ þ cosðϕ0Þ cosðδÞ cosðhÞ; (56) 19. C. Wu et al., “Phase and amplitude beam shaping with two deformable mirrors implementing input plane and Fourier plane phase modifica- tions,” Appl. Opt. 57(9), 2337–2345 (2018). Journal of Astronomical Telescopes, Instruments, and Systems 049003-13 Madurowicz et al.: Asymmetries in adaptive optics point spread functions Madurowicz et al.: Asymmetries in adaptive optics point spread functions 20. M. C. Roggemann and D. J. Lee, “Two-deformable-mirror concept for correcting scintillation effects in laser beam projection through the turbulent atmosphere,” Appl. Opt. 37(21), 4577–4585 (1998). 29. J. J. Wang et al., “pyKLIP: PSF subtraction for exoplanets and disks,” Astrophysics Source Code Library (2015). 30. L. Pueyo et al., “Application of a damped locally optimized combination of images method to the spectral characterization of faint companions using an integral field spectrograph,” Astrophys. J. Suppl. Ser. 199(1), 6 (2012). 21. B. Macintosh et al., “Extreme adaptive optics for the Thirty Meter Telescope,” Proc. SPIE 6272, 62720N (2006). 22. M. Troy et al., “Effects of diffraction and static wavefront errors on high-contrast imaging from the thirty meter telescope,” Proc. SPIE 6272, 62722C (2006). 31. M. Tallis et al., “Air, telescope, and instrument temperature effects on the Gemini planet imager’s image quality,” Proc. SPIE 10703, 1070356 (2018). 23. P. J. Borde and W. A. Traub, “High-contrast imaging from space: speckle nulling in a low-aberration regime,” Astrophys. J. 638(1), 488–498 (2006). 32. V. I. Tatarski, Wave Propagation in a Turbulent Medium, Dover Books on Physics and Mathematical Physics, Dover, New York (1961). 33. E. M. Johansson and D. T. Gavel, “Simulation of stellar speckle imag- ing,” Proc. SPIE 2200, 372–383 (1994). 24. A. Give’on, “The electric field conjugation—a unified formalism for wavefront correction algorithms,” in Front. Opt. 2009/Laser Sci. XXV/Fall 2009 OSA Opt. & Photonics Tech. Digest, p. AOWA3 (2009). 34. J. W. Hardy, Adaptive Optics for Astronomical Telescopes, Oxford Series in Optical and Imaging Sciences, Oxford University Press, New York (1998). 25. B. L. Gerard, C. Marois, and R. Galicher, “Fast coherent differential imaging on ground-based telescopes using the self-coherent camera,” Astron. J. 156, 106 (2018). 35. J. R. Males and O. Guyon, “Ground-based adaptive optics corona- graphic performance under closed-loop predictive control,” J. Astron. Telesc. Instrum. Syst. 4, 019001 (2018). 26. B. L. Gerard et al., “Fast focal plane wavefront sensing on ground-based telescopes,” Proc. SPIE 10703, 1070351 (2018). 36. R. B. Blackman and J. W. Tukey, “The measurement of power spectra from the point of view of communications engineering—part I,” Bell Syst. Tech. J. 37(1), 185–282 (1958). 27. C. Marois, D. W. Phillion, and B. Macintosh, “Exoplanet detection with simultaneous spectral differential imaging: effects of out-of-pupil-plane optical aberrations,” Proc. SPIE 6269, 62693M (2006). 28. C. Journal of Astronomical Telescopes, Instruments, and Systems 049003-13 Oct–Dec 2019 • Vol. 5(4) Oct–Dec 2019 • Vol. 5(4) Downloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 06 Jul 2023 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use Downloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 06 Jul 2023 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use Madurowicz et al.: Asymmetries in adaptive optics point spread functions Marois et al., “Angular differential imaging: a powerful high-contrast imaging technique,” Astrophys. J. 641(1), 556–564 (2006). Biographies of the authors are not available. 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https://openalex.org/W3037184705	http://izvestiya.asu.ru/article/download/%282020%292-09/6605	Russian	null	Construction of Belovskii Zinc Plant in 1928-1932	Izvestiâ Altajskogo gosudarstvennogo universiteta	2,020	cc-by	2,972	The Federal Research Center of Coal and Coal Chemistry of SB RAS (Kemerovo, Russia) The history of Belovsky distillation zinc plant in 1928–1932 — the first child of non-ferrous metallurgy of Soviet Siberia, the scale of construction of which at that time in Kuzbass was inferior only to Kuznetskstroi is considered. The stages of selecting the site of the future enterprise, its economic justification, design and creation of a raw material base in Salair in the form of mines and a enrichment factory have been studied. The difficulties that prevented the construction of production facilities are shown: repeated revision of the cover lists, lack of qualified construction personnel and metal specialists, shortage of construction materials, months of delay in the supply of metal structures and equipment, lag in the development of the social sphere in comparison with the production sector. The measures that mobilized the first initiative and labor enthusiasm of workers and engineers for commissioning of the plant in the scheduled time frame were considered. It was concluded that the organization of the design and construction of the Belovsky zinc plant did not go beyond the culture of inter-economic relations and intra-production relations characteristic in the USSR for the period of the I five-year plan. Рассматривается история Беловского дистилля- ционного цинкового завода в 1928–1932 гг. — пер- венца цветной металлургии советской Сибири, мас- штаб строительства которого в то время в Кузбассе уступал лишь Кузнецкстрою. Изучены этапы выбора площадки будущего предприятия, его экономическо- го обоснования, проектирования и создания сырье- вой базы в Салаире в виде рудников и обогатитель- ной фабрики. Показаны трудности, которые мешали возведению объектов производственного назначе- ния: неоднократный пересмотр титульных списков, отсутствие квалифицированных строительных ка- дров и специалистов-металлургов, дефицит строи- тельных материалов, многомесячная задержка по- ставок металлических конструкций и оборудования, отставание в развитии социальной сферы в сравне- нии производственной. Рассмотрены мероприятия, мобилизовавшие творческую инициативу и трудо- вой энтузиазм рабочих и инженеров на сдачу завода в эксплуатацию в намеченные сроки. Сделан вывод о том, что организация проектирования и строи- тельства Беловского цинкового завода не выходи- ла за пределы культуры межхозяйственных связей и внутрипроизводственных отношений, характер- ных в СССР для периода первой пятилетки. б Key words: Kuzbasstsinkstroy, Belovsky zinc plant, Salairsky mine, Guryevsk, Kuznetskstroy. р р Ключевые слова: Кузбассцинкстрой, Беловский цин- ковый завод, Салаирский рудник, Гурьевск, Кузнецк- строй. * Статья написана в рамках реализации научного проекта «Создание индустриальной базы на территории Кузбас- са в конце XIX — первой половине XX в.» (АААА-А17-117041410054-8). Construction of Belovskii Zinc Plant in 1928–1932 N.M. Morozov N.M. Morozov Строительство Беловского цинкового завода... Строительство Беловского цинкового завода... УДК 94(571.17).08+314 ББК 63.3 (2Рос-4Кем)5+60.7 Строительство Беловского цинкового завода... в 1928–1932 гг.* Н.М. Морозов Федеральный исследовательский центр угля и углехимии СО РАН (Кемерово, Россия) УДК 94(571.17).08+314 ББК 63.3 (2Рос-4Кем)5+60.7 Федеральный исследовательский центр угля и углехимии СО РАН (Кемерово, Россия) DOI 10.14258/izvasu(2020)2-09 Кузнецкстрою, обусловлен слабой изученностью практики проектирования и организации возведе- ния его объектов. Изучение опыта советской инду- стриализации всегда являлось актуальным для ис- Научный интерес к первым годам истории Беловского цинкового завода — первенца цветной металлургии советской Сибири, масштаб строитель- ства которого в то время в Кузбассе уступал лишь 57 Известия АлтГУ. Исторические науки и археология. 2020. №2 (112) следователей. История больших строек локализует проявление проблем и тенденций в экономике и со- циальной сфере, общих для формировавшегося в на- чале 1930-х гг. промышленного комплекса СССР [1; 2; 3, с. 60–63]. первых двух лет предстоящей работы рассматрива- лась обогащенная риддерская руда. Образцы сала- ирского сырья прошли проверку в Петроградском институте «Механобр». Результаты показали, что по- лученные концентраты легко поддавались обогаще- нию, среднее содержание цинка составило 45–50% и железа — 5%. Несколько строк о деятельности предприя- тия в годы первой пятилетки написано в изда- нии «История Кузбасса» [4, с. 36]. Основные вехи 75-летней деятельности Беловского цинкового за- вода отражены в исторических очерках краеведов [5, с. 146–175; 6; 7]. В перечисленных выше трудах фрагментарно рассмотрены проблемы, с которыми приходилось сталкиваться трудовому коллективу на рубеже 1920–1930-х гг. Между тем анализ более широкого круга источников региональных архивов существенно обогащает представление об организа- ции этой большой стройки. В декабре 1927 г. руководство треста обрати- лось к председателю Главметалла Высшего Совета Народного Хозяйства (ВСНХ) В.И. Межлауку с хода- тайством о финансировании строительства цинково- го завода в Кузбассе. В ответ было получено завере- ние о резервировании в 1928 г. 1 млн руб. из 12 млн, предназначенных на развитие всей цветной метал- лургии СССР. Ввиду отсутствия в стране инженеров, знако- мых с новейшими достижениями заграничной тех- ники и технологиями в области выплавки цинка, для разработки проекта завода пришлось обратиться к иностранным специалистам. Поступившие от за- граничных фирм предложения о составлении про- екта завода оказались дорогостоящими. Так, фир- ма «Бер-Зонд-Хаймер» запросила 200.000 долларов, а фирма «Эрцрест-Гезельшафт» — 50.000 долларов, при этом последняя поставила условием постав- ку печей Спирле, менее зарекомендовавших себя на производстве, чем предложенные советской сто- роной печи Веджа. Во второй половине 1920-х гг. производство цинка на заводе во Владикавказе, единственном в СССР, было в состоянии удовлетворить всего лишь около 10% потребности промышленности в этом металле, и это несмотря на то, что в стране имелись богатые месторождения цинково-свинцо- вых руд. В этой связи Совет труда и обороны 3 июня 1927 г. вынес постановление о постройке за Уралом специализированного металлургического предпри- ятия [8, л. 43]. В середине июня 1927 г. Строительство Беловского цинкового завода... предложенных цен в сторону экономии средств, по- этому впоследствии на строительной площадке не- однократно приходилось переделывать уже частич- но возведенные элементы конструкций. на досрочный пуск, правительство аннулировало ли- цензию на импорт цинка из Германии в размере бу- дущей производственной программы предприятия [12, л. 16]. В летние месяцы 1929 г. более 120 бри- гад включились в социалистическое соревнование за звание «Бригада ударного труда». От строитель- ной площадки до станции Белово была проложена железнодорожная ветка длинной в 2,5 км и допол- нительно 3,5 км внутризаводских путей. С выходом на центральную магистраль Кузбасса более интен- сивным стал входящий поток грузов с материалами и оборудованием. ру В конце января 1928 г. в Белово приехал С.В. Фаер, назначенный управляющим комбина- том «Кузбассцинкстрой», специально созданным для строительства цинкового завода и предприятий его рудной базы в Салаире. Первый управляющий уже имел опыт восстановления Макеевского метал- лургического комбината и Харьковского паровозо- строительного завода. В начале февраля прибыла группа металлургов и строителей в составе немец- кого инженера Хольтмана, инженера П.Г. Логинова, прораба А.М. Епанчинцева, механика Шмидта и др. В полутора километрах от д. Белово была выбрана подходящая площадка [5, с. 146–147]. Начался под- готовительный этап масштабной стройки, и уже весной законтрактованные крестьяне соседних де- ревень огородили территорию забором и присту- пили к земляным работам. Летом это место напо- минало «муравейник»: около 1200 человек рыли котлованы будущих цехов, возводили временные жилые дома для рабочих, хозяйственные строе- ния [8, л. 8]. Основным источником пополнения строитель- ных кадров являлись крестьяне местных деревень, молодежь, приехавшая по комсомольским путев- кам, командированные биржами труда по оргнабо- ру из Сибири и европейской части России. Для обе- спечения стройки квалифицированными кадрами и техническим персоналом были открыты вечер- ние школы и производственные кружки. Отработав восемь часов на стройплощадке, в вечернее время люди четыре часа учились различным специаль- ностям: каменщика, кровельщика, плотника, шту- катура, арматурщика и др. Одновременно готови- лись кадры для металлургического производства. Будущих плавильщиков с 1930 г. обучал молодой инженер Г.С. Полетаев, а также А.А. Романов — житель деревни Верхний Чумыш, который в годы Первой мировой войны военнопленным несколь- ко лет работал на цинковом заводе в Германии [6, с. 36]. Повышение квалификации осуществля- лось путем направления специалистов на курсы в Москву и Ленинград, во время практики на ме- таллургических заводах Алагира, Риддера и Урала [10, л. 70об.]. Первый вариант титульного списка объектов строительства, выполненных в чертежах объемом в 300 листов, был представлен на утверждение прав- лению треста в ноябре 1928 г. DOI 10.14258/izvasu(2020)2-09 с целью экономического обоснования выбора места для дистилляционного цинкового завода в Кузнецкий округ был направлен геолог треста «Алтайполиметалл» Иван Трифонович Гуштюк. В течение трех месяцев он изучал состояние трех Салаирских рудников с подготовленными запа- сами сульфидной руды в объеме 150 000 000 пудов, оценил близлежащие месторождения сырья и его ка- чество для производства необходимых строитель- ных материалов. В результате в Берлине было решено орга- низовать собственное проектное бюро во гла- ве с инженером Хольтманом, рекомендованным Союзом германских инженеров. Под его руко- водством и с привлечением авторитетных кон- сультантов — крупных немецких специалистов: профессора Энделя (исследование огнеупорных ма- териалов для цинковых печей), профессора Розина (теплотехника) и докторов Юрецки и Пауля (метал- лургия цветных металлов), проектное бюро с 1 сен- тября 1927 г. приступило к работе. В докладной записке, направленной 16 сентября 1927 г. в Сибкрайсовнархоз (Новосибирск), было за- явлено о непригодности городов Кузбасса для раз- мещения завода из-за предполагаемого масштабного ущерба населению от выпуска свободного сернисто- го газа в атмосферу. В свою очередь выделялась дерев- ня Белово благодаря соседству с одноименной узло- вой станцией, примыканием железнодорожной ветки на Гурьевск и перспективой снабжения стройки метал- лом Гурьевского завода, а также сбыта продукции пред- приятиям Кемеровского и Кузнецк-Прокопьевского промышленных районов [9, л. 2об.–4]. Уже к 1 января 1928 г. был готов черновик про- екта завода, который заказчику обошелся в 20.000 долларов. Его вместе с ориентировочными смета- ми Берлинское бюро в марте представило в Москву. До августа проект изучали советские специалисты, вносились изменения части конструкций с целью их упрощения и облегчения. Обновленный про- ект 24 августа 1928 г. был утвержден Коллегией Главметалла ВСНХ СССР [8, л. 44]. В сентябре 1928 г. проектное бюро треста «Алтайполиметалл» в составе 24 инженеров при- ступило к детальной разработке чертежей завод- ских зданий. В связи с неопределенностью с заказами иностранного оборудования габариты предприятия и соответствующие размеры производственных по- мещений не могли быть сразу точно установлены. Выбор поставщиков во многом определялся уровнем Первоначально трест предполагал построить опытный дистилляционный цинковый завод с го- довой производительностью в 4500 т, т.е. 15 т в сут- ки и эксплуатацией в течение16 лет. Затем проек- тировщики мощность увеличили до 12500 т в год. Основной сырьевой базой был определен Салаирский рудник № 2. В качестве источника сырья в течение 58 Строительство Беловского цинкового завода... Известия АлтГУ. Исторические науки и археология. 2020. №2 (112) Известия АлтГУ. Исторические науки и археология. 2020. №2 (112 лаире сырьевой базы завода: Первого и Второго цинковых рудников, рудника «Кварцитовая горка», обогатительной фабрики, а также железнодорожной ветки Гурьевск — Салаир протяженностью 21,5 км. В этой связи в первые годы завод снабжался дорого- стоящими из-за транспортировки по железной до- роге рудными концентратами Риддера. тульных списков каждый раз вызывало пересмотр плана работ, передвижку сроков получения креди- тов и выполнение на 72–75% годового плана возве- дения производственных зданий, подсобных и хо- зяйственных сооружений. Учитывая имеющиеся недостатки, в октяб- ре 1929 г. президиум Кузнецкого окрисполко- ма обязал руководство стройки в зимний период 1929–1930 гг. форсировать подготовительные меро- приятия для обеспечения в теплые месяцы 1930 г. высокие темпы работы на всех пусковых объектах. Исходя из практики прошедшего строительного се- зона проводились корректировки норм выработки и расценок, ускоренно возводилось жилье, впервые приобреталась спецодежда, средства индивидуаль- ной защиты и мыло. В мае 1932 г. Государственная комиссия под- писала акт о пуске Беловского цинкового заво- да в эксплуатацию. В течение 1930–1932 гг. рядом с предприятием появился соцгородок с 22 двух- и трехэтажными кирпичными домами, кирпичной поликлиникой, хирургическим и родильным от- делениями больницы, большими корпусами школ №1, №3 и ФЗУ. К началу 1932 г. была проложена же- лезнодорожная ветка от Гурьевска до Салаира, свя- завшая предприятие с Салаирской обогатительной фабрикой, сданной в эксплуатацию в 1934 г. В тече- ние пяти лет металлурги нового завода приобрета- ли знания и нарабатывали опыт выплавки цинка, прежде чем в 1935 г. выдали 12200 т металла и поч- ти достигли годовой проектной мощности пред- приятия [15, 16]. Взятое коллективом обязательство по досроч- ному пуску завода к октябрю 1930 г. вызвало необ- ходимость перехода с 1 ноября 1929 г. на непрерыв- ную шестидневную рабочую неделю без выходных. Основной упор в снабжении стройки был сделан на поставку материалов производственного назначе- ния, иногда в ущерб обеспечения социальной сферы. В этой связи случались частые перебои в снабжении рабочих предметами первой необходимости, продук- тами питания. Вместе с тем не хватало врачей, дети еще не были охвачены школьной сетью первой сту- пени [14, л. 12об.]. Как видим, организация проектирова- ния и строительства Беловского цинкового за- вода не выходила за пределы культуры межхо- зяйственных связей и внутрипроизводственных отношений, характерных для советской промыш- ленности периода первой пятилетки. Строительство Беловского цинкового завода... В связи с их поздней передачей на стройку возникла необходимость вне- сения ряда изменений в первоначально намеченный план работ [10, л. 20–21]. Одновременно в районе Салаира в течение 1928–1929 гг. продолжалась разведка цинковых руд. В 1928 г. месторождения проверялись на глуби- ну 30–40 саженей, были выявлены запасы на 8–9 лет эксплуатации завода. В 1929 г. началось бурение на больших глубинах (до 70 саженей) и открыты запасы для использования уже на десятки лет впе- ред [11, л. 72]. Строительный сезон 1929 г., в котором были за- няты около 2000 человек, проходил с преодолением трудностей, вызванных несвоевременным посту- плением из Москвы проектов части важных про- изводственных и коммунальных объектов: силовой станции, больницы, бани, школы, водопровода, кана- лизации и т.д. Чертежи доставлялись частями по эле- ментам, что вынуждало возводить без полного пред- ставления о строящемся сооружении. Отсутствовали плановые заявки на материалы. Насколько аккурат- но, по мнению С.В. Фаера, выполняли свои обяза- тельства по договорам поставки оборудования ино- странные фирмы, настолько плохо выполняли свои обязательства отечественные заводы. Несущие ме- таллоконструкции, например, поступали с опозда- нием на шесть и более месяцев, поэтому некоторые из них пришлось заменить кирпичной кладкой [13, л. 39–40]. 27 сентября, т.е. за три дня до окончания сезона, в Белово был получен пятый окончательный вариант титульного списка строящихся объектов. Пятикратное в течение двух лет перекраивание ти- Весной 1929 г. за деревней Белово зарабо- тали два временных полукустарных кирпич- ных заводика. Для обеспечения стройплощадки щебнем и песком были открыты карьеры в рай- оне окрестных деревень: Банново, Колмогорово, Менчерепе, Пестерево, Семенушкино, Большого Улуса и Бачаты. С апреля началась закладка фун- даментов и кладка стен механического и муфель- ного цехов, а осенью — шихтовочного, обжигово- го и дистилляционного. В мае 1929 г. в ответ на Обращение XVI партий- ной конференции ВКП(б) сделать социалистическое соревнование главным средством борьбы за выпол- нение первой пятилетки, рабочие взяли обязатель- ство построить завод не за четыре года, как ранее на- мечалось, а за три, т.е. к октябрю 1930 г. Расчитывая 59 Строительство Беловского цинкового завода... Строительство Беловского цинкового завода... Известия АлтГУ. Исторические науки и археология. 2020. №2 (112) Несмотря на трудности, связанные с многократным пере- смотром проекта, длительной задержкой черте- жей, поставок технологического оборудования, конструкций, острым дефицитом материалов, от- сутствием квалифицированных рабочих стро- ительных специальностей, нехваткой дипломи- рованных специалистов-металлургов, завод был возведен в первоначально намеченные четыре года. Мобилизационный характер строительства проявился в преодолении указанных выше про- блем благодаря трудовому энтузиазму людей, гра- мотным и нестандартным техническим решениям инженеров, энергичному руководству со сторо- ны директора С.В. Фаера. Завод положил нача- ло формированию в центральной части Кузбасса Беловского угольно-металлургического района. Со строительством его сырьевой базы на Салаирском руднике последний в 1932 г. был преобразован в рабочий поселок Салаир [17], предприятия ко- торого вновь обрели особое значение для эконо- мики страны. В зимний период 1929–1930 гг. инженеры П.Г. Логинов и А.М. Епанчинцев на бетонных ра- ботах вместо привычных тепляков предложили на- гревать воду на уличных кострах в котлах, а затем заливать в бетономешалки. Технология не снизила качество, но значительно ускорила бетонные рабо- ты. На месте этот передовой опыт изучил главный инженер Кузнецкстроя И.П. Бардин и впоследствии стал широко использовать при возведении цехов бу- дущего металлургического комбината. Первую, из десяти по проекту, дистилляцион- ную печь поставили на газ 22 декабря 1930 г. Уже 29 декабря были выплавлены первые плитки белов- ского цинка. В следующем 1931 г. намечалось по- лучить 2583 т серебристого металла. Впрочем, этот показатель удалось выполнить только на 56%, так как сдвинулись сроки ввода в эксплуатацию отдель- ных дистилляционных печей и пуск центральной электрической станции. Еще низкой оставалась ква- лификация производственного персонала. Только весной 1930 г. комбинат «Куз- бассцинкстрой» приступил к строительству в Са- 60 6. 50 огненных лет. К юбилею Беловского цинкового завода / под ред. И.Ф. Шнайдера, Б.А. Козлова, А.Г. Мар- ченко и др. Кемерово, 1980. 5. Белово / гл. ред. В.П. Щелканов. Новосибирск, 2000. 4. История Кузбасса. Ч. III: История Кузбасса в пери- од строительства социализма и коммунизма. Кемерово, 1970. Библиографический список 1. Верхотуров Д. Сталинская индустриализация. М., 2017. 1. Верхотуров Д. Сталинская индустриализация. М., 2017. 7. Козлов Б.А. Белово. Кемерово, 1974. 7. Козлов Б.А. Белово. Кемерово, 1974. 8. Государственный архив Новосибирской области (ГАНО). Ф.П-6. Оп. 1. Д. 813. 8. Государственный архив Новосибирской области (ГАНО). Ф.П-6. Оп. 1. Д. 813. 2. Сельская В.А. Становление химической промыш- ленности СССР: по материалам Ярославского региона 1928–1940 гг. : автореф. дис. ... канд. ист. наук. Мытищи, 2018. 2. Сельская В.А. Становление химической промыш- ленности СССР: по материалам Ярославского региона 1928–1940 гг. : автореф. дис. ... канд. ист. наук. Мытищи, 2018. 9. ГАНО. Ф.Р-22. Оп. 1. Д. 704. 10. Государственный архив Кемеровской области (ГАКО). Ф. П-8. Оп. 1. Д. 406. 10. Государственный архив Кемеровской области (ГАКО). Ф. П-8. Оп. 1. Д. 406. 3. Бельков В.И. Вопросы строительства Беловского цинкового завода в документах ГКУ КО «ГАКО» // ТЭК и ресурсы Кузбассе. 2012. № 6(65). 11. Из истории Салаира / под ред. А.М. Адаменко. Ке- мерово, 2007. 11. Из истории Салаира / под ред. А.М. Адаменко. Ке- мерово, 2007. 12. ГАКО. Ф.П-8. Оп. 1. Д. 480. 4. История Кузбасса. Ч. III: История Кузбасса в пери- од строительства социализма и коммунизма. Кемерово, 1970. 4. История Кузбасса. Ч. III: История Кузбасса в пери- од строительства социализма и коммунизма. Кемерово, 1970. 13. ГАНО. Ф. Р-22. Оп. 1. Д. 1570. 14. ГАКО. Ф. П-22. Оп. 1. Д. 293. 15. Знамя ударника. 1936. № 1. 1 янв. 15. Знамя ударника. 1936. № 1. 1 янв. 5. Белово / гл. ред. В.П. Щелканов. Новосибирск, 2000. 5. Белово / гл. ред. В.П. Щелканов. Новосибирск, 2000. 16. Знамя ударника. 1936. № 109. 7 ноября. 17. Собрание узаконений РСФСР. 1932. № 11. Ст. 53. 6. 50 огненных лет. К юбилею Беловского цинкового завода / под ред. И.Ф. Шнайдера, Б.А. Козлова, А.Г. Мар- ченко и др. Кемерово, 1980. 6. 50 огненных лет. К юбилею Беловского цинкового завода / под ред. И.Ф. Шнайдера, Б.А. Козлова, А.Г. Мар- ченко и др. Кемерово, 1980.
https://openalex.org/W3115157151	https://russjcardiol.elpub.ru/jour/article/download/4223/3133	Russian	null	Effect of ticagrelor in patients with acute coronary syndrome on the achievement of the targets of federal project on the prevention of cardiovascular diseases: 3-year perspective estimates	Rossijskij kardiologičeskij žurnal	2,020	cc-by	8,435	Российский кардиологический журнал 2020; 25 (12) Российский кардиологический журнал 2020; 25 (12) https://russjcardiol.elpub.ru doi:10.15829/1560-4071-2020-4223 ISSN 1560-4071 (print) ISSN 2618-7620 (online) ISSN 1560-4071 (print) ISSN 2618-7620 (online) Отношения и деятельность. Исследование выполнено при поддержке ком- пании АстраЗенека. 1ФГБУ НЦЭСМП Минздрава России, Москва; 2ФГАОУ ВО Первый МГМУ им. И. М. Сеченова Минздрава России (Сеченовский Университет), Москва; 3ФГБУ НМИЦ Кардиологии Минздрава России, Москва; 4ФГАОУ ВО Россий- ский университет дружбы народов, Москва; 5ФГБУ НМИЦ им. В. А. Алмазова Минздрава России, Санкт-Петербург, Россия. Материал и методы. В качестве целевой популяции в первый год лечения рассматривались все взрослые российские пациенты, с подтвержденным диагнозом острого коронарного синдрома (ОКС), которым показана ДАТ, на второй и третий годы лечение продолжали только пациенты высокого риска атеротромботических событий. Расчет количества смертей, которые воз- можно предотвратить используя ДАТ “тикагрелор 90 мг + АСК” вместо “кло- пидогрел + AСК” в течение первого года с даты постановки диагноза, произ- водился на основе данных клинической эффективности рандомизированного клинического исследования (РКИ) PLATO, а для расчета количества смертей, которые возможно предотвратить, используя ДАТ тикагрелор в дозировке 60 мг + АСК — вместо монотерапии AСК были использованы данные клиниче- ской эффективности РКИ PEGASUS для пациентов с ИМ в анамнезе не более двух лет назад. Журавлева М. В.* — д.м.н., профессор; кафедра клинической фармаколо- гии и пропедевтики внутренних болезней, ORCID: 0000-0002-9198-8661, Палеев Ф. Н. — член-корр. РАН, д.м.н., профессор, заместитель генерально- го директора, ORCID: 0000-0001-9481-9639, Зырянов С. К. — д.м.н., профес- сор, зав. кафедрой общей и клинической фармакологии, ORCID: 0000-0002- 6348-6867, Яковлев А. Н. — к.м.н., доцент кафедры НИЛ острого коронар- ного синдрома, ORCID: 0000-0001-5656-3978, Марин Т. В. — к.м.н., доцент кафедры клинической фармакологии и пропедевтики внутренних болезней, ORCID: 0000-0002-8974-4457, Гагарина Ю. В. — ассистент кафедры клини- ческой фармакологии и пропедевтики внутренних болезней, ORCID: 0000- 0002-4459-3034. В дальнейшем рассчитывалось, какую долю ЦП ФП “Борьба с ССЗ” в 2021- 2023гг удалось бы достичь при применении ДАТ с тикагрелором вместо кло- пидогрела или монотерапии АСК. В дальнейшем рассчитывалось, какую долю ЦП ФП “Борьба с ССЗ” в 2021- 2023гг удалось бы достичь при применении ДАТ с тикагрелором вместо кло- пидогрела или монотерапии АСК. В дальнейшем рассчитывалось, какую долю ЦП ФП “Борьба с ССЗ” в 2021- 2023гг удалось бы достичь при применении ДАТ с тикагрелором вместо кло- пидогрела или монотерапии АСК. Автор, ответственный за переписку (Corresponding author): doc@pharmset.ru Автор, ответственный за переписку (Corresponding author): doc@pharmset.ru Результаты. Применение тикагрелора в составе ДАТ для лечения пациен- тов с ОКС в соответствии с клиническими рекомендациями позволит (по сравнению с ДАТ АСК с клопидогрелом или антитромбоцитарной терапией АСК): Результаты. Отношения и деятельность. Исследование выполнено при поддержке ком- пании АстраЗенека. Применение тикагрелора в составе ДАТ для лечения пациен- тов с ОКС в соответствии с клиническими рекомендациями позволит (по сравнению с ДАТ АСК с клопидогрелом или антитромбоцитарной терапией АСК): АСК — ацетилсалициловая кислота, БСК — болезни системы кровообраще- ния, ДАТ — двойная антитромбоцитарная терапия, ИМ — инфаркт миокарда, ИМпST — инфаркта миокарда c подъемом сегмента ST, ЛП — лекарственный препарат, МЗ — Министерство здравоохранения, ОКС — острый коронарный синдром, ОКСбпST — острый коронарный синдром без подъема сегмента ST, ОНМК — острое нарушение мозгового кровообращения, РКИ — рандомизи- рованное клиническое исследование, РФ — Российская Федерация, ССЗ — сердечно-сосудистые заболевания, ССО — сердечно-сосудистые осложне- ния, ТЛТ — тромболитическая терапия, ФП — федеральный проект, ЦП — це- левые показатели. — в 2021-2023гг дополнительно предотвратить 5389, 5702 и 6009 смертей в указанные годы, что обеспечит прирост в достижении ЦП “снижение смерт- ности от БСК” ФП “Борьба с ССЗ” в Российской Федерации на 18,4%, 19,4% и 20,5%, соответственно. — в 2021-2023гг дополнительно предотвратить 5389, 5702 и 6009 смертей в указанные годы, что обеспечит прирост в достижении ЦП “снижение смерт- ности от БСК” ФП “Борьба с ССЗ” в Российской Федерации на 18,4%, 19,4% и 20,5%, соответственно. Кроме того, применение тикагрелора в составе ДАТ для лечения пациентов с ОКС, по сравнению с клопидогрелом или антитромбоцитарной терапией АСК, также позволит: Кроме того, применение тикагрелора в составе ДАТ для лечения пациентов с ОКС, по сравнению с клопидогрелом или антитромбоцитарной терапией АСК, также позволит: Кроме того, применение тикагрелора в составе ДАТ для лечения пациентов с ОКС, по сравнению с клопидогрелом или антитромбоцитарной терапией АСК, также позволит: — в 2021-2023гг дополнительно предотвратить 957, 1013 и 1067 смертей от ИМ в указанные годы, что обеспечит прирост в достижении ЦП “снижение смертности от ИМ” ФП “Борьба с ССЗ” в Российской Федерации на 50,2%, 49,3% и 51,9%, соответственно. — в 2021-2023гг дополнительно предотвратить 957, 1013 и 1067 смертей от ИМ в указанные годы, что обеспечит прирост в достижении ЦП “снижение смертности от ИМ” ФП “Борьба с ССЗ” в Российской Федерации на 50,2%, 49,3% и 51,9%, соответственно. Рукопись получена 27.11.2020 Рукопись получена 27.11.2020 Оценка влияния применения лекарственного препарата тикагрелор у пациентов с острым коронарным синдромом на выполнение целевых показателей федерального проекта “Борьба с сердечно-сосудистыми заболеваниями” — в перспективе трех лет Журавлева М. В.1,2, Палеев Ф. Н.3, Зырянов С. К.4, Яковлев А. Н.5, Марин Т. В.2, Гагарина Ю. В.2 Отношения и деятельность. Исследование выполнено при поддержке ком- пании АстраЗенека. Цель. Оценка дополнительного влияния применения тикагрелора в составе двойной антитромбоцитарной терапии (ДАТ) по сравнению с ДАТ клопидо- грелом и антитромботической терапией ацетилсалициловой кислотой (АСК) на выполнение целевых показателей (ЦП) федеральной программы (ФП) “Борьба с сердечно-сосудистыми заболеваниями” (“Борьба с ССЗ”): “сни- жение смертности от болезней систем кровообращения (БСК)” и “снижение смертности от инфаркта миокарда (ИМ)”. Received: 27.11.2020 Revision Received: 07.12.2020 Accepted: 15.12.2020 Received: 27.11.2020 Revision Received: 07.12.2020 Accepted: 15.12.2020 In addition, the use of ticagrelor within DAPT for patients with ACS compared with clopidogrel or ASA monotherapy will also allow: In addition, the use of ticagrelor within DAPT for patients with ACS compared with clopidogrel or ASA monotherapy will also allow: For citation: Zhuravleva M. V., Paleev F. N., Zyryanov S. K., Yakovlev A. N., Ma rin T. V., Gagarina Yu. V. Effect of ticagrelor in patients with acute coronary syndrome on the achievement of the targets of federal project on the prevention of cardio- vascular diseases: 3-year perspective estimates. Russian Journal of Cardiology. 2020;25(12):4223. (In Russ.) doi:10.15829/1560-4071-2020-4223 — to prevent an additional 957, 1013 and 1067 deaths due to MI in 2021, 2022, and 2023, respectively, achieving the target of federal project on the prevention of cardiovascular diseases “reduction of mortality from MI” by 50,2%, 49,3% and 51,9%, respectively. — to prevent an additional 957, 1013 and 1067 deaths due to MI in 2021, 2022, and 2023, respectively, achieving the target of federal project on the prevention of cardiovascular diseases “reduction of mortality from MI” by 50,2%, 49,3% and 51,9%, respectively. к 2024г, кроме того целью данного ФП является сни- жение смертности от ИМ с 40,0 случаев на 100 тыс. населения в 2018г до 30,6 случаев на 100 тыс. населе- ния к 2024г [4]. Болезни системы кровообращения (БСК) явля- ются основной причиной смерти в России, по дан- ным за 2018г, смертность от БСК составила 573 слу- чая на 100 тыс. населения, что составило 44,6% от всех причин смерти [1]. В России в 2018г с диагнозом острого коронарного синдрома (ОКС) было госпи- тализировано 531019 человек, из них в 147375 случаях (27,8%) был поставлен диагноз — инфаркт миокарда (ИМ) с подъемом сегмента ST (ИМпST) и в 383644 случаях (72,2%) — ОКС без подъема сегмента ST (ОКСбпST) [2]. Согласно данным ФГБУ “ЦНИИОИЗ” Мин здрава России, реализация целей и задач ФП “Борь- ба с ССЗ” по снижению смертности от ИМ и остро- го нарушения мозгового кровообращения (ОНМК) к 2024г позволит сохранить порядка 180800 жизней пациентов (22,6% от всех сохраненных жизней от смертей по причине всех БСК) [5]. Снижение смертности от БСК является важ- ным государственным приоритетом Российской Федерации (РФ). Так, одной из четырех целей нацио нального проекта “Здравоохранение” является сни- жение смертности от БСК до 450 случаев на 100 тыс. населения к 2024г [3]. Принята к публикации 15.12.2020 Заключение. Применение тикагрелора у пациентов с ОКС позволит снизить смертность от БСК и будет способствовать выполнению целевых показателей ФП "Борьба с ССЗ". Заключение. Применение тикагрелора у пациентов с ОКС позволит снизить смертность от БСК и будет способствовать выполнению целевых показателей ФП "Борьба с ССЗ". Для цитирования: Журавлева М. В., Палеев Ф. Н., Зырянов С. К., Яковлев А. Н., Марин Т. В., Гагарина Ю. В. Оценка влияния применения лекарственного препарата тикагрелор у пациентов с острым коронарным синдромом на вы- полнение целевых показателей федерального проекта “Борьба с сердечно- сосудистыми заболеваниями” — в перспективе трех лет. Российский кардио логический журнал. 2020;25(12):4223. doi:10.15829/1560-4071-2020-4223 Ключевые слова: тикагрелор, вторичная профилактика сердечно-сосудис тых заболеваний, целевые показатели, смертность от болезней системы кровообращения, смертность от инфаркта миокарда, федеральный проект “Борьба с сердечно-сосудистыми заболеваниями”. “Борьба с сердечно-сосудистыми заболеваниями”. 134 134 ПЕРЕДОВАЯ СТАТЬЯ КЛИНИКА И ФАРМАКОТЕРАПИЯ Relationships and Activities. The study was supported by AstraZeneca. 1Scientific Center for Expert Evaluation of Medicinal Products, Moscow; 2I. M. Sechenov First Moscow State Medical University, Moscow; 3National Medical Research Center of Cardiology, Moscow; 4Peoples’ Friendship University of Russia, Moscow; 5Almazov National Medical Research Center, St. Petersburg, Russia. Zhuravleva M. V.* ORCID: 0000-0002-9198-8661, Paleev F. N. ORCID: 0000-0001- 9481-9639, Zyryanov S. K. ORCID: 0000-0002-6348-6867, Yakovlev A. N. ORCID: 0000-0001-5656-3978, Marin T. V. ORCID: 0000-0002-8974-4457, Gagarina Yu. V. ORCID: 0000-0002-4459-3034. Then the proportion of goals that can be achieved in 2021-2023 was calculated when using DAPT with ticagrelor instead of clopidogrel or ASA monotherapy. Results. The use of ticagrelor within DAPT for patients with ACS will allow (com- pared to DAPT with clopidogrel and ASA monotherapy): Results. The use of ticagrelor within DAPT for patients with ACS will allow (com- pared to DAPT with clopidogrel and ASA monotherapy): — to prevent an additional 5389, 5702 and 6009 deaths in 2021, 2022, and 2023, respectively, achieving the target of federal project on the prevention of cardiovas- cular diseases “reduction of cardiovascular mortality” by 18,4, 19,4 and 20,5%, respectively. Corresponding author: doc@pharmset.ru Effect of ticagrelor in patients with acute coronary syndrome on the achievement of the targets of federal project on the prevention of cardiovascular diseases: 3-year perspective estimates Zhuravleva M. V.1,2, Paleev F. N.3, Zyryanov S. K.4, Yakovlev A. N.5, Marin T. V.2, Gagarina Yu. V.2 ravleva M. V.1,2, Paleev F. N.3, Zyryanov S. K.4, Yakovlev A. N.5, Marin T. V.2, Gagarina Yu. V.2 Aim. To assess the additional effect of ticagrelor as part of dual antiplatelet therapy (DAPT) compared with DAPT with clopidogrel and acetylsalicylic acid (ASA) mono- therapy on the achievement of goals of federal project on the prevention of cardio- vascular diseases: “reduction of cardiovascular mortality” and “reduction of mortal- ity from myocardial infarction (MI)”. Conclusion. The use of ticagrelor in patients with ACS will reduce cardiovascular mortality and contribute on the achievement of goals of federal project on the pre- vention of cardiovascular diseases. Key words: ticagrelor, secondary prevention of cardiovascular disease, targets, cardiovascular mortality, mortality from myocardial infarction, federal project on the prevention of cardiovascular diseases. Material and methods. In the first year of treatment, all adult Russian patients with documented acute coronary syndrome (ACS), who had indications for DAPT, were considered as the target population. In the second and third years, treat- ment was continued only by patients with a high risk of thrombotic events. The calculation of the number of deaths that can be prevented using DAPT with ti- cagrelor 90 mg + ASA instead of clopidogrel + ASA during the first year was based on the data of the PLATO trial. To calculate the number of deaths that can be prevented using DAPT with ticagrelor 60 mg + ASA instead of ASA monotherapy, we used the data of PEGASUS trial for patients with a history of MI within the past 2 years. Relationships and Activities. The study was supported by AstraZeneca. Received: 27.11.2020 Revision Received: 07.12.2020 Accepted: 15.12.2020 Для обеспечения выполнения национального проекта “Здравоохранение” и дости- жения соответствующих целевых показателей (ЦП) был разработан ряд федеральных проектов, в т. ч. фе- деральный проект (ФП) “Борьба с сердечно-сосудис тыми заболеваниями” (“Борьба с ССЗ”), основным ЦП которого является снижение смертности от БСК от базового значения в 587,6 случаев на 100 тыс. на- селения в 2017г до 450 случаев на 100 тыс. населения Для обеспечения целей и результатов ФП “Борьба с ССЗ” в 2020г на плановый период 2021 и 2022гг бюджетам субъектов РФ были выделены субсидии на обеспечение профилактики развития ССЗ и сердеч- но-сосудистых осложнений (ССО) в амбулаторных условиях у пациентов высокого риска, перенесших ОНМК, ИМ, а также лиц, которым были выполне- ны аортокоронарное шунтирование, ангиопластика коронарных артерий со стентированием и катетер- ная аблация по поводу ССЗ. Правила предостав- ления и распределения субсидий на обеспечение профилактики развития ССЗ и ССО у пациентов высокого риска, находящихся на диспансерном на- блюдении, определяются постановлением прави 135 135 Российский кардиологический журнал 2020; 25 (12) тельства РФ № 1569 от 30 ноября 2019г “О вне- сении изменений в государственную программу Российской Федерации “Развитие здравоохранения” [6]. Лекарственные препараты (ЛП), доступные для вторичной профилактики ССЗ и ССО в рамках по- становления правительства 1569, регламентируются перечнем доступных ЛП, утвержденным приказом Министерства здравоохранения (МЗ) РФ от 9 ян- варя 2020г № 1н “Об утверждении перечня лекар- ственных препаратов для медицинского применения для обеспечения в течение одного года в амбулатор- ных условиях лиц, которые перенесли ОНМК, ИМ, а также которым были выполнены аортокоронарное шунтирование, ангиопластика коронарных арте- рий со стентированием и катетерная аблация по по- воду сердечно-сосудистых заболеваний” (далее — Перечень) [7]. жения риска неблагоприятных исходов и рассмот реть возможность продления ДАТ в виде сочетания АСК с уменьшенной дозой тикагрелора (60 мг 2 раза/сут. внутрь) на дополнительные 36 мес. с целью дополнительного снижения риска неблагоприятных исходов (сумма случаев сердечно-сосудистой смерти, ИМ и инсульта) [8, 9]. Данное исследование представляет из себя продол- жение и расширение научной работы по оценке влия- ния применения блокаторов рецептора P2Y12 тромбо- цитов у пациентов с ОКС на целевые показатели ФП “Борьба с ССЗ”, в рамках настоящего исследования оценивается дополнительное снижение риска небла- гоприятных исходов на протяжении 3 лет у популя- ции пациентов с ОКС, расширенной за счет включе- ния пациентов с высоким ишемическим риском, для которых рассматривается возможность продления ДАТ (сочетание АСК с ингибитором Р2Y12-рецептора тромбоцитов) на более длительный срок [10]. Received: 27.11.2020 Revision Received: 07.12.2020 Accepted: 15.12.2020 В настоящем исследовании рассматривается вли- яние на ЦП “снижение смертности от БСК” и “сни- жение смертности от ИМ” ФП “Борьба с ССЗ” при- менения блокаторов рецептора P2Y12 тромбоцитов, включенных в Перечень, в составе двойной антитром- боцитарной терапии (ДАТ) для терапии ОКС [4, 7]. Цель исследования: оценка дополнительного вли- яния применения тикагрелора в составе ДАТ с АСК по сравнению с ДАТ клопидогрелом и АСК и анти- тромботицитарной терапией АСК на выполнение ЦП ФП “Борьба с ССЗ”: “снижение смертности от БСК” и “снижение смертности от ИМ”. р р (Д ) р [ , ] Антиагреганты занимают прочное место в кли- нических рекомендациях по ведению пациентов с ОКСбпST и ИМпST, и относятся к препаратам, влияющим на прогноз жизни и частоту повторных атеротромботических событий [8, 9]. Согласно дей- ствующим клиническим рекомендациям, для сни- жения риска смерти, сосудистой смерти, повторного ИМ и ишемического инсульта рекомендуется дли- тельный (неопределенно долгий) прием ацетилсали- циловой кислоты (АСК) всем пациентам с ИМпST, не имеющим противопоказаний, вне зависимости от исходной стратегии реперфузии [8]. Также неопреде- ленно долгий прием АСК рекомендуется всем паци- ентам с ОКСбпST, не имеющим противопоказаний, вне зависимости от стратегии лечения для сниже- ния риска суммы случаев смерти и ИМ [9]. При этом всем пациентам с ИМпST, не имеющим высокого риска кровотечений, в добавление к АСК на протя- жении 12 мес. рекомендуется прием блокатора Р2Y12- рецептора тромбоцитов (для снижения суммарного риска смерти, ИМ и ишемического инсульта) [8]. У пациентов с ОКСбпST, не имеющих высокого ри- ска кровотечений, также рекомендуется продолжать ДАТ (сочетание АСК с ингибитором P2Y12-рецептора тромбоцитов) на протяжении 12 мес. вне зависи- мости от тактики лечения и типа установленного стента для коронарных артерий, с целью уменьшить риск ишемических осложнений [9]. У пациентов с ОКСбпST, высоким риском коронарных осложне- ний и низким риском кровотечений, рекомендуется рассмотреть возможность продления ДАТ (сочетание АСК с ингибитором Р2Y12-рецептора тромбоцитов) на более длительный срок для дополнительного сни- Материал и методы р Определение характеристик и численности целе- вой популяции пациентов с ОКС, которым показана ДАТ. В качестве целевой популяции в первый год лечения рассматривались все взрослые российские пациенты с подтвержденным диагнозом ОКС, ко- торым показана ДАТ, на второй и третий годы ле- чение продолжали только пациенты с подтвержден- ным диагнозом ИМ высокого коронарного риска, т. е. лица 50 лет и старше, у которых присутствует как минимум один из следующих факторов риска: возраст 65 лет, наличие сахарного диабета, требую- щего медикаментозного лечения, два и более пере- несенных ИМ в прошлом, многососудистый коро- нарный атеросклероз, хроническая болезнь почек с расчетной скоростью клубочковой фильтрации <60 мл/мин/1,73 м2 [11]. Согласно данным мони- торинга ФГБУ “ЦНИИОИЗ”, представленного на профильной комиссии главными внештатными спе- циалистами-кардиологами МЗ РФ [5], за 2018г бы- ло зарегистрировано 530098 случаев госпитализа- ции пациентов в стационары с диагнозом ОКС. Из настоящего исследования исключались пациенты с ОКС, которым была проведена тромболитическая терапия (ТЛТ), ввиду отсутствия таких пациентов в исследовании PLATO [12]. Количество больных c ОКС, которым в 2018г была выполнена ТЛТ, составило 40176 человек [2]. Таким образом, популяция пациентов в первый год терапии составила 489922 человека (табл. 1). 136 136 Российский кардиологический журнал 2020; 25 (12) Таблица 1 Таблица 1 Расчет целевой популяции пациентов с ОКС, которым показана антитромбоцитарная терапия в течение трех лет после установки диагноза Показатель Значение Источник Расчет целевой популяции пациентов, получающих антитромбоцитарную терапию тикагрелором 90 мг в течение первого года после постановки диагноза Число больных с ОКС, поступивших в стационары субъекта 530098 [5] Число больных c ОКС, которым выполнен тромболизис 40176 [2] Всего пациентов с ОКС (за исключением пациентов с ТЛТ) 489922 Расчет целевой популяции пациентов, получающих антитромбоцитарную терапию тикагрелором 60 мг в течение второго и третьего года после постановки диагноза Количество пациентов, которым впервые установлены диагнозы “Острый ИМ” и “Повторный ИМ”, 2018г 186636 [13] Доля пациентов, соответствующих критериям высокого коронарного риска, % 41,1% [14] Количество пациентов высокого коронарного риска, перенесших ИМ не менее одного и не более двух лет назад 76707 Сокращения: ИМ — инфаркт миокарда, ОКС — острый коронарный синдром, ТЛТ — тромболитическая терапия. Та Расчет целевой популяции пациентов с ОКС, Для расчета количества смертей, которые воз- можно предотвратить используя ДАТ тикагрелор 90 мг + АСК вместо клопидогрел + AСК, в течение первого года с даты постановки диагноза использо- вались данные клинической эффективности рандо- мизированного клинического исследования (РКИ) PLATO c учетом ограничений исследования в части оценки доли пациентов с ОКСбпST высокого риска [12]. Вероятности исходов терапии пациентов с ОКС оценивались в % к 360 дню по методу Каплана- Мейера. Расчетные значения доли умерших от лю- бой причины, среди пациентов получающих тикагре- лор 90+АСК составила 4,5%, среди получавших ДАТ клопидогрел + АСК — 5,9%, p<0,001, из них доля исхода “сердечно-сосудистая смерть” среди пациен- тов получавших ДАТ с тикагрелором составила 4,0%, а среди пациентов, получавших ДАТ с клопидогре- лом — 5,1%, p=0,001. Таблица 2 Вероятность наступления события “сердечно-сосудистая смерть” Год терапии Тикагрелор 60+АСК АСК 1-й год 0,84% 1,25% 2-й год 1,67% 2,48% 3-й год 2,50% 3,70% Сокращение: АСК — ацетилсалициловая кислота. Таблица 4 Таблица 4 Оценка влияния применения препарата тикагрелор у пациентов с ОКС на достижение ЦП “снижение смертности от ИМ” в 2021-2023гг в РФ Показатель 2020 2021 2022 2023 ЦП “снижение смертности от БСК” (на 100 тыс. населения) 36 34,4 33 31,6 ЦП “снижение смертности от БСК” (абсолютное значение) 52389 50482 48427 46373 Количество смертей, которое необходимо предотвратить для достижения ЦП 1908 2054 2054 Количество смертей, которое возможно дополнительно предотвратить при добавлении тикагрелора к стандартной терапии 957 1013 1067 % достижения ЦП при применении тикагрелора 50,2% 49,3% 51,9% Сокращения: БСК — болезни систем кровообращения, ЦП — целевой показатель. Оценка влияния применения препарата тикагрелор у пациентов с ОКС на достижение ЦП “снижение смертности от ИМ” в 2021-2023гг в РФ Оценка влияния применения препарата тикагрелор у пациентов с ОКС на достижение ЦП “снижение смертности от ИМ” в 2021-2023гг в РФ лись показатели смертности при лечении целевой популяции пациентов ДАТ с тикагрелором в дози- ровке 60 мг по сравнению с терапией АСК. На осно- вании полученных данных рассчитывалась доля ЦП ФП “Борьба с ССЗ”, которую возможно достичь при применении ДАТ с тикагрелором в 2021-2023гг по сравнению с альтернативной терапией. Для целей данного анализа было сделано предпо- ложение, что доля смертей от ИМ среди смертнос ти от ССЗ в используемых РКИ соответствует доле смертей от ИМ среди смертности от ИМ и инсуль- та в РФ. Согласно данным Федеральной службы го- сударственной статистики в 2018г в России от ИМ умерло 56904 пациента, от инсульта — 263573 паци- ента [10]. Таким образом, доля смертности от ИМ составила 17,8% — это консервативная оценка, учи- тывающая реалии российской клинической практи- ки, включает в себя исходы вне зависимости от вре- мени наступления ОКС. Таблица 3 Оценка возможного вклада применения препарата тикагрелор у пациентов с ОКС на достижение ЦП “снижение смертности от БСК” в 2021-2023гг в РФ Оценка возможного вклада применения препарата тикагрелор у пациентов с ОКС на достижение ЦП “снижение смертности от БСК” в 2021-2023гг в РФ Вероятность наступления события населения) 545 525 505 485 ЦП “снижение смертности от БСК” (абсолютное значение) 770430 741080 711731 682381 Количество смертей, которое необходимо предотвратить для достижения ЦП 29350 29350 29350 Количество смертей, которое возможно дополнительно предотвратить при добавлении тикагрелора к стандартной терапии 5389 5702 6009 % достижения ЦП при применении тикагрелора 18,4% 19,4% 20,5% Сокращения: БСК — болезни систем кровообращения, ЦП — целевой показатель. Таблица 4 Оценка влияния применения препарата тикагрелор у пациентов с ОКС на достижение ЦП “снижение смертности от ИМ” в 2021-2023гг в РФ Показатель 2020 2021 2022 2023 ЦП “снижение смертности от БСК” (на 100 тыс. населения) 36 34,4 33 31,6 ЦП “снижение смертности от БСК” (абсолютное значение) 52389 50482 48427 46373 Количество смертей, которое необходимо предотвратить для достижения ЦП 1908 2054 2054 Количество смертей, которое возможно дополнительно предотвратить при добавлении тикагрелора к стандартной терапии 957 1013 1067 % достижения ЦП при применении тикагрелора 50,2% 49,3% 51,9% Сокращения: БСК — болезни систем кровообращения, ЦП — целевой показатель. Таблица 3 Оценка возможного вклада применения препарата тикагрелор у пациентов с ОКС на достижение ЦП “снижение смертности от БСК” в 2021-2023гг в РФ Показатель 2020 2021 2022 2023 ЦП “снижение смертности от БСК” (на 100 тыс. населения) 545 525 505 485 ЦП “снижение смертности от БСК” (абсолютное значение) 770430 741080 711731 682381 Количество смертей, которое необходимо предотвратить для достижения ЦП 29350 29350 29350 Количество смертей, которое возможно дополнительно предотвратить при добавлении тикагрелора к стандартной терапии 5389 5702 6009 % достижения ЦП при применении тикагрелора 18,4% 19,4% 20,5% Сокращения: БСК — болезни систем кровообращения, ЦП — целевой показатель. Вероятность наступления события В течение первого года сравниваемыми схемами терапии являются ДАТ в составе АСК + тикагре- лор 90 мг 2 раза/сут. и АСК + клопидогрел 75 мг 1 раз/сут. Во второй и третий годы ДАТ получали только пациенты высокого коронарного риска, с подтверж- денным диагнозом ИМ. Согласно данным МЗ РФ за 2018г было зарегистрировано 186636 пациентов, перенесших ИМ или повторный ИМ [13]. Исходя из доли пациентов, соответствующих критериям высо- кого коронарного риска, количество пациентов, пе- ренесших ИМ более одного, но менее двух лет назад, составило 76707 человек (табл. 1). При этом срав- ниваемыми схемами терапии являются тикагрелор 60 мг 2 раза/сут. в комбинации с низкой дозой АСК (75-150 мг) в соответствии с инструкцией по приме- нению по сравнению с монотерапией АСК в дози- ровке 75-150 мг [15]. В свою очередь, расчет количества смертей, ко- торые возможно предотвратить, используя ДАТ “Тикагрелор в дозировке 60 мг + АСК” вместо моно- терапии AСК, производился на основе данных кли- нической эффективности РКИ PEGASUS для па- циентов с ИМ в анамнезе [17]. Вероятности исходов антитромбоцитарной терапии пациентов высокого коронарного риска, перенесших ИМ не менее од- ного и не более двух лет назад, оценивались в про- центном отношении к 3-му году лечения по методу Каплана-Мейера. В настоящей статье сделано допущение о неиз- менной заболеваемости ИМ и ОКС в последующие 2 года. Таким образом, делается предположение о равномерном ежегодном приросте популяции. Расчетные значения доли умерших по причине сердечно-сосудистой смерти среди пациентов, полу- чающих Тикагрелор 60+АСК, составили 2,5%, среди получавших антитромбоцитарную терапию АСК — 3,7%, отношение рисков 0,68 (95% доверительный интервал); p<0,019. Методика расчета достижения ЦП “снижение смерт- ности от БСК” и “снижение смертности от ИМ” ФП “Борьба с ССЗ”. Основываясь на данных о числен ности постоянного населения в РФ на 1 января 2020г [16] и ЦП ФП “Борьба с ССЗ” на 2020-2023гг [3, 4], рассчитывались абсолютные ЦП смертности от БСК и ИМ в каждом регионе страны. Для расчета вероятности наступления события “сердечно сосудистая смерть” в течение первого и второго года лечения использовалась методология Fleurence RL, et al. [18]. Результаты расчетов пред- ставлены в таблице 2. 138 138 ПЕРЕДОВАЯ СТАТЬЯ КЛИНИКА И ФАРМАКОТЕРАПИЯ Таблица 3 Оценка возможного вклада применения препарата тикагрелор у пациентов с ОКС на достижение ЦП “снижение смертности от БСК” в 2021-2023гг в РФ Показатель 2020 2021 2022 2023 ЦП “снижение смертности от БСК” (на 100 тыс. Результаты Оценка дополнительного влияния применения тикагрелора в составе ДАТ с АСК по сравнению с ДАТ клопидогрелом и АСК и антитромбоцитар- ной терапией АСК на выполнение ЦП ФП “Борьба с ССЗ” в 2021-2023гг у пациентов с ОКС была про- ведена на основании данных клинической эффек- тивности РКИ PLATO, где сравнивались влияние на сердечно-сосудистую смертность и частоту возник- новения сердечно-сосудистых событий при приме- нении тикагрелора в дозировке 90 мг и клопидогре- ла в составе ДАТ у пациентов с ОКС, а также РКИ PEGASUS, где сравнивались те же показатели при применении тикагрелора в дозировке 60 мг в комби- нации с низкими дозами АСК (75-150 мг) по сравне- нию с монотерапией АСК. Расчет количества смертей, которые возможно предотвратить в первый год при применении ДАТ с тикагрелором, основывался на данных мониторин- га ФГБУ “ЦНИИОИЗ” МЗ РФ для каждого регио- на. Исходя из количества пациентов с ОКС без ТЛТ, рассчитывались показатели смертности для целевой популяции пациентов ДАТ с тикагрелором 90 мг по сравнению с клопидогрелом. Далее, основываясь на данных по регионам о количестве случаев перене- сенного ИМ или повторного ИМ в 2018г, рассчиты- вались показатели смертности при лечении целевой популяции пациентов ДАТ с тикагрелором в дози- ровке 60 мг по сравнению с АСК. В таблицах 3 и 4 приведен расчет влияния препа- рата тикагрелор у пациентов с ОКС на достижение ЦП “снижение смертности от БСК” и “снижение смертности от ИМ” ФП “Борьба с ССЗ” в 2021- 2023гг, соответственно. Результаты Продолжение Оценка влияния применения препарата тикагрелор у пациентов с ОКС на достижение ЦП “снижение смертности от БСК” в 2021-2023гг в регионах РФ Таблица 5. Результаты П Оценка влияния применения препарата тикагрелор у пациентов с ОКС на достижение ЦП “снижение смертности от БСК” в 2021-2023гг в регионах РФ Таблица Оценка влияния применения препарата тикагрелор у пациентов с ОКС на достижение ЦП “снижение смертности от БСК” в 2021-2023гг в регионах РФ Регион 2021 2022 2023 Количество смертей, которое возможно дополнительно предотвратить % достижения ЦП** при применении тикагрелора Количество смертей, которое возможно дополнительно предотвратить* % достижения ЦП при применении тикагрелора Количество смертей, которое возможно дополнительно предотвратить* % достижения ЦП при применении тикагрелора Республика Бурятия 27 33,6% 29 34,7% 30 36,2% Республика Дагестан 48 309,6% 49 397,6% 51 326,4% Республика Ингушетия 18 144,4% 19 149,6% 20 154,7% Республика Калмыкия 13 41,4% 13 43,2% 14 45,0% Республика Карелия 24 17,7% 26 19,0% 28 20,4% Республика Коми 22 18,6% 24 19,9% 27 21,0% Республика Крым 56 8,0% 61 8,6% 65 9,2% Республика Марий Эл 14 10,4% 16 11,3% 17 12,2% Республика Мордовия 31 259,0% 32 291,5% 34 284,8% Республика Саха (Якутия) 27 18,0% 28 19,7% 29 27,0% Республика Северная Осетия — Алания 19 14,6% 20 15,2% 21 15,8% Республика Татарстан 144 18,9% 151 19,7% 157 20,6% Республика Тыва 4 10,7% 4 11,6% 4 12,4% Республика Хакасия 32 23,1% 34 24,3% 35 25,3% Ростовская область 163 21,3% 174 22,2% 185 23,0% Рязанская область 33 14,2% 37 15,6% 40 17,0% Самарская область 150 25,0% 158 26,5% 166 27,7% Санкт-Петербург 193 18,2% 198 18,6% 203 19,1% Саратовская область 140 38,6% 147 40,5% 154 42,4% Сахалинская область 11 99,8% 12 109,6% 13 119,1% Свердловская область 301 32,2% 312 33,4% 323 34,7% Севастополь 12 5,4% 13 5,8% 14 7,7% Смоленская область 22 12,2% 24 14,2% 26 15,5% Ставропольский край 149 18,8% 156 19,8% 164 20,7% Тамбовская область 35 18,8% 38 20,5% 41 22,0% Тверская область 62 9,1% 67 9,9% 72 10,6% Томская область 40 22,5% 42 32,5% 44 25,8% Тульская область 52 56,7% 56 59,6% 59 64,3% Тюменская область без автономных округов 48 25,3% 51 26,9% 54 28,4% Удмуртская Республика 66 43,3% 70 45,9% 73 48,4% Ульяновская область 55 16,8% 58 18,6% 61 20,5% Хабаровский край 58 15,2% 61 15,9% 64 16,5% Ханты-Мансийский автономный округ — Югра 61 92,8% 64 100,7% 67 103,3% Челябинская область 154 22,9% 162 24,2% 170 25,3% Чеченская Республика 22 146,1% 23 152,4% 23 158,5% Чувашская Республика 37 26,4% 39 28,1% 41 29,8% Чукотский автономный округ 1 1006,2% 1 382,1% 1 641,9% Ямало-Ненецкий автономный округ 12 222,4% 13 78,7% 14 124,7% Ярославская область 39 15,1% 43 16,8% 47 18,1% Москва 349 6,8% 364 14,4% 379 15,0% Примечание: * — при применении тикагрелора в составе ДАТ с АСК по сравнению с ДАТ клопидогрелом и АСК и антитромбоцитарной терапией АСК, ** — ЦП — целевой показатель. Результаты Расчет проводился по сце- нарию I: в качестве целевой популяции в первый год Во втором сценарии исследовалась только попу- ляция пациентов, перенесших ИМ не менее одного и не более двух лет назад, таким образом, исходя из количества случаев перенесенного ИМ или повтор- ного ИМ в 2018г в каждом субъекте РФ рассчитыва- 139 139 Российский кардиологический журнал 2020; 25 (12) Оценка влияния применения препарата тикагрелор у пациентов с ОКС на достижение ЦП “снижение смертности от БСК” в 2021-2023гг в регионах РФ Оценка влияния применения препарата тикагрелор у пациентов с ОКС на достижение ЦП “снижение смертности от БСК” в 2021-2023гг в регионах РФ на достижение ЦП “снижение смертности от БСК” в 2021-2023гг в регионах РФ Регион 2021 2022 2023 Количество смертей, которое возможно дополнительно предотвратить* % достижения ЦП** при применении тикагрелора Количество смертей, которое возможно дополнительно предотвратить* % достижения ЦП при применении тикагрелора Количество смертей, которое возможно дополнительно предотвратить* % достижения ЦП при применении тикагрелора Российская Федерация 5389 18,4% 5702 19,4% 6009 20,5% Алтайский край 85 18,3% 92 20,0% 100 21,6% Амурская область 37 471,7% 39 549,3% 41 516,5% Архангельская область 36 15,1% 38 16,3% 41 17,5% Астраханская область 28 19,0% 30 11,8% 33 10,9% Белгородская область 47 12,5% 50 13,4% 53 14,3% Брянская область 59 19,8% 62 20,8% 65 21,7% Владимирская область 80 24,2% 84 25,3% 88 26,3% Волгоградская область 57 12,4% 63 13,7% 68 14,8% Вологодская область 43 15,5% 46 16,5% 49 17,6% Воронежская область 79 28,5% 84 30,2% 88 31,7% Еврейская автономная область 4 7,6% 5 4,1% 5 9,0% Забайкальский край 17 10,1% 19 11,5% 21 12,7% Ивановская область 52 84,1% 56 89,8% 59 95,5% Иркутская область 104 18,1% 109 18,9% 113 19,7% Кабардино-Балкарская Республика 33 349,2% 35 337,9% 37 355,4% Калининградская область 27 12,8% 29 13,8% 30 14,5% Калужская область 37 15,9% 39 16,4% 40 17,1% Камчатский край 18 33,5% 18 35,3% 19 37,1% Карачаево-Черкесская Республика 17 40,5% 18 42,4% 18 43,3% Кемеровская область 136 26,7% 144 29,3% 152 32,1% Кировская область 58 18,3% 62 16,9% 66 21,7% Костромская область 42 28,5% 45 30,2% 47 31,9% Краснодарский край 121 13,2% 131 14,5% 142 15,5% Красноярский край 102 89,0% 107 93,5% 112 97,8% Курганская область 44 26,7% 47 28,3% 49 29,8% Курская область 45 11,6% 47 10,6% 49 8,9% Ленинградская область 60 20,8% 65 22,2% 69 11,4% Липецкая область 47 20,4% 50 21,7% 53 23,0% Магаданская область 3 11,8% 3 13,9% 4 16,9% Московская область 192 12,3% 203 13,0% 215 13,7% Мурманская область 40 26,8% 42 28,1% 44 29,4% Ненецкий автономный округ 1 15,3% 1 17,0% 1 18,6% Нижегородская область 130 15,7% 142 17,0% 153 18,4% Новгородская область 16 7,8% 17 8,9% 19 10,2% Новосибирская область 110 13,7% 114 14,2% 118 14,7% Омская область 85 80,1% 91 87,9% 98 92,3% Оренбургская область 76 19,4% 80 20,4% 84 21,4% Орловская область 37 18,8% 39 20,2% 42 21,5% Пензенская область 61 27,3% 66 29,2% 70 31,4% Пермский край 101 19,4% 109 21,0% 117 22,6% Приморский край 86 17,1% 89 11,7% 92 10,9% Псковская область 26 11,1% 28 12,0% 31 13,0% Республика Адыгея 20 17,7% 22 18,7% 23 19,8% Республика Алтай 3 12,7% 4 14,2% 4 15,7% Республика Башкортостан 142 22,2% 150 23,3% 157 24,4% 140 140 ПЕРЕДОВАЯ СТАТЬЯ КЛИНИКА И ФАРМАКОТЕРАПИЯ Таблица 5. Примечание: * — при применении тикагрелора в составе ДАТ с АСК по сравнению с ДАТ клопидогрелом и АСК и антитромбоцитарной терапией АСК, ** — ЦП — целевой показатель. ение: ЦП — целевой показатель. Результаты на достижение ЦП “снижение смертности от БСК” в 2021-2023гг в регионах РФ 141 141 Российский кардиологический журнал 2020; 25 (12) в целях обеспечения достижения целей, показателей и результатов ФП “Борьба с ССЗ” согласно перечню ЛП, утвержденному специальным приказом МЗ РФ. Данный перечень содержит 23 ЛП. Таким образом, настоящая работа представляет пример оценки влия- ния применения препаратов из Перечня на достиже- ние ЦП ФП “Борьба с ССЗ” на временном отрезке от 1 до 3-х лет. лечения рассматривались взрослые российские па- циенты с подтвержденным диагнозом ОКС, которым показана ДАТ, во второй и третий годы — лечение продолжали только пациенты высокого коронарного риска. Таким образом, применение тикагрелора, по сравнению с другой антитромбоцитарной терапией для лечения пациентов с ОКС, позволит обеспечить прирост в достижении ЦП “снижение смертности от БСК” ФП “Борьба с ССЗ” в РФ: Выбор препарата для конкретного пациента ос- новывается на положениях соответствующих клини- ческих рекомендаций и видении клинической ситу- ации лечащим врачом. Однако на уровне субъектов РФ, в рамках реализации ФП с выделенным государ- ственным финансированием лекарственного обе- спечения, при выборе подходов к лекарственному обеспечению пациентов, перенесших ИМ или коро- нарное вмешательство, и пациентов группы высоко- го коронарного риска, представляется целесообраз- ным руководствоваться также целями ФП “Борьба с ССЗ”. Такой подход позволит обеспечить целевое расходование выделяемых на лекарственное обеспе- чение средств, а также будет способствовать скорей- шему достижению целей и показателей ФП в субъек- тах РФ. — в 2021г — на 18,4% (дополнительно предотвра- тить 5389 смертей по причине БСК), — в 2022г — на 19,4% (дополнительно предотвра- тить 5702 смертей по причине БСК), — в 2023г — на 20,5% (дополнительно предот- вратить 6009 смертей по причине БСК). Кроме того, применение тикагрелора в составе ДАТ для лечения пациентов с ОКС по сравнению с клопидогрелом, также позволит обеспечить прирост в достижении ЦП “снижение смертности от ИМ” ФП “Борьба с ССЗ” в РФ: — в 2021г — на 50,2% (дополнительно предотвра- тить 957 смертей от ИМ), — в 2022г — на 49,3% (дополнительно предотвра- тить 1013 смертей от ИМ), В данном исследовании, втором из серии научных работ, мы продолжаем применять подход, базирую- щийся на сравнительной оценке влияния клиниче- ской эффективности лекарственных альтернатив на показатели смертности от БСК. Рациональным пред- ставляется выбор препарата, способного внести бо- лее значимый вклад в достижение ЦП ФП “Борьба с ССЗ”. Результаты Результаты предшествующего исследования показали существенное влияние применения ти- кагрелора в составе ДАТ как на ЦП по снижению смертности от БСК (25%), так и на ЦП по снижению смертности от повторного ИМ (63%) на временном промежутке в 1 год. В настоящей работе представлена оценка влияния применения препаратов из Перечня на достижение ЦП ФП “Борьба с ССЗ” на времен- ном отрезке от 1 до 3 лет, и продемонстрирована пре- емственность полученного ранее результата. В группе антиагрегантов, включенных в Перечень, проведен- ная оценка влияния тикагрелора в сравнении с кло- пидогрелом на достижение ЦП продемонстрировала, что применение тикагрелора в составе ДАТ для лече- ния пациентов с ОКС в течение года позволит: — в 2023г — на 51,9% (дополнительно предотвра- тить 1067 смертей от ИМ). Расчеты влияния тикагрелора у пациентов с ОКС на достижение ЦП “снижение смертности от БСК” и “снижение смертности от ИМ” ФП “Борьба с ССЗ” в 2021-2023гг для каждого региона в отдельности приведены в таблицах 5 и 6, соответственно. Анализ показал существенные различия между регионами по величине эффекта применения тика- грелора на выполнение ЦП “снижение смертности от БСК” и “снижение смертности от ИМ”, что объяс- няется различиями в эпидемиологической ситуации, базовых и плановых значениях показателей, опреде- ленных для каждого региона в паспорте ФП “Борьба с ССЗ”. В рамках данного исследования для ряда ре- гионов было продемонстрировано достижение ЦП “снижение смертности от БСК” и “снижение смерт- ности от ИМ” свыше 100%. Это означает, что приме- нение ДАТ с тикагрелором в течение года у пациентов с ОКС позволит предотвратить больше смертей, чем требуется согласно паспорту ФП “Борьба с ССЗ” для достижения соответствующих ЦП. — в 2021г дополнительно предотвратить 5389 смер- тей по причине БСК и 957 смертей от ИМ, что обеспе- чит прирост в достижении ЦП “снижение смертности от БСК” и “снижение смертности от ИМ” ФП “Борьба с ССЗ” на 18,4% и 50,2%, соответственно, Обсуждение Постановление правительства № 1569 “О вне- сении изменений в государственную программу Российской Федерации “Развитие здравоохранения” открывает возможность обеспечить пациентов вы- сокого риска, перенесших ОНМК, ИМ или некото- рые виды оперативных вмешательств на сердце, ЛП для вторичной профилактики ССО и заболеваний, — в 2022г дополнительно предотвратить 5702 смер- тей по причине БСК и 1013 смертей от ИМ, что обе- спечит прирост в достижении ЦП “снижение смерт- ности от БСК” и “снижение смертности от ИМ” ФП “Борьба с ССЗ” на 19,4% и 49,3%, соответственно, 142 142 ПЕРЕДОВАЯ СТАТЬЯ КЛИНИКА И ФАРМАКОТЕРАПИЯ ние: ЦП — целевой показатель. Примечание: * — при применении тикагрелора в составе ДАТ с АСК по сравнению с ДАТ клопидогрелом и АСК и антитромбоцитарной терапией АСК, ** — ЦП — целевой показатель. Таблица 6 Т Оценка влияния применения тикагрелора у пациентов с ОКС на достижение целевого показателя “снижение смертности от ИМ” в 2021-2023гг в регионах РФ на достижение целевого показателя снижение смертности от ИМ в 2021-2023гг в регионах РФ Регион 2021 2022 2023 Количество смертей, которое возможно дополнительно предотвратить* % достижения ЦП** при применении тикагрелора Количество смертей, которое возможно дополнительно предотвратить* % достижения ЦП при применении тикагрелора Количество смертей, которое возможно дополнительно предотвратить* % достижения ЦП при применении тикагрелора Российская Федерация 957 50,2% 1013 49,3% 1067 51,9% Алтайский край 15 59,1% 16 59,1% 18 69,7% Амурская область 7 76,1% 7 73,1% 7 83,4% Архангельская область 6 36,1% 7 36,7% 7 41,8% Астраханская область 5 27,2% 5 29,7% 6 32,2% Белгородская область 8 67,1% 9 71,9% 9 68,1% Брянская область 10 54,9% 11 54,2% 11 60,2% Владимирская область 14 65,7% 15 73,3% 16 76,4% Волгоградская область 10 21,5% 11 23,6% 12 24,3% Вологодская область 8 32,7% 8 34,9% 9 39,1% Воронежская область 14 85,9% 15 79,9% 16 84,5% Еврейская автономная область 1 29,5% 1 32,4% 1 35,1% Забайкальский край 3 17,7% 3 18,8% 4 22,2% Ивановская область 9 102,8% 10 109,9% 10 131,4% Иркутская область 18 51,5% 19 50,4% 20 52,4% Кабардино-Балкарская Республика 6 136,4% 6 144,0% 7 151,5% Калининградская область 5 60,2% 5 63,1% 5 66,0% Калужская область 7 36,7% 7 38,2% 7 39,6% Камчатский край 3 70,9% 3 74,8% 3 73,4% Карачаево-Черкесская Республика 3 163,7% 3 135,4% 3 174,8% Кемеровская область 24 56,9% 26 56,6% 27 63,3% Кировская область 10 62,5% 11 66,9% 12 66,2% Костромская область 7 56,2% 8 56,8% 8 59,8% Краснодарский край 21 31,4% 23 34,2% 25 37,0% Красноярский край 18 24,3% 19 25,5% 20 25,7% Курганская область 8 79,1% 8 83,8% 9 96,4% Курская область 8 42,4% 8 47,2% 9 46,4% Ленинградская область 11 43,9% 11 47,0% 12 50,0% Липецкая область 8 73,8% 9 78,4% 9 75,5% Магаданская область 1 17,3% 1 20,8% 1 23,4% Московская область 34 26,0% 36 29,3% 38 31,0% Мурманская область 7 73,2% 7 71,3% 8 74,5% Ненецкий автономный округ 0 19,0% 0 21,1% 0 23,2% Нижегородская область 23 55,6% 25 56,2% 27 65,3% Новгородская область 3 28,9% 3 34,5% 3 35,8% Новосибирская область 20 58,3% 20 60,4% 21 62,4% Омская область 15 48,9% 16 52,7% 17 56,3% Оренбургская область 13 43,1% 14 42,6% 15 47,4% Орловская область 7 40,4% 7 43,2% 7 46,0% Пензенская область 11 59,2% 12 63,7% 12 68,1% Пермский край 18 53,1% 19 57,4% 21 66,8% Приморский край 15 25,2% 16 25,2% 16 26,8% Псковская область 5 27,3% 5 29,7% 5 30,9% Республика Адыгея 4 49,0% 4 55,4% 4 54,8% Республика Алтай 1 39,5% 1 44,3% 1 48,9% Республика Башкортостан 25 89,4% 27 109,6% 28 98,5% 143 143 Российский кардиологический журнал 2020; 25 (12) Сокращение: ЦП — целевой показатель. Таблица 6 Табл Оценка влияния применения тикагрелора у пациентов с ОКС на достижение целевого показателя “снижение смертности от ИМ” в 2021-2023гг в регионах РФ Регион 2021 2022 2023 Количество смертей, которое возможно дополнительно предотвратить* % достижения ЦП** при применении тикагрелора Количество смертей, которое возможно дополнительно предотвратить* % достижения ЦП при применении тикагрелора Количество смертей, которое возможно дополнительно предотвратить* % достижения ЦП при применении тикагрелора Республика Бурятия 5 61,8% 5 57,5% 5 67,5% Республика Дагестан 9 137,4% 9 282,4% 9 144,9% Республика Ингушетия 3 641,0% 3 332,1% 3 686,8% Республика Калмыкия 2 76,2% 2 87,4% 2 82,7% Республика Карелия 4 47,2% 5 47,7% 5 54,6% Республика Коми 4 39,8% 4 44,1% 5 48,3% Республика Крым 10 52,3% 11 56,3% 12 60,2% Республика Марий Эл 3 46,9% 3 58,5% 3 55,4% Республика Мордовия 5 86,2% 6 90,6% 6 84,3% Республика Саха (Якутия) 5 54,0% 5 50,7% 5 52,8% Республика Северная Осетия — Алания 3 44,7% 4 42,7% 4 44,3% Республика Татарстан 26 38,6% 27 40,4% 28 42,1% Республика Тыва 1 18,9% 1 20,5% 1 24,3% Республика Хакасия 6 70,9% 6 74,3% 6 77,5% Ростовская область 29 49,1% 31 52,6% 33 52,2% Рязанская область 6 35,4% 6 36,6% 7 39,9% Самарская область 27 69,9% 28 73,7% 30 84,4% Санкт-Петербург 34 39,7% 35 38,4% 36 41,8% Саратовская область 25 64,3% 26 67,4% 27 70,5% Сахалинская область 2 22,9% 2 25,2% 2 27,4% Свердловская область 53 82,6% 55 85,7% 57 95,1% Севастополь 2 39,6% 2 42,7% 2 45,8% Смоленская область 4 27,5% 4 30,4% 5 33,3% Ставропольский край 26 62,8% 28 66,0% 29 69,1% Тамбовская область 6 44,7% 7 45,2% 7 52,2% Тверская область 11 38,0% 12 43,1% 13 44,4% Томская область 7 27,7% 7 27,7% 8 28,7% Тульская область 9 52,9% 10 52,1% 11 60,0% Тюменская область без автономных округов 8 61,4% 9 73,3% 10 68,9% Удмуртская Республика 12 86,3% 12 82,3% 13 86,8% Ульяновская область 10 32,9% 10 36,5% 11 38,6% Хабаровский край 10 46,4% 11 45,8% 11 50,5% Ханты-Мансийский автономный округ — Югра 11 128,5% 11 169,8% 12 143,0% Челябинская область 27 49,2% 29 51,9% 30 51,2% Чеченская Республика 4 64,9% 4 67,6% 4 56,3% Чувашская Республика 7 44,6% 7 47,4% 7 50,3% Чукотский автономный округ 0 12,8% 0 14,5% 0 15,2% Ямало-Ненецкий автономный округ 2 49,4% 2 52,4% 2 55,3% Ярославская область 7 34,8% 8 35,9% 8 41,4% Москва 62 61,0% 65 56,6% 67 59,0% Примечание: * — при применении тикагрелора в составе ДАТ с АСК по сравнению с ДАТ клопидогрелом и АСК и антитромбоцитарной терапией АСК, ** — ЦП — целевой показатель на достижение целевого показателя снижение смертности от ИМ в 2021 2023гг в регионах РФ 144 144 ПЕРЕДОВАЯ СТАТЬЯ КЛИНИКА И ФАРМАКОТЕРАПИЯ кагрелором при лечении данных пациентов. Заключение Результаты настоящего исследования проде- монстрировали значительный потенциал приме- нения тикагрелора в составе ДАТ для вторичной профилактики ССЗ у пациентов высокого риска с целью снижения смертности и достижения ЦП ФП “Борьба с ССЗ”. В частности, терапия тикагре- лором в составе ДАТ пациентов высокого риска на срок >1 года от коронарного события оказала су- щественное влияние на прирост ЦП по снижению смертности от БСК и в особенности ЦП по сни- жению смертности от повторного ИМ, что свиде- тельствует о важности непрерывности терапии ти- Отношения и деятельность. Исследование выпол- нено при поддержке компании АстраЗенека. Отношения и деятельность. Исследование выпол- нено при поддержке компании АстраЗенека. Таблица 6 Таким образом, применение тикагрелора у пациентов с ОКС будет значимо способствовать достижению целей как ФП “Борьба с ССЗ”, так и национально- го проекта “Здравоохранение” в целом. Препарат тикагрелор может быть рекомендован для макси- мально широкого применения в рамках реализации программы “Развитие здравоохранения” в субъек- тах РФ, лечения пациентов с ОКС в рамках терри- ториальных программ государственных гарантий бесплатного оказания гражданам медицинской помощи, а также для лечения пациентов высокого ишемического риска, продолжающих терапию пос ле года от коронарного события в рамках регио нальных программ льготного лекарственного обе- спечения. — в 2023г дополнительно предотвратить 6009 смер- тей по причине БСК и 1067 смертей от ИМ, что обе- спечит прирост в достижении ЦП “снижение смерт- ности от БСК” и “снижение смертности от ИМ” ФП “Борьба с ССЗ” на 20,5% и 51,9%, соответственно. Литература/References https://www.rosminzdrav.ru/poleznye-resursy/natsproektzdravoohranenie/bssz. 5. Data from the Central research Institute for health organization and Informatization of the Ministry of health of the Russian Federation. (In Russ.) Данные Центрального научно- исследовательского института организации и информатизации здравоохранения Министерства здравоохранения Российской Федерации. https://box.almazovcentre. ru/index.php/s/EYpf3hPX5OrViHq?fbclid=IwAR3jBOaHp-tf3P13ztI7uUBAhjo1Jem6MYsF Z9RubSQbegl1wgrqp5uDVfs. 4. Passport of the Federal project “Fight against cardiovascular diseases”. (In Russ.) Паспорт федерального проекта “Борьба с сердечно-сосудистыми заболеваниями”. 2019. https://www.rosminzdrav.ru/poleznye-resursy/natsproektzdravoohranenie/bssz. 5. Data from the Central research Institute for health organization and Informatization of the Ministry of health of the Russian Federation. (In Russ.) Данные Центрального научно- исследовательского института организации и информатизации здравоохранения Министерства здравоохранения Российской Федерации. https://box.almazovcentre. ru/index.php/s/EYpf3hPX5OrViHq?fbclid=IwAR3jBOaHp-tf3P13ztI7uUBAhjo1Jem6MYsF Z9RubSQbegl1wgrqp5uDVfs. 12. 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https://openalex.org/W1987952892	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0081553&type=printable	English	null	Reconstructing Coherent Networks from Electroencephalography and Magnetoencephalography with Reduced Contamination from Volume Conduction or Magnetic Field Spread	PloS one	2,013	cc-by	14,869	Abstract Funding: Funding was provided by the University of Manchester School of Psychological Sciences and the University of Manchester Alumni Fund. 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: drakesmithm@cardiff.ac.uk Introduction propose a physiologically valid method to reduce the effects of VC and MFS in estimating coherent source networks. Before introducing the proposed method, we review coherence as a method of inferring functional connectivity and dynamic imaging of coherent sources (DICS) [2] as a method of estimating coherence between reconstructed sources. The issues of VC and MFS are discussed further along with the obstacles they present to uncovering true source level coherences, as well as previous attempts to solve the problem. As cognitive function arises from the dynamic interaction between brain regions, there is an increasing interest in moving cognitive brain imaging beyond the identification of anatomical loci of functional processes, to the detection of the underlying functionally connected networks. Functional relationships between brain regions can be inferred from correlated haemodynamic or electrophysiological signals. The shortcomings of using the haemodynamic response as the basis of detecting correlated regions due to poor temporal resolution have been documented [3]. The millisecond temporal resolution of electroencephalogra- phy (EEG) and magnetoencephalography (MEG) should make them ideal tools to measure functional connectivity, since the time scales of neural interaction are also of this order. However, two challenges arise from using scalp recording to infer interacting networks: Volume conduction (VC) and magnetic field spread (MFS) cause smearing of the effect of the neural generators at the surface and result in poor spatial resolution. Further, VC and MFS introduce spurious and erroneous correlation in the recorded signals such that the estimated networks are inaccurate. Here we Mark Drakesmith1,2, Wael El-Deredy1, Stephen Welbourne1 Mark Drakesmith1,2, Wael El-Deredy1, Stephen Welbourne1 1 School of Psychological Sciences, University of Manchester, Manchester, United Kingdom, 2 Cardiff University Brain Imaging Researc Cardiff, United Kingdom 1 School of Psychological Sciences, University of Manchester, Manchester, United Kingdom, 2 Cardiff University Brain Imaging Research Centre (CUBRIC), Cardiff University, Cardiff, United Kingdom Abstract Volume conduction (VC) and magnetic field spread (MFS) induce spurious correlations between EEG/MEG sensors, such that the estimation of functional networks from scalp recordings is inaccurate. Imaginary coherency [1] reduces VC/MFS artefacts between sensors by assuming that instantaneous interactions are caused predominantly by VC/MFS and do not contribute to the imaginary part of the cross-spectral densities (CSDs). We propose an adaptation of the dynamic imaging of coherent sources (DICS) [2] - a method for reconstructing the CSDs between sources, and subsequently inferring functional connectivity based on coherences between those sources. Firstly, we reformulate the principle of imaginary coherency by performing an eigenvector decomposition of the imaginary part of the CSD to estimate the power that only contributes to the non-zero phase-lagged (NZPL) interactions. Secondly, we construct an NZPL-optimised spatial filter with two a priori assumptions: (1) that only NZPL interactions exist at the source level and (2) the NZPL CSD at the sensor level is a good approximation of the projected source NZPL CSDs. We compare the performance of the NZPL method to the standard method by reconstructing a coherent network from simulated EEG/MEG recordings. We demonstrate that, as long as there are phase differences between the sources, the NZPL method reliably detects the underlying networks from EEG and MEG. We show that the method is also robust to very small phase lags, noise from phase jitter, and is less sensitive to regularisation parameters. The method is applied to a human dataset to infer parts of a coherent network underpinning face recognition. ation: Drakesmith M, El-Deredy W, Welbourne S (2013) Reconstructing Coherent Networks from Electroencephalography and Magneto duced Contamination from Volume Conduction or Magnetic Field Spread. PLoS ONE 8(12): e81553. doi:10.1371/journal.pone.0081553 Editor: Gareth Robert Barnes, University College of London - Institute of Neurology, United Kingdom Editor: Gareth Robert Barnes, University College of London - Institute of Neurology, United Kingdom Editor: Gareth Robert Barnes, University College of London - Institute of Neurology, United Kingdom Received October 15, 2012; Accepted October 21, 2013; Published December 2, 2013 Received October 15, 2012; Accepted October 21, 2013; Published December 2, 2013 Copyright: 2013 Drakesmith 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. Dynamic Imaging of Coherent Sources y g g Dynamic imaging of coherent sources (DICS) [2] is a beamformer technique that operates under the same principle as LCMV, but with two principle differences: (1) The covariance matrix in the calculation of the spatial filter is replaced by the sensor-level CSD matrix. (2) The filter is applied to the sensor-level CSD to reconstruct the source-level CSDs of all combinations of pairwise voxels. From these the source-level coherences between sources can be estimated. Thus DICS differs from other beamforming methods in that it directly estimates the interactions between sources as well as their individual powers. Another issue concerning beamformer methods is that of regularisation. The spatial filter requires regularisation to prevent overfitting. Higher regularisation is preferred to reduce the chance of false positives, but this results in the smoothing of sources. Lower regularisation allows for more focal sources to be reconstructed, but is more prone to false positives [10,11]. This is described mathematically in the theory section. DICS is advantageous over separate source-reconstruction and functional connectivity combinations because functional interac- tions are reconstructed as source pairs. The assumptions underlying the localisation and the estimation of coherence of source pairs are the same. This reduces the confounds of systematic bias on the coherence estimates. Another significant benefit is the massive reduction in computational demands. The prospect of whole-brain source-level connectivity inference is more computationally tractable when the step of reconstructing the source-level time-series is bypassed. A common application of the beamformer technique is to estimate the source-level time-series. This type of approach is often referred to as ‘‘virtual electrode’’ methods, because they can be conceptualised as placing virtual electrodes into voxels. The most obvious method of investigating functional connectivity at the source level would be to estimate time-series for pairs of voxels and measure the functional connectivity between them. While superficially very attractive, this approach needs to be treated with some caution when used in the context of functional connectivity estimation, because uncertainties in the reliability of the reconstructions can give rise to systematic errors, which will contaminate subsequent connectivity estimates [12]. Our prefer- ence is to use DICS, a modified beamformer method (described later), which allows for coherence based source connectivity estimates to be computed in a single step. A full description of DICS is given in [2,10]. Dynamic Imaging of Coherent Sources DICS has been applied to MEG data to investigate coherence in a number of cognitive phenomena such as reading [20], motor control [21] and binocular rivalry [22]. For EEG, there are some recent examples of applying DICS to EEG data [23,24] although these did not reconstruct cortico-cortical coherence, but rather sources coherent with an external EMG signal. To our knowledge, only one study has successfully inferred cortico-cortical network using DICS with EEG recordings [25]. They implemented a variation of the standard DICS method where the real-valued spatial filter in the direction of maximum variance was used to estimate source CSDs. Generally, VC limits the applicability of DICS to EEG as detailed below. In it’s current form, DICS has no means of reducing artefactual connectivity that arises due to VC. The present study is the first to use DICS to infer cortico-cortical connections using EEG, by explicitly minimising the effects of VC. Coherence Coherence is a statistic that often increases when activity in neural assemblies is functionally synchronised and as such can be taken as a measure of functional connectivity in electrophysiolog- ical data (see [13] for a review). Coherence is thought to be the mechanism by which percepts are bound together: The ‘‘binding hypothesis’’ [14,15]. Electrophysiological studies recording multi- unit activity and local field potentials have illustrated that coherence accurately reflects both intra-cortical and inter-cortical communication (see [16–18], for reviews). Volume Conduction (VC) and Magnetic Field Spread (MFS) Coherent Networks from EEG/MEG without VC/MSF solution [6]. Solutions such as the linearly constrained minimum variance (LCMV) beamformer [7] do not explicitly try to solve the inverse problem, unlike dipole fitting methods [8], but instead rely on spatial filters that weight the estimated sources as a function of the covariance matrix of the time-series. A spatial filter is constructed for each source point (voxel) such that the variance of the total source power is minimal while keeping the output of the filtered lead field constant. This maximises the beamformer output for the target source while contributions from other sources are attenuated (however signals from strong, nearby sources may subdue output from the target source). Beamformers have been shown to be a useful method for identifying EEG/MEG sources (see [9] for a review). However, the fact that beamformers assume that distinct sources are uncorrelated may pose problems when trying to infer functional connectivity between reconstructed sources. This issue will be addressed in more detail in the discussion. manifests. This provides a strong motivation for looking at connectivity at the source-level. Using the ‘virtual-electrode’ method described above is an obvious means of achieving this. This however is computationally demanding and can suffer a number of pitfalls detailed in the sections below. Beamformers as a Solution to the Inverse Problem Overcoming the poor spatial resolution of EEG/MEG has been the focus of a great deal of research (see [4,5] for reviews). This generally involves the calculation of a linear forward solution, or lead field, which describes the transformation of the signal from the neural generators to the surface sensors. This is followed by an attempt to reverse the calculation to solve its inverse. The inverse solution is problematic as it attempts to describe a complex dynamical system from a relatively small number of observations. The inverse problem is described as ‘ill-posed’ as it has no unique December 2013 \| Volume 8 \| Issue 12 \| e81553 1 PLOS ONE \| www.plosone.org Coherent Networks from EEG/MEG without VC/MSF December 2013 \| Volume 8 \| Issue 12 \| e81553 Coherent Networks from EEG/MEG without VC/MSF examining source dynamics with imaginary coherency is that of Marzetti et al [41]. Here, the imaginary CSD is approximated by a model in a modified principle component analysis (PCA) technique to separate contributions of interacting sources. This is a qualitatively different approach to beamformer, as it does not scan each source (or source pair) independently for their contribution to the sensor data. Other recent studies [42–44] have adopted a two-step approach to the problem, by estimating source level time-series using the virtual electrode method and then computing imaginary coherency between the estimated source-level time-series. This approach reduces spurious interac- tions that arises from smoothing of the source space inherent in source localisation algorithms. This approach suffers the same limitations as other connectivity methods based on reconstructed source time-series. It also requires additional processing time due to the separate generation of source time-series and coherence calculations. This makes it unfeasible to do whole-brain network analysis [45] As a result a priori selection of regions of interest (ROIs) has to be performed which can be subjective. In addition to these drawbacks, VC/MFS artefacts at the sensor-level, if not accurately modelled, can lead to source mislocalisations [29,32]. While computing imaginary coherency from source-level time- series will reduce artefactual connectivity estimates, it can not resolve any effects VC/MFS may be projected to the source space. To reduce the likelihood of these errors, it would be more prudent to remove VC/MFS artefacts at the sensor-level prior to source reconstruction. have very large effects. For example, even very small holes (less than 1 mm) in the skull can drastically alter the flow of volume currents [31], resulting in inaccurate localisation of sources as well as false positives when reconstructing source-level coherence. MEG is generally considered less susceptible to VC [27,28,32]. Secondary volume currents can theoretically induce equivalent magnetic fields confounding the primary neuromagnetic fields; however, these effects are negligible in comparison to EEG [33]. Head tissues are permeable to magnetic fields, so the neuromag- netic fields are less dependent on anatomy than EEG. However, due to the effects of magnetic field spread (MFS), artefacts can still arise. These artefacts are maximal over short distances but can extend over large areas of the topographic surface, and therefore can manifest even at long-range connections. Unlike VC, however, MFS is not dependent on specific tissue conductivities. Source localisation can theoretically be attained by simple inversion of the Biot-Savart law. Coherent Networks from EEG/MEG without VC/MSF A volume conductor model is still necessary to account for the impressed currents of a dipole [34]. A basic model-free method of overcoming the problem of VC in EEG in determining surface coherences is to take the Laplacian of the surface potentials [35,36]. This acts as a high-pass spatial filter, emphasizing sources at smaller spatial scales. This method is heavily dependent on the spatial distances between coherent sources. As such, valid, short-range coherences may be removed while erroneous, long-range volume currents may be retained. Imaginary coherency (described below) offers an improvement on this providing a non-spatially dependent way of removing VC. Another approach to overcoming VC/MFS at the sensor-level is the phase lag index (PLI) [46]. This attempts to overcome the issue with imaginary coherency that it is sensitive to the size of the phase lag as well as the strength of coupling. For example, if two interacting pairs of sources have equal coupling strength but one has a larger phase difference than the other, imaginary coherency will be biased towards the connection with the larger phase lag. PLI overcomes this issue by measuring the asymmetry of the distribution of phase lags, as symmetrical phase lag distributions are more likely to arise where the phase lag is at 0 or p. As a result, this measure is less sensitive to the degree of phase lag, only the presence of phase lag. So far, one study has extended this principle to infer connectivity between virtual electrode time-series [47]. Imaginary Coherency Nolte et al [1] proposed the concept of imaginary coherency as a model-free way of dealing with VC artefacts when calculating coherence at the sensor level (this is detailed in the theory section below). It is based on two critical assumptions: Firstly, that true neural interactions must have some phase lag and, secondly, that VC coherences are always instantaneous with zero phase lag. The first assumption can be justified in that even when studies describe phase lags between populations as instantaneous, there is still a phase lag of a few milliseconds or even microseconds [37]. There may be chance instances of phase difference too small to give a meaningful imaginary component, but if the CSD is calculated for a sufficiently long time-series, or is averaged over a sufficient number of event-related epochs, this effect will be negligible. There is also the possibility of sustained reciprocal interactions where there is zero phase lag and the phase lag does not vary at all over time. Imaginary coherence would not be able to detect this type of interaction (this issue is elaborated on further in the discussion). Volume Conduction (VC) and Magnetic Field Spread (MFS) ( ) Source reconstruction is generally poorer for EEG than MEG due to the low conductivity of the skull, which leads to attenuation and spatial smearing of source currents. A more specific confound exists with regards to coherence, in that the brain tissue, being highly conductive in comparison to the skull, can result in currents being conducted to distal electrodes [26,27]. The current flow is highly dependant on anatomical factors, including structural discontinuities in the skull, lesions [28], and anisotropic conduc- tivity of white matter [29]. Although these currents can be modelled during source analysis, EEG sensors are generally maximally sensitive to the region directly beneath the electrode. Therefore, additional contribution to the EEG signal from distal sources will significantly impact on the mapping of these signals to source space. The calculation of the lead field requires precise tissue segmentation and assigning accurate conductivity values to each compartment, using boundary element method (BEM) or more recently, finite element method (FEM) models (see [30] for a review). This introduces several levels of potential errors that can In practice, coherence describes how closely related the spectral densities of two signals are, and so is equivalent to a correlation coefficient in the frequency domain. Coherence is the absolute value of coherency, the complex-valued ratio between the cross- spectral density (CSD) and the individual auto-spectra (or power) of two signals, i.e. a covariance matrix in the frequency domain. This can be calculated by Fourier transformation or wavelet convolution of the cross-covariance of the two time-series [19]. Since the ratio between the individual auto-spectra and the cross- spectra are complex, they embody both correlation of power amplitude and phase synchrony of the signals. While coherence analysis is simple and physiologically-valid, the interpretation of its spatial characteristics at the sensor level is difficult, due to the irregular way in which the source activity December 2013 \| Volume 8 \| Issue 12 \| e81553 PLOS ONE \| www.plosone.org December 2013 \| Volume 8 \| Issue 12 \| e81553 2 Coherent Networks from EEG/MEG without VC/MSF PLOS ONE \| www.plosone.org Imaginary Coherency This section describes the motivation for using the imaginary coherency [1] to quantify true neural interactions from sensor- level time-series. The Fourier transformed sensor time-series p from sensor i, is given by: ~Yk~XWk ð2Þ pi f ð Þ~ x i tð Þe{2pitf dt ð7Þ pi f ð Þ~ x i tð Þe{2pitf dt ð7Þ ð7Þ ð2Þ In the LCMV and DICS beamformers, Wk is estimated by solving the constrained minimisation problem: For simplicity and clarity, the notation f is removed from here on; all variables are still functions of frequency. i denotes the imaginary unit. As per the convolution theorem, the cross-spectra can be calculated directly from the complex conjugate product of the two Fourier transformed signals. These are averaged across a number, ne, of epochs to estimate the true cross-spectral density (CSD) matrix. Wk~ min Wk E Wkp j j2 n o zc Wk j j2 h i , subject to WT k Lk~I3 ð3Þ Where E denotes the expected value, p is the Fourier transformed data and c is the Tikhonov regularisation parameter. cij~Spip j Tne ð8Þ ð8Þ The solution to this is given by [7]: The solution to this is given by [7]: The complex coherency function between two signals i and j is the ratio of the cross-spectra to the auto-spectra: Wk~ LT kC{1 X Lk {1LkC{1 X ð4Þ ð4Þ mij~ cij ﬃﬃﬃﬃﬃﬃﬃﬃﬃ ciicjj p ð9Þ For DICS, the spatial filter for two source points are multiplied with the sensor level CSD (sCSD), CX to create a 363 matrix of reconstructed source CSDs (rCSD) between the 3 Cartesian components of sources k and l. ð9Þ The absolute value of which is usually taken as a measure of coherence, while the imaginary part is a measure of the phase- lagged coherence. The Fourier transformed data can be expressed in polar form: ~CYkl~WT kCXWl ð5Þ ð5Þ The highest singular value of this matrix is treated as the amplitude of the rCSD between k and l [2] when this is significantly larger than the next singular value. Otherwise, the trace of the matrix is taken. Source Reconstruction All reconstruction methods estimate the solution to the inverse problem: X~ X k LkYT k ð1Þ ð1Þ The lead field, Lk, is an ns63 matrix, which describes the contribution of source k to the sensors in 3 directions in Cartesian space where ns is the number of sensors. X is the sensor data in the time or frequency domain. The beamformer method estimates a solution to the inverse problem by using a spatial filter Wk to create an estimate of the source activity ~Y. Theory of sources. More focal sources are favoured by low regularisation, but risks introducing false positives, while sources that are more diffuse are favoured by high regularisation, but risks making false negatives. Larger a, therefore reduces the acuity of the spatial filter, so given data of sufficient quality, lower regularisation parameters are preferred. Until recently there was no robust benchmark for choosing an appropriate regularisation for spatial filtering. However, there is a recent method for estimating the optimal regularisation parameter from the condition number of the matrix [11]. Aims This study proposes an adaptation of the DICS method, in order to reduce the effect of VC and MFS artefacts in reconstructing source-level coherent networks, and therefore improve recovery of source-level coherences. In contrast to the methods cited in the section above, this will allow source-level CSDs to be computed directly, making the process more computationally efficient and negating the potential pitfalls of a separate stage of reconstructing the source-level time series. Our approach is two-fold. First, we reformulate the approach of imaginary coherency to increase its sensitivity, by constructing a non-zero phase-lagged (NZPL) CSD matrix. Secondly, we use the same approach of DICS to solve a problem analogous to the standard M/EEG inverse problem, with an additional a priori assumption that true sources have non-zero phase lags. The second assumption is valid given that VC can be described by the quasi-static approximation of Maxwell’s equations [38]. The approximation describes the dynamics of VC without time- derivatives: They are treated as effectively instantaneous and hence do not contribute any phase lag. There is experimental evidence that the approximation is justified for frequencies below 1 kHz [39], which is well within the frequency range typically analysed in human EEG/MEG. The theory for the NZPL CSD and its application to DICS is presented first, followed by the results from experimental simulations comparing network reconstruction using DICS with the full CSD to that using the NZPL CSD. Simulations were carried out to comparing the performance of these two methods at different levels of phase jitter, phase lags and regularisation parameters. Finally, the applicability of the method is illustrated on a set of real EEG recordings. Nolte et al [1] successfully used imaginary coherency to identify interhemispheric coherence between electrodes on the motor regions during finger movement. Imaginary coherency has been used in a number of other EEG studies. For example, [40] correlated imaginary coherency with certain phases of brain maturation. A few recent studies have attempted to utilise this to uncover phase-lagged coherences at the source level. A recent method for December 2013 \| Volume 8 \| Issue 12 \| e81553 PLOS ONE \| www.plosone.org 3 Coherent Networks from EEG/MEG without VC/MSF Non-Zero Phase-Lagged (NZPL) CSD and Coherence In this section we reformulate the principle of imaginary coherency to improve its sensitivity to non-zero phase lags. A term representing the power that arises due to the phase- lagged interactions can therefore be obtained by using the analogous terms from the imaginary decomposition ( q_02 1 l _ 1 ). By populating the diagonal of the imaginary CSD with this term, we obtain: A term representing the power that arises due to the phase- lagged interactions can therefore be obtained by using the l f h i i d i i ( _02 l _ ) While instantaneous and phase-lagged components can be easily separated in the off-diagonal elements, the contribution of these two components to the full power is still unknown. The existing imaginary coherency approach does not take into account the dependence of phase lag on the imaginary CSD. This results in a biased estimate of non-instantaneous interactions (see appendix S1). We therefore wish to identify the components in the power that contribute only to the imaginary part of the CSD. To demonstrate this, we consider the eigenvector decomposition of the full CSD, for each sample to give the full power, p. analogous terms from the imaginary decomposition ( q 1 l 1 ). By populating the diagonal of the imaginary CSD with this term, we obtain: C _ ~SIm pp{ z q_02 1 l _ 1 D Tne ð17Þ ð17Þ The CSD calculation in equation 8 can be expressed as an outer product of two vectors, averaged across samples: Where the superscript D indicates a vector expressed as a diagonal matrix. In this version of the CSD, a more accurate estimate of the auto-spectra contributing to the non-instantaneous interaction within that sample is used. The NZPL coherence can then be computed in the same way as standard coherence: C~Spp{Tne ð12Þ ð12Þ The decomposition of a single sample is given by: The decomposition of a single sample is given by: m _ ij~ c_ ij ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ c_ ii c_ jj q ð18Þ pp{~QLQ{1 ð13Þ ð18Þ ð13Þ Where Q = [q1…qns] is a matrix of column eigenvectors and L = [l1…lns]D is a diagonal matrix of corresponding eigenvalues. This decomposition yields one non-zero eigenvector/eigenvalue, which satisfies: This results in a more sensitive measure of non-instantaneous interactions and is less susceptible to bias from phase lag. Non-Zero Phase-Lagged (NZPL) CSD and Coherence This approach as the same advantage as the PLI method [46], however, by manipulating the CSD directly, the NZPL approach can be extended to source localisation directly, without the need to reconstruct virtual electrodes [47] (see below). p02 ~ q02 1 l1 ð14Þ ð14Þ Coherent Networks from EEG/MEG without VC/MSF Coherent Networks from EEG/MEG without VC/MSF affect the imaginary coherency (unless the amplitude is constant across all trials, which is unlikely). In the case of the symmetric real part, there are two non-zero eigenvector/eigenvalues ( q^ 1, l ^ 1and q^ 2, l ^ 2), which represent the instantaneous contributions to the power. In the case of the anti-symmetric imaginary part, there are two non-zero eigenvec- tor/eigenvalues. These eigenvectors are purely imaginary and exist as a complex conjugate pair (i.e. q_ 1~ q_ 2 and l _ 1~ l _ 2). The eigenvectors yielded by the real decomposition relate to the full power in a similar way to equation 14, by satisfying: The real and imaginary parts of coherency can, in this case, be treated as representing the proportion of the CSD with zero phase lag and that with maximal phase difference (p/2). Independent signals will lead to random wi but also small ri so these will not contribute significantly to imaginary coherency. As VC/MFS coherence is only instantaneous, the removal of the real part removes the contribution of VC/MFS (at least the first order effects) to the connectivity inferred from the coherence calcula- tions. exist as a complex conjugate pair (i.e. q_ 1~ q_ 2 and l 1~ l 2). The eigenvectors yielded by the real decomposition relate to the full power in a similar way to equation 14, by satisfying: p02 ~ q^02 1 l ^ 1 z q^02 2 l ^ 2 ð16Þ ð16Þ Imaginary Coherency The spatial filter accommodates Tikhonov regularisation by substituting CX for: pi~rieiwi ð10Þ ð10Þ Where ri is the amplitude and wi is the phase, the CSD for a single sample can be expressed as: CX?CXzcI where c~a CX j j ð6Þ ð6Þ pip j ~rirjeiDwij~rirj cos Dwijzi sin Dwij ð11Þ ð11Þ Where c is the absolute regularisation parameter, which is a multiple of the Euclidian norm of the sCSD matrix, while a is the relative regularisation [10]. This parameter prevents over-fitting in a minimum norm solution to an ill-posed problem. It adjusts the sensitivity of the spatial filter according to the expected distribution Where Dwij~wi{wj is the phase lag between signals i and j. When there is no phase lag (Dw = 0), cos Dw = 1 while sin Dw = 0 and when the lag is maximum (Dw = p/2), cos Dw = 0, while sin Dw = 1. When averaged across samples, the amplitudes rirj will also December 2013 \| Volume 8 \| Issue 12 \| e81553 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org December 2013 \| Volume 8 \| Issue 12 \| e81553 4 Coherent Networks from EEG/MEG without VC/MSF December 2013 \| Volume 8 \| Issue 12 \| e81553 Higher-Order Artefacts It should be noted that imaginary coherency, PLI and the NZPL method are restricted to removing first order artefacts, that is artefacts that arise due to spurious interactions between the true signal and its VC ‘echo’. While this interaction will be instantaneous, if there is a true (phase-lagged) interaction with a second source, this will inevitably be phase-lagged with respect to the VC echo of the first source and, therefore, produce a phase- lagged coherence. We will refer to this interaction as a second order artefact. In addition, if a VC echo of the second source arises, then there will also be phase-lagged coherence with the first VC echo. This we call a third-order artefact. The issue is represented graphically in figure 1. The superscript u2 represents the Hadamard (element-wise) square function. We can obtain an equivalent representation of the power contributing only to the imaginary part of the CSD by performing the same decomposition on the imaginary CSD: Any matrix can be uniquely decomposed into one symmetric (or Hermetian) and one anti-symmetric (or anti-Hermetian) matrix. In this case, these equate to the real and imaginary parts, respectively. These two matrices can then be decomposed further into two sets of eigenvectors/eigenvalues. While the NZPL CSD in itself does not deal with this issue, its application to spatial filtering can minimise the effects of higher order artefacts. Assuming a VC echo is always smaller in magnitude than the originating signal, the higher-order pp{~Re pp{ ziIm pp{ ~Q ^ L ^ Q ^{1 ziQ _ L _ Q _{1 ð15Þ ð15Þ PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org December 2013 \| Volume 8 \| Issue 12 \| e81553 December 2013 \| Volume 8 \| Issue 12 \| e81553 December 2013 \| Volume 8 \| Issue 12 \| e81553 5 Coherent Networks from EEG/MEG without VC/MSF Figure 1. An illustration of the effects of higher order artefacts. Contralateral 1st order artefacts are instantaneous, but there are smaller 2nd order artefacts from the true sources and the ipsilateral VC ‘source’ that are phase-lagged. In addition, there are also much weaker 3rd order artefacts between the VC ‘sources’. This is a simplified representation of the coherence patterns reported in supplementary material. doi:10.1371/journal.pone.0081553.g001 Figure 1. An illustration of the effects of higher order artefacts. Higher-Order Artefacts Contralateral 1st order artefacts are instantaneous, but there are smaller 2nd order artefacts from the true sources and the ipsilateral VC ‘source’ that are phase-lagged. In addition, there are also much weaker 3rd order artefacts between the VC ‘sources’. This is a simplified representation of the coherence patterns reported in supplementary material. doi:10.1371/journal.pone.0081553.g001 interactions will likely be small enough to be adequately suppressed by the correlation minimisation constraint of the spatial filter. This approach is detailed in the section below. C _ X~L Im CY ð ÞzS q _02 1Y l _ 1Y DTne " # LT ð21Þ ð21Þ It should be noted that while an imaginary component in the CSD in sensor space implies a phase-lagged interaction in source space, the absence of the imaginary component in sensor space does not necessarily imply the lack of a phase-lagged interaction in source space. Therefore there are many potential source interactions that will be invisible to the sensors. This issue is inherited from the general ill-posed nature of the EEG/MEG inverse problem. The projection of the imaginary cross-spectra of the formula can be derived easily from the imaginary sCSD. C _ X~Im CX ð ÞzL S q_02 1Y l _ 1Y DTne " # LT ð22Þ ð22Þ Reconstructing NZPL Sources Having established a method to more accurately identify non- instantaneous interactions, we apply the new NZPL CSD matrix to the problem of reconstructing coherent sources. We impose additional assumptions to that of the standard DICS approach (see [48–52] for similar approaches). Using the a priori assumption that truly interacting sources will always have non-zero phase lag, we create a representation of these signals as projected to the sensor level. To estimate this we need representations of both the auto- spectra and cross-spectra of these signals. However, the NZPL auto-spectra part of the equation cannot be easily derived from the sensor data as it requires a priori knowledge of the separation of instantaneous cross-talk from the real part of the phase-lagged interaction. We therefore make a second assumption that the NZPL operation applied to the sCSD will sufficiently model the projection of the NZPL source power to the sensor level. The projection of the full CSD from sources to sensors can be expressed as: C _ X&Im CX ð ÞzS q_02 1X l _ 1X DTne ð23Þ ð23Þ CX~LCYLT ð19Þ ð19Þ This formulation comes at the expense of omitting the cross-talk that inevitably arises in this projection of the auto-spectra. Our assumption is that there is sufficient information about this cross- talk in the higher-order artefacts (see above) that remain in the imaginary part of the sCSD. The imaginary part of the sCSD can be obtained using the same projection. Im CX ð Þ~LIm CY ð ÞLT ð20Þ ð20Þ This estimate of the projected source NZPL CSD is then used to compute a spatial filter optimised to recover NZPL interactions, using the same covariance minimisation constraint as per the existing DICS method. The NZPL CSD at the source level can also be projected in the same way. PLOS ONE \| www.plosone.org December 2013 \| Volume 8 \| Issue 12 \| e81553 6 Coherent Networks from EEG/MEG without VC/MSF Figure 2. Positions of sources used in all simulations. MNI coordinates: [240.5 272.5 17] and [42 272.5 17]. Colour bar indicates normalised source strength. doi:10.1371/journal.pone.0081553.g002 W _ k~ LT k C _{1 X Lk {1 Lk C _{1 X ð24Þ ð24Þ The use of the NZPL sCSD in the spatial filter calculation provides a more optimal reconstruction of the rCSD than the standard spatial filter. Reconstructing NZPL Sources The NZPL filter selectively supresses signals that do not arise from non-instantaneous interacting sources, compared to the standard filter, which treats instantaneous and non-instantaneous interactions equally. This results in more signal from non-instantaneous interacting sources being attributed to the target sources (essentially treating a pair of phase-lagged interact- ing sources as a single source). This has two advantages: (1) It will lead to a beneficial overestimation of the non-instantaneous interactions when projecting sensor data to source space. (2) It will lead to an increase in the apparent SNR of the signal, resulting in greater spatial acuity of the filter [11]. The NZPL filter is still able to supress the higher-order artefacts that remain present in the imaginary part of the sCSD. These artefacts are much weaker than the true instantaneous interactions and therefore more readily suppressed by the filter. Appendix S2 uses a simple simulation, based on more straightforward mapping between sensors and sources, to illustrate the properties and benefits of the NZPL spatial filter compared to the standard filter. Figure 2. Positions of sources used in all simulations. MNI coordinates: [240.5 272.5 17] and [42 272.5 17]. Colour bar indicates normalised source strength. doi:10.1371/journal.pone.0081553.g002 selected as nodes for the coherent network (MNI coordinates: [240.5 272.5 17] and [42 272.5 17] separated by a distance of 82.5 mm). One voxel was selected as the centre of the node and activity in that node was smoothed to surrounding voxels with a FWHM of 5 mm3 (see figure 2). Equal pre- and post- stimulus periods of 1000 ms were used. In the post-stimulus period, activity in the two occipital nodes was simulated using a 33 Hz sinusoidal wave. The two sources were synchronised with a jittered phase lag sampled from a von Mises distribution (a Gaussian distribution across a circle) with mean of 0.5p radians and a FWHM of 0.25p radians. In the pre stimulus period, noise was generated from the same oscillatory activity used in the post-stimulus period, but with a completely random phase lag sampled from a uniform circular distribution. This ensured the pre- and post- stimulus periods has the same power in the frequency band of interest. Additional Gaussian noise was superimposed on the signal with a signal-to-noise proportion (SNP) of 0.9, equivalent to an SNR of 9.54 dB, which is in the range of SNRs typically seen in evoked EEG responses [53]. Reconstructing NZPL Sources The amplitude of the total signal was constant at 1 nA. These simulations were repeated over 100 epochs. Once the NZPL spatial filter is constructed, it is straightforward to project the NZPL sCSD to source space in the same way as DICS (as per equation 5): ~CYkl~W _T k C _ XW _ l ð25Þ ð25Þ This creates an rCSD where non-instantaneous interactions are overestimated. 1st order artefacts are removed completely by the NZPL manipulation while higher-order artefacts are reduced by the minimisation constraint of the spatial filter. This approach significantly reduces the projection of artefactual VC/MFS interactions into the rCSD, improving the accuracy of the inferred connectivity. Forward Calculations Lead fields for both EEG and MEG were calculated using FieldTrip [54]. The EEG lead field was based on a VC model created using the boundary element method (BEM) [30]. The MNI template brain was segmented into brain, skull and scalp compartments defining 3 homogeneous conductive mediums with conductivity values of 33 mS/m, 0.41 mS/m and 33 mS/m, respectively. The scalp potentials were calculated using the forward solution (equation 1) and sampled by 64 scalp electrodes based on a Biosemi64 scalp electrode array with the reference electrode placed at infinity. General Method A simulation of a simple coherent network was used to test the performance of the NZPL DICS method. From this, the forward solution was used to calculate simulated EEG and MEG recordings. These were used to reconstruct a source-level coherent network using an sCSD calculated in the standard way (full sCSD) or the NZPL sCSD. A number of experimental manipulations were carried out to test the effects of phase-jitter, phase lag and regularisation on the performance of DICS in each case. A quantification of the effect of phase lag and noise on coherence computed with NZPL CSDs is also made. The MEG lead field was based on a single-shell model from a grey-matter segmentation of the same MRI template brain. The lead field was calculated using the quasi-static approximation of the magnetic forward model, described by the Biot–Savart law [34]. The forward solution was used to calculate simulated readings for 148 axial gradiometer sensors based on the configuration of a 4D MAGNES 2500 WH scanner. The sensor data for both modalities was obtained from the linear forward solution in equation 1. Example forward solutions for one simulation are shown in appendix S3. Effects of Phase Lag Method. To understand the effects of phase lag, simulations were repeated with the mean of the phase lag distribution varied. Phase lags of 0, 0.0625p, 0.125p, 0.25p, 0.5p, p, 1.5p and 2p radians were tested. Phase jitter and regularisation were fixed at FWHM = p/4 and a = 1026, respectively. Results. AUCs for all phase lags are shown in figure 6 and t- test results are summarised in table 2. AUCs for all reconstructions with the full sCSD were not significantly higher than the critical AUC. For the NZPL CSD all non-instantaneous phase lags were significantly above the critical AUC (although the AUC for 0.0625p in EEG was borderline). In EEG all instantaneous phase lags (0, p and 2p) were non significant and in MEG all instantaneous phase lags were borderline significant. 1-way ANOVAs showed there was a significant effect of phase lag in EEG (F(7,72) = 134.41, p,10238) and MEG (F(7,72) = 14.32, p,10211). The effect is much stronger in EEG than MEG. Testing for differences in between the methods all showed a significant increase in AUC for the NZPL method at all phase lags in both EEG and MEG (all significant at p,1024). Reconstruction Performance and Effects of Phase Jitter The corre- sponding log ROC curves are shown in figure 4. For the EEG full sCSD, there is very poor reconstruction of the original network in both jittered and unjittered conditions. There was improvement when using the NZPL sCSDs, which appears to provide good reconstruction of the original network in both conditions. For MEG, the full sCSD without jitter partially reconstructs the correct network but with some false positives. The jittered conditions show poor performance as with EEG. In both cases, the NZPL CSD provides a very accurate reconstruction. Source Coherences This shows that DICS with the NZPL sCSD is much more robust to phase jitter. For paired-sampled t-tests comparing the two methods, NZPL sCSD performed significantly better than the full sCSD in all cases, with the exception of unjittered MEG reconstruction (t(9) = 2.31, p = 0.023). Performance Measurement The performance of the whole-brain network reconstruction was evaluated by comparing the ‘‘true’’ matrix with the reconstructed matrix across a range of thresholds. The ‘‘true’’ matrix consisted of the signal from the active source pair smoothed out across the connection space using a Gaussian kernel with a FWHM of 5 mm3. The Gaussian-smoothed network deals with spatial inaccuracies across a gradient of distance by progressively penalising reconstruction performance as it moves further away from the centre of true activity. The noise-contrasted reconstruct- ed network was thresholded at 120 equally spaced values from the lowest to the highest values in the matrix. The true positive rate (TPR) and false positive rate (FPR) were calculated from the proportions of all sources, which are identified as true or false at the given threshold. The TPR and log FPR provided the points for the log receiver-operator characteristic (ROC) curve. The log ROC curve was used in preference to a standard ROC curve because of the large number of negative values. This was proposed as a method of scaling the ROC curve where there is a large ratio of negatives to positives, as there is here [55]. Finally, the area under the log ROC curve (AUC) was calculated by integrating the log ROC curve using the trapezium rule. The AUC measure gives a single value that quantifies the accuracy of each reconstruction. 10 runs of each experiment were carried out to provide a measure of variance for these AUC values. Reconstruction Performance and Effects of Phase Jitter Method. To assess the effects of phase jitter (i.e. the FWHM of the phase lag distribution) the simulations were repeated where the phase jitter between the two oscillatory sources was varied. FWHMs of 0, p/32, p/16, p/8, p/4, p/2 and p radians were tested. The Fast Fourier transform was applied to the full length of the time-series to obtain the complex spectra for each channel. The full sCSD, and the NZPL sCSD were calculated as per equations 12 and 17, respectively. The sCSD was averaged over the frequency band of interest (25–40 Hz) and across the 100 epochs. tested. Results. Examples of reconstructed networks for unjittered and jittered (FWHM = p/4) conditions are shown in figure 3 for each reconstruction method and imaging modality. The corre- sponding log ROC curves are shown in figure 4. For the EEG full sCSD, there is very poor reconstruction of the original network in both jittered and unjittered conditions. There was improvement when using the NZPL sCSDs, which appears to provide good reconstruction of the original network in both conditions. For MEG, the full sCSD without jitter partially reconstructs the correct network but with some false positives. The jittered conditions show poor performance as with EEG. In both cases, the NZPL CSD provides a very accurate reconstruction. The AUCs for reconstructions across all jitter FWHMs are shown in figure 5 and summarised in table 1. Single sample t-tests show that in EEG and MEG, all reconstructions using the full sCSD were not significantly above the critical AUC, with the exception of the unjittered MEG reconstruction (t(9) = 17.74, p,1027), indicating DICS with the full sCSD is very intolerant of phase jitter. In contrast, the AUCs for reconstructions using the NZPL sCSD were significantly above the critical AUC across all jitter FWHMs, with the exception of the largest FWHM (p radians), where EEG reconstruction was non-significant and MEG reconstruction was borderline significant (t(9) = 2.86, p = 0.0094). This shows that DICS with the NZPL sCSD is much more robust to phase jitter. For paired-sampled t-tests comparing the two methods, NZPL sCSD performed significantly better than the full sCSD in all cases, with the exception of unjittered MEG reconstruction (t(9) = 2.31, p = 0.023). Results. Examples of reconstructed networks for unjittered and jittered (FWHM = p/4) conditions are shown in figure 3 for each reconstruction method and imaging modality. Statistical Analysis Log ROC AUCs for each condition were tested for significant increase from a critical AUC value using one-tailed single sample t-tests. The critical AUC was defined as the log ROC AUC computed for an overlap between the FPR and TPR distributions of p = 0.05 (with the assumption that the FPR and TPR distributions are Gaussian). The critical AUC is approximately 3.84. Paired sample t-tests were also performed to test for significant increases in AUCs for the reconstructions with the NZPL sCSD compared to the reconstructions for the full sCSD. Bonferroni correction was applied to all t-tests. Source Coherences DICS [2] was performed to reconstruct source coherence across all voxel combinations, using the same source positions defined in the source simulations (1454 sources restricted to superficial grey matter). Spatial filters were calculated using either the full, or NZPL sCSDs, as per equations 4 and 24, respectively. A relative regularisation parameter of a = 1026 was used. The rCSDs corresponding to the two sCSD types were then estimated using equations 5 and 25, respectively. Source-level coherences for both rCSD types were calculated in the standard way using equation 9 for the pre and post stimulus matrices. Systematic bias and filter leakage was removed by subtracting the pre-stimulus reconstruc- tion from the post-stimulus reconstruction. This noise-contrasted matrix was used as the measure of reconstructed source-level coherences. The AUCs for reconstructions across all jitter FWHMs are shown in figure 5 and summarised in table 1. Single sample t-tests show that in EEG and MEG, all reconstructions using the full sCSD were not significantly above the critical AUC, with the exception of the unjittered MEG reconstruction (t(9) = 17.74, p,1027), indicating DICS with the full sCSD is very intolerant of phase jitter. In contrast, the AUCs for reconstructions using the NZPL sCSD were significantly above the critical AUC across all jitter FWHMs, with the exception of the largest FWHM (p radians), where EEG reconstruction was non-significant and MEG reconstruction was borderline significant (t(9) = 2.86, p = 0.0094). This shows that DICS with the NZPL sCSD is much more robust to phase jitter. For paired-sampled t-tests comparing the two methods, NZPL sCSD performed significantly better than the full sCSD in all cases, with the exception of unjittered MEG reconstruction (t(9) = 2.31, p = 0.023). The AUCs for reconstructions across all jitter FWHMs are shown in figure 5 and summarised in table 1. Single sample t-tests show that in EEG and MEG, all reconstructions using the full sCSD were not significantly above the critical AUC, with the exception of the unjittered MEG reconstruction (t(9) = 17.74, p,1027), indicating DICS with the full sCSD is very intolerant of phase jitter. In contrast, the AUCs for reconstructions using the NZPL sCSD were significantly above the critical AUC across all jitter FWHMs, with the exception of the largest FWHM (p radians), where EEG reconstruction was non-significant and MEG reconstruction was borderline significant (t(9) = 2.86, p = 0.0094). Network Simulation Neural activity was simulated in a regular 7.5 mm3 grid of 1454 dipoles restricted to superficial grey matter in a standard MNI T1- weighted MRI template. All dipoles were orientated orthogonally to the head surface. All dipole time-series were simulated with Gaussian white noise to simulate the presence of non-correlated activity. Two cortical regions in opposite occipital lobes were December 2013 \| Volume 8 \| Issue 12 \| e81553 PLOS ONE \| www.plosone.org 7 Coherent Networks from EEG/MEG without VC/MSF Reconstruction Performance and Effects of Phase Jitter Sensor-Level Cross Spectral Density (sCSD) Sensor-Level Cross Spectral Density (sCSD) Quantifying Phase Lag Tolerance Method. A more detailed quantification of the expected phase lag tolerance of the NZPL method was obtained across a range of SNPs. Given that the full coherence between two signals with no noise is equal to 1, the coherences obtained from these simulations are a measure of the level of coherence that is retained as phase lag reduces, which we use as a metric for phase lag tolerance. Two sinusoidal waves with the same amplitude and PLOS ONE \| www.plosone.org December 2013 \| Volume 8 \| Issue 12 \| e81553 8 Coherent Networks from EEG/MEG without VC/MSF Figure 3. Reconstructed network with phase jitter FWHM of 0 and p/4 calculated from full and NZPL sCSDs for EEG and MEG. Colour code indicates the proportion of trials in which a voxel pair lies in the top 0.01% of the coherence matrix. Blue indicates location of the original network nodes. doi:10.1371/journal.pone.0081553.g003 Figure 3. Reconstructed network with phase jitter FWHM of 0 and p/4 calculated from full and NZPL sCSDs for EEG and MEG. Colour code indicates the proportion of trials in which a voxel pair lies in the top 0.01% of the coherence matrix. Blue indicates location of the original network nodes. doi:10.1371/journal.pone.0081553.g003 Effects of Regularisation Parameter frequency were generated, with the same parameters described previously. SNP and phase lag were systematically manipulated across different simulations: SNP was varied between 0 (all noise, no signal) and 1 (all signal, no noise); phase lags were varied between 0 and 0.5p radians. Coherence (equation 9) was calculated from the NZPL sCSD (equation 17) for each SNP and phase lag. For comparison, the imaginary coherency was also computed between the two signals. frequency were generated, with the same parameters described previously. SNP and phase lag were systematically manipulated across different simulations: SNP was varied between 0 (all noise, no signal) and 1 (all signal, no noise); phase lags were varied between 0 and 0.5p radians. Coherence (equation 9) was calculated from the NZPL sCSD (equation 17) for each SNP and phase lag. For comparison, the imaginary coherency was also computed between the two signals. DICS is sensitive to regularisation: An optimal regularisation parameter should be chosen based on the expected spatial distribution of sources. Lower regularisation increases the sensi- tivity of the beamformer but also is more likely to result in false positives, whereas a higher regularisation decreases sensitivity, while reducing false positives [10]. The reduction of VC/MFS artefacts in NZPL sCSD has been shown to improve the localisation of the coherent network and reduce the number of false positives in reconstructed source-level networks that would ordinarily arise in standard DICS. By eliminating these confound- ing factors prior to the spatial filter calculation, the dependency of reconstruction accuracy on regularisation should be less pro- nounced. Here, the performance of the sCSD types was tested on a range of regularisation parameters with the expectation that the NZPL method will be less sensitive to the exact value of the parameters. Results. Figure 7 shows the coherences estimated with NZPL sCSD across variation in phase lags and SNP. NZPL is clearly quite tolerant of small phase lags and low SNPs. Applying a suitable threshold to these coherence values allows the identifica- tion of minimum acceptable SNPs and phase lags. For example, the white line in figure 7 delineates the region where the simulated activity generates coherences of at least 0.9. This region encloses most of the available space indicating that the method performs well over a wide range of values. This contrasts with the coherence estimates using imaginary coherency, which is much less tolerant of variation in phase lag and SNP. Results. Effects of Regularisation Parameter Figure 7 shows the coherences estimated with NZPL sCSD across variation in phase lags and SNP. NZPL is clearly quite tolerant of small phase lags and low SNPs. Applying a suitable threshold to these coherence values allows the identifica- tion of minimum acceptable SNPs and phase lags. For example, the white line in figure 7 delineates the region where the simulated activity generates coherences of at least 0.9. This region encloses most of the available space indicating that the method performs well over a wide range of values. This contrasts with the coherence estimates using imaginary coherency, which is much less tolerant of variation in phase lag and SNP. Method. The simulations were repeated with phase lag fixed at Dw = p/2 and jitter FWHM fixed at p/4 radian and the December 2013 \| Volume 8 \| Issue 12 \| e81553 PLOS ONE \| www.plosone.org 9 Coherent Networks from EEG/MEG without VC/MSF Figure 4. Log ROC curves for performance of the DICS reconstruction for noise-normalised coherences for EEG and MEG reconstructions, plotted for full and NZPL sCSDs, for zero and non-zero (p/4) phase jitters. FPR = false positive rate, TPR = true positive rate. doi:10.1371/journal.pone.0081553.g004 Figure 4. Log ROC curves for performance of the DICS reconstruction for noise-normalised coherences for EEG and MEG reconstructions, plotted for full and NZPL sCSDs, for zero and non-zero (p/4) phase jitters. FPR = false positive rate, TPR = true positive rate. doi:10.1371/journal.pone.0081553.g004 doi:10.1371/journal.pone.0081553.g004 regularisation parameter was varied using a-values between 0 and log10-8 in log-increments of log100.5. The AUCs for NZPL sCSD are uniformly high and significantly above the critical AUC for EEG (all p,10214) and MEG (all p,10223) with the exception of a$0.1, where the AUCs shows a sudden drop off. Results. Figure 8 shows the log ROC AUCs for reconstruct- ed networks from full and NZPL sCSDs. For EEG, AUCs for full sCSD are low across much of the range with small, but still insignificant peaks at a = 1 and a = 0.01 (log100 and log10-2 on the x-axis). Full CSD for MEG showed a similar pattern with a small but insignificant peak at a = log102.5 with a mean AUC of 5.01. Application to Human Data To demonstrate the applicability of the NZPL modification to DICS in real human EEG recordings, the method was applied to Figure 5. Log ROC AUCs calculated for noise-normalised coherence for EEG and MEG reconstructions, across phase jitter FWHMs. doi:10.1371/journal.pone.0081553.g005 Figure 5. Log ROC AUCs calculated for noise-normalised coherence for EEG and MEG reconstructions, across phase jitter FWHMs. doi:10.1371/journal.pone.0081553.g005 Figure 5. Log ROC AUCs calculated for noise-normalised coherence for EEG and MEG reconstructions, across phase jitter FWHMs. doi:10.1371/journal.pone.0081553.g005 Figure 5. Log ROC AUCs calculated for noise-normalised coherence for EEG and MEG reconstructions, across phase jitter FWHMs. doi:10.1371/journal.pone.0081553.g005 December 2013 \| Volume 8 \| Issue 12 \| e81553 PLOS ONE \| www.plosone.org 10 Coherent Networks from EEG/MEG without VC/MSF Table 1. Results of all t-tests comparing AUCs with critical AUC, and comparing AUCs between sCSD types for all phase jitter FWHMs. EEG MEG Jitter FWHM (p radians) Full sCSD. Critical AUC NZPL CSD. critical AUC NZPL CSD. Full CSD NZPL CSD. critical AUC Full sCSD. Critical AUC NZPL CSD. Full CSD 0 t(9) = 255.70, t(9) = 65.59, t(9) = 101.78, t(9) = 17.74, t(9) = 2535.28, t(9) = 2.31, n.s. p,10212 p,10214 p,1027 p,10227 p = 0.023 0.03125 t(9) = 254.15, t(9) = 162.10, t(9) = 105.54, t(9) = 27.52, t(9) = 2994.72, t(9) = 30.93, n.s. p,10216 p,10214 n.s. p,10227 p,10210 0.0625 t(9) = 2132.45, t(9) = 311.74, t(9) = 242.35, t(9) = 228.92, t(9) = 2195.15, t(9) = 83.63, n.s. p,10219 p,10218 n.s. p,10226 p,10213 0.125 t(9) = 2137.70, t(9) = 88.20, t(9) = 124.89, t(9) = 218.00, t(9) = 3350.15, t(9) = 54.02, n.s. p,10214 p,10215 n.s. p,10228 p,10212 0.25 t(9) = 2121.67, t(9) = 83.23, t(9) = 120.49, t(9) = 238.38, t(9) = 1484.72, t(9) = 113.44, n.s. p,10213 p,10215 n.s. p,10225 p,10215 0.5 t(9) = 2210.40, t(9) = 10.01, t(9) = 30.84, t(9) = 240.73, t(9) = 497.42, t(9) = 123.77, n.s. p,1025 p,10210 n.s. p,10220 p,10215 1 t(9) = 2126.51, t(9) = 25.96, t(9) = 7.52, t(9) = 249.22, t(9) = 2.86, t(9) = 7.59, n.s. n.s. p,1024 n.s. p = 0.0094 p,1024 doi:10.1371/journal.pone.0081553.t001 Table 1. Results of all t-tests comparing AUCs with critical AUC, and comparing AUCs between sCSD types for all phase jitter FWHMs. doi:10.1371/journal.pone.0081553.t001 identify coherent network components that underpin face recog- nition during the primary visual response that takes place in the first 400 ms of visual face presentation. Application to Human Data processing was carried out using FieldTrip [54]. Data was epoched, band pass filtered at 2-45 Hz, baseline corrected and de-trended. Source reconstruction was carried out using the same procedure described for the simulations. NZPL sCSDs were calculated in a 0–400 ms, theta band (4–8 Hz) time-frequency Hanning-window for both conditions and averaged across all epochs. The BEM model and lead fields were computed from a subject-specific anatomical T1-weighted MRI using the same procedure to generate the head model for the EEG simulations. Method. 128-channel BioSemi EEG Data for visual presen- tation of faces and scrambled images in a single subject [56] were obtained from the SPM website (downloaded from http://www.fil. ion.ucl.ac.uk/spm/data/mmfaces/). The paradigm consisted of 85 randomised trials of 500 ms fixation cross, followed by 600 ms presentation of either a face or a scrambled image [57]. The pre- Figure 6. Log ROC AUCs calculated for noise-normalised coherence for EEG and MEG reconstructions, across phase lags. doi:10.1371/journal.pone.0081553.g006 Figure 6. Log ROC AUCs calculated for noise-normalised coherence for EEG and MEG reconstructions, across phase lags. doi:10 1371/journal pone 0081553 g006 AUCs calculated for noise-normalised coherence for EEG and MEG reconstructions, across phase lags. one.0081553.g006 Figure 6. Log ROC AUCs calculated for noise-normalised coherence for EEG and MEG reconstructions, across phase lags. doi:10.1371/journal.pone.0081553.g006 December 2013 \| Volume 8 \| Issue 12 \| e81553 PLOS ONE \| www.plosone.org 11 Coherent Networks from EEG/MEG without VC/MSF Table 2. Results of all t-tests comparing AUCs with critical AUC, and comparing AUCs between sCSD types for all phase lags. Table 2. Results of all t-tests comparing AUCs with critical AUC, and comparing AUCs between sCSD types for all phase lags. Table 2. Results of all t-tests comparing AUCs with critical AUC, and comparing AUCs between sCSD types for all phase lags. EEG MEG Phase lag (p radians) Full sCSD. Critical AUC NZPL CSD. critical AUC NZPL CSD. Full CSD NZPL CSD. critical AUC Full sCSD. Critical AUC NZPL CSD. Full CSD 0 t(9) = 2113.25, t(9) = 25.89, t(9) = 6.50, t(9) = 2149.70, t(9) = 2.76, t(9) = 7.30, n.s. n.s. p,1024 n.s. p = 0.011 p,1024 0.0625 t(9) = 2118.36, t(9) = 2.71, t(9) = 17.05, t(9) = 2186.32, t(9) = 33.5, t(9) = 55.32, n.s. p = 0.012 p,1027 n.s. p,10210 p,10212 0.125 t(9) = 2121.51, t(9) = 23.36, t(9) = 55.70, t(9) = 2167.98, t(9) = 595.81, t(9) = 521.39, n.s. Discussion This study aimed to optimise the dynamic imaging of coherent sources [2] method to reconstruct only non-zero phase-lagged (NZPL) interactions using a variation of imaginary coherency [1]. This approach reduces the impact of spurious interactions arising due to volume conduction (VC) and magnetic field spread (MFS) on the reconstructed source networks. To generate an unbiased symmetrical estimate of the sensor cross-spectral density (sCSD) that only embodies phase-lagged interactions, the diagonal of the imaginary part of the sCSD matrix was replaced with an estimate of NZPL-only components of the power, using eigenvector decomposition of the imaginary sCSD. This CSD is an approximation of the phase-lagged interactions with reduced phase lag bias. Using the NZPL sCSD as an approximation of the projected NZPL source interactions, an NZPL-optimised spatial filter was constructed. The projection of the NZPL sCSD using this filter significantly reduces the confounding effects of VC/MFS on source localisation are reduced. The application of the NZPL sCSD to the filter calculation is advantageous as it deliberately overestimates the signal arising from phase-lagged interactions while suppressing weaker interactions. This improves the spatial acuity of the filter. This method offers significant improvement compared to using the full sCSD for both EEG and MEG. NZPL significantly improves accuracy of the source reconstruction compared to using the full sCSD. This is shown consistently for a range of non-instantaneous phase lags and noise levels (in the form of phase jitter) and is true for both MEG and EEG data. In the case of phase jitter, using DICS with the full CSD is highly The study has also shown that the NZPL sCSD performance is more invariant to regularisation compared to the full sCSD. Regularisation reduces the sensitivity of DICS to false positives, but also increases the probability of false negatives. Higher regularisation comes at the expense of lower spatial acuity. The removal of spurious interactions by the NZPL manipulation before computation of the filter reduces the need for regularisation. NZPL therefore allows the use of spatial filters with the highest possible spatial acuity (i.e. where regularisation is set to, or close to zero). An interesting point to note is that across all experiments the performance of the NZPL sCSD in EEG reaches a maximum AUC of about 6, while for MEG this was about 9. In addition, EEG was slightly less tolerant of small phase lags and high degrees of jitter than MEG. Application to Human Data doi:10.1371/journal.pone.0081553.g008 for noise normalised coherences for EEG and MEG reconstructions, across regularisation parameters a. 1553.g008 cortex and the right superior temporal region. The most strongly connected source pairs (the highest 0.01% of the connectivity matrix) within this contrast are those between the primary visual cortex and right superior temporal gyrus (figure 9c). This suggests that perceiving faces engages a direct coherent interaction between the primary visual cortex and the superior temporal gyrus. This region has been implicated in face recognition and more specifically in detecting gaze and emotion propensity [58–60]. intolerant of phase jitter, unlike DICS with the NZPL CSD, which shows tolerance to even very wide distributions of phase jitter (up to FWHM = p/2). The intolerance of standard coherence measures to phase jitter has been reported previously [61]. In the case of instantaneous phase lag, the accuracy is much more inconsistent in NZPL, which is to be expected, although in the case of MEG at least, the accuracy is still good. The presence of phase jitter in these simulations means that the coherent activity can still be detected, but with much more variable accuracy. The small variances of AUCs across repetitions for NZPL compared with the full sCSD (figures 5,6 and 8) also suggest the NZPL method is more robust to noise as there is more consistency in the responses across noise-varied trials. This is consistent with the principle of imaginary coherency where the imaginary component of coherency is reduced with increasing noise [1]. Noise is attenuated by the loss of the real components and hence will not be modelled in the spatial filter or the subsequent rCSD. However, the amplitude of the phase-lagged interactions will be reduced. This may introduce scaling issues if making contrasts between networks with different levels of noise [45]. December 2013 \| Volume 8 \| Issue 12 \| e81553 Application to Human Data p,1028 p,10212 n.s. p,10221 p,10221 0.25 t(9) = 2144.07, t(9) = 100.00, t(9) = 183.74, t(9) = 265.67, t(9) = 502.80, t(9) = 172.20, n.s. p,10214 p,10216 n.s. p,10220 p,10216 0.5 t(9) = 2106.39, t(9) = 142.72, t(9) = 198.92, t(9) = 222.55, t(9) = 909.55, t(9) = 63.04, n.s. p,10215 p,10217 n.s. p,1023 p,10212 1 t(9) = 281.51, t(9) = 23.37, t(9) = 16.64, t(9) = 219.53, t(9) = 4.43, t(9) = 7.80, n.s. n.s. p,1027 n.s. p = 0.0008 p,1024 1.5 t(9) = 2152.60, t(9) = 103.81, t(9) = 174.85, t(9) = 255.59, t(9) = 1076.97, t(9) = 156.91, n.s. p,10214 p,10216 n.s. p,10223 p,10216 2 t(9) = 2124.59, t(9) = 25.89, t(9) = 6.56, t(9) = 2150.05, t(9) = 2.76, t(9) = 7.31, n.s. n.s. p,1024 n.s. p = 0.011 p,1024 doi:10.1371/journal.pone.0081553.t002 doi:10.1371/journal.pone.0081553.t002 DICS was performed using this lead field and the epoch-averaged NZPL sCSD to obtain the source-level rCSDs. The coherence between all source pairs was then calculated. To obtain contrasts, analogous noise coherences were estimated using the same procedure applied to a pre-stimulus period of equal length. The true and noise coherences were Fisher transformed and then contrasted. Results. Results are shown in figure 9. The highest noise contrasted coherences are plotted to visualise the most highly coherent regions. The results show that the strongest coherences in both conditions take place in primary visual cortex and the right superior temporal gyrus and precentral gyrus (figure 9a). Contrasts between the two conditions (figure 9b) show faces elicit higher coherences in the left lateralised occipital and temporo-occipital Figure 7. Estimated coherence calculated from imaginary coherency and NZPL coherence for a pair of sources with varying phase lag and SNR. White lines indicates estimated thresholds for coherences of 0.9. doi:10.1371/journal.pone.0081553.g007 Figure 7. Estimated coherence calculated from imaginary coherency and NZPL coherence for a pair of sources with varying phase lag and SNR. White lines indicates estimated thresholds for coherences of 0.9. doi:10.1371/journal.pone.0081553.g007 December 2013 \| Volume 8 \| Issue 12 \| e81553 December 2013 \| Volume 8 \| Issue 12 \| e81553 PLOS ONE \| www.plosone.org 12 12 Coherent Networks from EEG/MEG without VC/MSF Figure 8. Log ROC AUCs for noise normalised coherences for EEG and MEG reconstructions, across regularisation parameters a. doi:10.1371/journal.pone.0081553.g008 Figure 8. Log ROC AUCs for noise normalised coherences for EEG and MEG reconstructions, across regularisation parameters a. Discussion This is to be expected given the greater spatial acuity MEG offers in comparison to EEG and the fact that in MEG, there is less distortion of the magnetic field compared to the smearing of electrical potentials in EEG. In our simulations, this is December 2013 \| Volume 8 \| Issue 12 \| e81553 December 2013 \| Volume 8 \| Issue 12 \| e81553 PLOS ONE \| www.plosone.org 13 Coherent Networks from EEG/MEG without VC/MSF Figure 9. Source Coherence estimates from human EEG using DICS with NZPL sCSD. (a) shows the maximum value for each voxel value in the noise-normalised coherence matrix for faces (top) and scrambled (bottom) images at 0–400 ms, 4–8 Hz. (b) shows the maximum of the contrast between faces and scrambled connectivity matrices. (c) shows the top 0.01% of the face-scrambled contrast matrix. doi:10.1371/journal.pone.0081553.g009 Figure 9. Source Coherence estimates from human EEG using DICS with NZPL sCSD. (a) shows the maximum value for each voxel value in the noise-normalised coherence matrix for faces (top) and scrambled (bottom) images at 0–400 ms, 4–8 Hz. (b) shows the maximum of the contrast between faces and scrambled connectivity matrices. (c) shows the top 0.01% of the face-scrambled contrast matrix. doi:10.1371/journal.pone.0081553.g009 not an issue as the NZPL method was able to reconstruct EEG networks with a high degree of accuracy. However, there may be conditions where the spatial distribution of the sensor data is too smooth to permit sufficiently accurate reconstruction. A quanti- fication of the data smoothness such as the condition number [11] may be used as a criterion for the feasibility of source reconstruction in such cases. phase-lagged coherences, both between true sources and VC artefacts, and between different artefacts. In the data presented here, this problem was not observed to any great extent, so it can be reasonably assumed that higher order artefacts are sufficiently small in NZPL as to not give rise to any false positives when reconstructing the source network. However, for more complex networks it may be more of a problem. This issue of mixing phase- lagged signals has been previously raised by Lachaux et al [63] in response to the assumption that VC/MFS coherences have no phase lag. In the data from the simulations presented here a small effect can be seen in the simulated sensor data (appendix S3, figure 2). December 2013 \| Volume 8 \| Issue 12 \| e81553 Coherent Networks from EEG/MEG without VC/MSF same way it does in standard DICS analysis. Additionally, the relaxation of the suppression of signals for phase-lagged sources increases the apparent SNR of the sCSD, improving its spatial accuracy (see also appendix S2). Higher-order artefacts are substantially smaller in magnitude than first-order artefacts (see appendix S3, figure 2b). However, the importance of the accuracy or complexity of the VC model to the calculation of an accurate spatial filter in the presence of higher-order artefacts remains a question. Further investigation is required to quantify this relationship. the distribution of phase lags. The method presented here offers a similar advantage over the original imaginary coherency method. We have shown the bias in variability of the imaginary CSD arises from the size of the phase lag (appendix S1). Removal of this bias by eigenvector decomposition therefore results in CSD estimates that remain consistent across a range of phase lags. Only when SNR is low is there an increased bias from the size of the phase lag on the inferred coherence. The reduction in SNR manifests in the case where the phase lag distribution is centred around 0 or p radians. The absence of an imaginary component at the peak of the distribution means the spatial filter has to rely on the signal obtained from the much weaker imaginary components at the periphery of the distribution. The effect is also seen in the EEG reconstruction where small phase lags are tolerated less than for MEG. However, as only two sources were simulated, we are making a generalisation from the two-source case to one with multiple sources, which may be less tolerant on small phase lags due to increased mixing between multiple sources. Further study is required to quantify the effect of number of interacting sources on phase lag tolerance. Of course beamformers are not the only reconstruction method that can be used to explore source level connectivity and coherence is not the only functional connectivity measure that could be employed. Any functional connectivity measure can theoretically be applied to reconstructed sources to infer functional networks in source space. For instance source time-series could be estimated using the ‘virtual electrode’ method, and this could then be used to calculate coherence, synchronisation, Granger causality or transfer entropy. However, making deterministic connectivity inferences between reconstructed sources should be done with caution, as there is uncertainty about the accuracy of these reconstructions. Coherent Networks from EEG/MEG without VC/MSF Each reconstruction method carries with it a set of assumptions, which will give rise to some systematic error, which can contaminate connectivity estimates [12]. In particular, VC/ MFS artefacts if not accounted for in the spatial filter will lead to mislocalisations of sources [29,32]. There is also the issue of increase computational demands of separate source time-series reconstruction and coherence estimates and the issue of VC/MFS artefacts. DICS overcomes these issues as source connectivity estimates are made in a single step without the intermediary step of reconstructing the time-series. However, a particular point of concern that has arisen in connection with DICS (and any other connectivity analysis based on beamformer data) is that the covariance minimisation constraint on the spatial filter appears contradictory to the aim of identifying coherence in the source activity. The consequence of this is that reconstructed coherences are likely to be attenuated by the spatial filter. Hipp et al [25] regard this as an advantage because the false positive rate for reconstructed coherence is markedly reduced. This is apparent in this study by the absence of the higher-order artefact from the source reconstruction using NZPL shown in figure 3 (also see appendix S3, figure 2). It is certainly true that in the cases reported here, the covariance minimisation has not prevented the reliable detection of coherent sources, both from simulated and human data. The over-estimation of phase-lagged interaction on the NZPL CSD, compared to the full CSD, resulted in their preservation when projected to source space. In addition to the tolerance to small phase lags, averaging the sCSD over sufficient event-related epochs will ensure functionally relevant phase-lagged interactions are retained. This raises an issue about assumptions made concerning how neural populations interact. As noted previously, one assumption is that the probabilistic nature of encoding within stochastic firing patterns means that it is unlikely that two functionally connected neural populations are ever perfectly in phase within a given epoch. By averaging sCSDs across epochs, a different assumption is made, based on the same premise: that there is sufficient phase lag variability between epochs to render the chance occurrence of zero-lagged coherences trivial. Some other functional connectivity methods, such as phase locking value (PLV) [63], which treat phase lag consistency as a measure of functional connectivity will not detect these type of interactions. Appendix S2 Properties of the spatial filter. (PDF) Appendix S2 Properties of the spatial filter. (PDF) Appendix S3 Surface-level coherence. (PDF) Appendix S3 Surface-level coherence. (PDF) Appendix S3 Surface-level coherence. (PDF) Coherent Networks from EEG/MEG without VC/MSF Fortunately, NZPL sCSD averaged across epochs will be sensitive to both types of interaction, with only one exception - when there is exactly zero phase lag with very low phase lag variability. It is assumed this type of interaction is sufficiently rare as to not significantly increase the false negative rate of the NZPL method. In conclusion, NZPL DICS offers a method of significantly improving localisation of coherent networks. The method is also less computationally demanding than separately reconstructing source time-series and inferring functional networks, making it feasible to compute and perform statistical inferences on whole brain networks. This modification allows DICS to be a much more accurate tool for inferring functional connectivity from EEG and MEG recordings. As previously discussed, the inference of connectivity in the current study depends on there being some phase lag between sources. Based on previous experimental evidence [39], it is assumed that true neural interactions would never have instanta- neous phase. Given that the improvement in performance is maintained even at very small phase lags tested, Dw = 0.0625p it seems likely that this assumption will only rarely be violated. Indeed, the data in figure 7 demonstrates the robustness of NZPL to even smaller phase lags. Treating these coherence values as a metric for retention of the true coherence by NZPL for decreasing SNPs and phase lags, it can be shown that even at SNP = 0.5 (equal signal and noise), NZPL will tolerate (with 90% of coherence retained) a phase difference as small as Dw = 0.025p (approx. 4.5u). This feature addresses an issue previously raised with imaginary coherency, which is the sensitivity to the size of the phase lag. One other method shown to overcome this drawback is the phase lag index (PLI) [46], which measures the asymmetry of Discussion The EEG full coherence plot shows first-order VC artefacts between the occipital bilateral electrodes to frontal bilateral (both contralateral and ipsilateral) electrodes. This is due to the source activity conducting to opposite sides of the head. In the NZPL coherence plot, higher order-artefacts can be seen, where occipital bilateral electrodes are weakly coherent with ipsilateral frontal electrodes, but not contralateral electrodes. The contralateral 1st order artefacts were removed by NZPL while the ipsilateral 2nd order artefacts remain. This is the same effect illustrated graphically in figure 1. In addition to tests on simulated data sets, the NZPL DICS analysis was tested using human data that compares the identification of faces with scrambled images. This appeared to elicit increased coherence between primary visual cortex and the right superior temporal cortex in the first 400 ms. The superior temporal cortex has previously been implicated in analysis of facial features, which is prominently right-lateralised [60,62], suggesting this is a plausible subcomponent of a face recognition network. Other parts of the network may be uncovered by examining other time windows and frequency bands. An attractive aspect of imaginary coherency is that it offers a model-free method of reducing VC/MFS artefacts [1]. This feature emerges from the fact that spurious VC/MFS interactions will always have zero phase lag. The contribution of these interactions can therefore be reduced by considering only the imaginary components. We anticipate that this attractive feature, when applied to DICS, will help to prevent the reconstruction of artefactual interactions in source space that can arise from inaccurate VC models, or from sources of electromagnetic interference external to the brain. An issue that remains however is that of higher-order VC artefacts. In any mixing of sources where there are non-zero phase lags, there will be artefactual The issue described above is fundamentally the same as the EEG/MEG inverse problem, which the spatial filter resolves. The artefacts not eliminated by NZPL are still attenuated by the spatial filter. It is therefore reasonable to conclude that while the NZPL manipulation eradicates first order artefacts, higher order artefacts will remain and are still dependant on the performance of the spatial filter, and hence the accuracy of the forward model. 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Oostenveld R, Fries P, Maris E, Schoffelen J-M (2011) FieldTrip: Open source software for advanced analysis of MEG, EEG, and invasive electrophysiological data. Comput Intell Neurosci 2011: 156869. 27. Winter WR, Nunez PL, Ding J, Srinivasan R (2007) Comparison of the effect of volume conduction on EEG coherence with the effect of field spread on MEG coherence. Stat Med 26: 3946–3957. p 55. Clark RD, Webster-Clark DJ (2008) Managing bias in ROC curves. J Comput Aided Mol Des 22: 141–146. December 2013 \| Volume 8 \| Issue 12 \| e81553 PLOS ONE \| www.plosone.org 16 63. Lachaux J-P, Rodriguez E, Martinerie J, Varela FJJ (1999) Measuring phase synchrony in brain signals. Hum Brain Mapp 8: 194–208. Coherent Networks from EEG/MEG without VC/MSF Coherent Networks from EEG/MEG without VC/MSF Coherent Networks from EEG/MEG without VC/MSF 56. Henson RN, Mouchlianitis E, Friston KJ (2009) MEG and EEG data fusion: simultaneous localisation of face-evoked responses. Neuroimage 47: 581–589. p g 57. Henson RN, Goshen-Gottstein Y, Ganel T, Otten LJ, Quayle A, et al. (2003) Electrophysiological and haemodynamic correlates of face perception, recogni- tion and priming. Cereb Cortex 13: 793–805. 61. Brookes MJ, Hale JR, Zumer JM, Stevenson CM, Francis ST, et al. (2011) Measuring functional connectivity using MEG: methodology and comparison with fcMRI. Neuroimage 56: 1082–1104. g 62. Haxby JV V, Hoffman EAA, Gobbini MII (2000) The distributed human neural system for face perception. Trends Cogn Sci 4: 223–233. 63. Lachaux J-P, Rodriguez E, Martinerie J, Varela FJJ (1999) Measuring phase synchrony in brain signals. Hum Brain Mapp 8: 194–208. PLOS ONE \| www.plosone.org December 2013 \| Volume 8 \| Issue 12 \| e81553 PLOS ONE \| www.plosone.org 17
https://openalex.org/W2994835903	https://iris.unipa.it/bitstream/10447/405599/2/Odontogenic%20Cysts%3a%20A%2030-Year%20Retrospective%20Clinicopathological%20Study.pdf	English	null	Odontogenic Cysts: A 30-Year Retrospective Clinicopathological Study	null	2,019	cc-by	1,301	Marco Mascitti 1,, Lucrezia Togni 1, Lorenzo Lo Muzio 2, Giuseppina Campisi 3, Federico Mazzoni 1 and Andrea Santarelli 1 1 Department of Clinical Specialistic and Dental Sciences, Marche Polytechnic University, 60126 Ancona, Italy; togni.lucrezia@gmail.com (L.T.); edottor@libero.it (F.M.); andrea.santarelli@staff.univpm.it (A.S.) 1 Department of Clinical Specialistic and Dental Sciences, Marche Polytechnic University, 60126 Ancona, Italy; togni.lucrezia@gmail.com (L.T.); edottor@libero.it (F.M.); andrea.santarelli@staff.univpm.it (A.S.) 2 De a t e t of Cli i al a d E e i e tal Medi i e U i e ity of Fo ia 71122 Fo ia Italy Italy; togni.lucrezia@gmail.com (L.T.); edottor@libero.it (F.M.); andrea.santarelli@staff.univpm.it (A.S.) 2 Department of Clinical and Experimental Medicine, University of Foggia, 71122 Foggia, Italy; lorenzo.lomuzio@unifg.it 2 Department of Clinical and Experimental Medicine, University of Foggia, 71122 Foggia, Italy; lorenzo.lomuzio@unifg.it 3 Department of Surgical, Oncological and Oral Sciences (DICHIRONS), University of Palermo, 90127 Palermo, Italy; campisi@odonto.unipa.it 3 Department of Surgical, Oncological and Oral Sciences (DICHIRONS), University of Palermo, 90127 Palermo, Italy; campisi@odonto.unipa.it C d h l l y p p * Correspondence: marcomascitti86@hotmail.it; Tel.: +39-071-220-6226 † Presented at the XV National and III International Congress of the Italian Society of Oral Pathology and Medicine (SIPMO), Bari, Italy, 17–19 October 2019. † Presented at the XV National and III International Congress of the Italian Society of Oral Pathology and Medicine (SIPMO), Bari, Italy, 17–19 October 2019. † Presented at the XV National and III International Congress of the Italian Society of Oral Pathology and Medicine (SIPMO), Bari, Italy, 17–19 October 2019. g y gy Medicine (SIPMO), Bari, Italy, 17–19 October 2019. Medicine (SIPMO), Bari, Italy, 17–19 October 2 Published: 11 December 2019 Published: 11 December 2019 Odontogenic cysts (OC) are one of the most frequent lesions affecting the jaws. These lesions are characterized by a pathologic cavity, either completely or partially covered by an epithelial tissue of odontogenic origin. OCs share similar features; therefore, the differential diagnosis requires a combination of clinical, radiological, and histological findings [1]. This study aims to perform an epidemiologic analysis of OCs treated from 1990 to 2019 at the “Ospedali Riuniti” General Hospital, Ancona, Italy, according to 4th Edition of WHO Classification of Head and Neck Tumours. The present study considered all the patients who underwent surgery for jaw cysts from January 1990 to August 2019. Data were retrieved and catalogued from clinical records and from the archive of the Institute of Pathology, Marche Polytechnic University, Italy. Extended Abstract Extended Abstract Proceedings 2019, 35, 31; doi:10.3390/proceedings2019035031 Marco Mascitti 1,, Lucrezia Togni 1, Lorenzo Lo Muzio 2, Giuseppina Campisi 3, Federico Mazzoni 1 and Andrea Santarelli 1 Because of the 30-year period considered, histological slides of OCs were re-evaluated to confirm the diagnosis, according to the current WHO criteria [2]. From each case, they were extrapolated the following information: age, sex, diagnosis, site distribution, and relapses. Overall, 1942 patients were treated for jaw cysts, corresponding to 1862 patients with OC, of which 98 showing multiple OCs at the time of diagnosis, and 80 patients with nonodontogenic cysts (NOC). Furthermore, 50 patients showed at least one OC recurrence during follow-up. 2126 surgical specimens were retrieved, corresponding to 2046 OCs and 80 NOCs. 50 patients developed 69 recurrences, mainly Odontogenic keratocysts (OKC). Mean age of occurrence for primary OC was 46.9 ± 17.1 years, with a higher frequency in males (M:F ratio of 1.79). Regarding localization, posterior mandibular and anterior maxillary regions were the most commonly affected sites (Mandible:Maxilla ratio of 1.42). Mean size of primary OC was 1.9 ± 1.0 cm (Table 1). Radicular cysts were the most frequently diagnosed, with 815 cases (39.83%), followed by Dentigerous cysts (21.51%), and OKC (13.54%) (Figure 1). All other OCs showed a very low frequency, reaching a total of 83 cases (4.06%) (Figure 2). Noteworthy, in 431 cases the clinicopathological data were insufficient to establish a certain diagnosis (21.07%). Proceedings 2019, 35, 31; doi:10.3390/proceedings2019035031 www.mdpi.com/journal/proceedings www.mdpi.com/journal/proceedings 2 of 3 Proceedings 2019, 35, 31 Table 1. Demographic and clinical data of OCs (1990–2019). Clinical Presentation N° of Cysts - Primary OCs - Recurred OCs 1977 69 Site Distribution - Mandible 792 - Maxilla 557 - Not specified 697 Sex (n° of Patients) - Males 1194 - Females 668 Age (years) 46.9 ± 17.1 Size (cm) 1.9 ± 1.0 Figure 1. Annual frequency of diagnosed OCs in “Ospedali Riuniti” General Hospital, Ancona, Italy (red line). Radicular cysts (RC, orange line) were the most frequently diagnosed, followed by Dentigerous cysts (DC, blue line) and Odontogenic keratocysts (OKC, green line). Table 1. Demographic and clinical data of OCs (1990–2019). ble 1. Demographic and clinical data of OCs (1990–2019 Clinical Presentation N° of Cysts - Primary OCs - Recurred OCs 1977 69 Site Distribution - Mandible 792 - Maxilla 557 - Not specified 697 Sex (n° of Patients) - Males 1194 - Females 668 Age (years) 46.9 ± 17.1 Size (cm) 1.9 ± 1.0 Figure 1. Annual frequency of diagnosed OCs in “Ospedali Riuniti” General Hospital, Ancona, Italy (red line). Marco Mascitti 1,, Lucrezia Togni 1, Lorenzo Lo Muzio 2, Giuseppina Campisi 3, Federico Mazzoni 1 and Andrea Santarelli 1 Radicular cysts (RC, orange line) were the most frequently diagnosed, followed by Dentigerous cysts (DC, blue line) and Odontogenic keratocysts (OKC, green line). Figure 2. Relative frequency of diagnosed OCs according to 3rd and 4th Edition of WHO Classification, respectively. In 2017 there was a significative simplification of OC classification; the most important changes regard the reintroduction of Odontogenic keratocyst (OKC) and Calcifying Odontogenic cyst (COC). DC = Dentigerous cyst; EC = Eruptive cyst; RC = Radicular cyst; ReC = Residual cyst; IPC = Inflammatory paradental cyst; ICC = Inflammatory collateral cyst; GCI = Gingival cyst of infant; GCA = Gingival cyst of adult; GC = Gingival cyst; GOC = Glandular odontogenic cyst; LPC = Lateral periodontal cyst; OOC = Orthokeratinized odontogenic cyst. Figure 2. Relative frequency of diagnosed OCs according to 3rd and 4th Edition of WHO Classification, respectively. In 2017 there was a significative simplification of OC classification; the most important changes regard the reintroduction of Odontogenic keratocyst (OKC) and Calcifying Odontogenic cyst (COC). DC = Dentigerous cyst; EC = Eruptive cyst; RC = Radicular cyst; ReC = Residual cyst; IPC = Inflammatory paradental cyst; ICC = Inflammatory collateral cyst; GCI = Gingival cyst of infant; GCA = Gingival cyst of adult; GC = Gingival cyst; GOC = Glandular odontogenic cyst; LPC = Lateral periodontal cyst; OOC = Orthokeratinized odontogenic cyst. 3 of 3 Proceedings 2019, 35, 31 Proceedings 2019, 35, 31 Proceedings 2019, 35, 31 Although limited in its retrospective nature, these findings could be useful to determine the incidence and prevalence of OCs. Prevalence studies related to OCs should be conducted in each tertiary referral center, in order to improve current epidemiological data. ts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References 1. Lo Muzio, L.; Mascitti, M. Cystic lesions of the jaws: a retrospective clinicopathologic study of 2030 cases. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. 2017, 124, 128–138, doi:10.1016/j.oooo.2017.04.006. 1. Lo Muzio, L.; Mascitti, M. Cystic lesions of the jaws: a retrospective clinicopathologic study of 2030 cases. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. 2017, 124, 128–138, doi:10.1016/j.oooo.2017.04.006. 2. El-Naggar, A.; Chan, J. WHO Classification of Head and Neck Tumours, 4th ed.; IARC: Lyon, France, 2017; pp. 232–242. 2. El-Naggar, A.; Chan, J. WHO Classification of Head and Neck Tumours, 4th ed.; IARC: Lyon, France, 2017; pp. 232–242. © 2019 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/). © 2019 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/W4389793057	https://aacr.figshare.com/articles/journal_contribution/Supplementary_Figure_S4_from_CD24_sup_hi_sup_CD27_sup_sup_Bregs_within_Metastatic_Lymph_Nodes_Promote_Multidrug_Resistance_in_Breast_Cancer/24826155/1/files/43667367.pdf	English	null	Supplementary Figure S4 from CD24<sup>hi</sup>CD27<sup>+</sup> Bregs within Metastatic Lymph Nodes Promote Multidrug Resistance in Breast Cancer	null	2,023	cc-by	682	Fig. S4. CD24hiCD27+ Breg-induced drug resistance in BC is independent of small molecule substances. (A) The survival rate of BT474 cells treated with or without conditioned medium (CM) from the direct coculture system of BT474 and CD24hiCD27+ Bregs under the treatment of the indicated concentration of pyrotinib or paclitaxel. CM without protein with a molecular weight greater than 5-kDa (CM (< 5-kDa)) was obtained by Sartorius spin columns with a cut-off of 5-kDa. (B) The proportion of apoptotic BT474 cells treated with or without CM under the indicated concentration of pyrotinib or paclitaxel. (CM: conditioned medium from the direct coculture system of BT474 and CD24hiCD27+ Bregs in contact. Data are represented as the mean ± SEM; * p < 0.05, p < 0.01, and * p < 0.001). Fig. S4. CD24hiCD27+ Breg-induced drug resistance in BC is independent of small molecule substances. (A) The survival rate of BT474 cells treated with or without conditioned medium (CM) from the direct coculture system of BT474 and CD24hiCD27+ Bregs under the treatment of the indicated concentration of pyrotinib or paclitaxel. CM without protein with a molecular weight greater than 5-kDa (CM (< 5-kDa)) was obtained by Sartorius spin columns with a cut-off of 5-kDa. (B) The proportion of apoptotic BT474 cells treated with or without CM under the indicated concentration of pyrotinib or paclitaxel. (CM: conditioned medium from the direct coculture system of BT474 and CD24hiCD27+ Bregs in contact. Data are represented as the mean ± SEM; * p < 0.05, p < 0.01, and * p < 0.001). Fig. S4. CD24hiCD27+ Breg-induced drug resistance in BC is independent of small molecule substances. (A) The survival rate of BT474 cells treated with or without Fig. S4. CD24hiCD27+ Breg-induced drug resistance in BC is independent of small molecule substances. (A) The survival rate of BT474 cells treated with or without conditioned medium (CM) from the direct coculture system of BT474 and CD24hiCD27+ Bregs under the treatment of the indicated concentration of pyrotinib or paclitaxel. CM without protein with a molecular weight greater than 5-kDa (CM (< 5-kDa)) was obtained by Sartorius spin columns with a cut-off of 5-kDa. (B) The proportion of apoptotic BT474 cells treated with or without CM under the indicated concentration of pyrotinib or paclitaxel. (CM: conditioned medium from the direct coculture system of BT474 and CD24hiCD27+ Bregs in contact. Data are represented as the mean ± SEM; * p < 0.05, p < 0.01, and * p < 0.001). Fig. S4. CD24hiCD27+ Breg-induced drug resistance in BC is independent of small molecule substances. (A) The survival rate of BT474 cells treated with or without conditioned medium (CM) from the direct coculture system of BT474 and CD24hiCD27+ Bregs under the treatment of the indicated concentration of pyrotinib or paclitaxel. CM without protein with a molecular weight greater than 5-kDa (CM (< 5-kDa)) was obtained by Sartorius spin columns with a cut-off of 5-kDa. (B) The proportion of apoptotic BT474 cells treated with or without CM under the indicated concentration of pyrotinib or paclitaxel. (CM: conditioned medium from the direct coculture system of BT474 and CD24hiCD27+ Bregs in contact. Fig. S4. CD24hiCD27+ Breg-induced drug resistance in BC is independent of small molecule substances. (A) The survival rate of BT474 cells treated with or without conditioned medium (CM) from the direct coculture system of BT474 and CD24hiCD27+ Bregs under the treatment of the indicated concentration of pyrotinib or paclitaxel. CM without protein with a molecular weight greater than 5-kDa (CM (< 5-kDa)) was obtained by Sartorius spin columns with a cut-off of 5-kDa. (B) The proportion of apoptotic BT474 cells treated with or without CM under the indicated concentration of pyrotinib or paclitaxel. (CM: conditioned medium from the direct coculture system of BT474 and CD24hiCD27+ Bregs in contact. Data are represented as the mean ± SEM; * p < 0.05, p < 0.01, and * p < 0.001). Data are represented as the mean ± SEM; * p < 0.05, p < 0.01, and * p < 0.001). Fig. S4. CD24hiCD27+ Breg-induced drug resistance in BC is independent of small
W3100128270.txt	http://diposit.ub.edu/dspace/bitstream/2445/134703/1/687012.pdf	en		Detection of the blazar S4 0954+65 at very-high-energy with the MAGIC telescopes during an exceptionally high optical state	Astronomy & astrophysics	2,018	public-domain	13,316	Astronomy & Astrophysics A&A 617, A30 (2018) https://doi.org/10.1051/0004-6361/201832624 © ESO 2018 Detection of the blazar S4 0954+65 at very-high-energy with the MAGIC telescopes during an exceptionally high optical state MAGIC Collaboration: M. L. Ahnen1 , S. Ansoldi2,20 , L. A. Antonelli3 , C. Arcaro4 , D. Baack5 , A. Babić6 , B. Banerjee7 , P. Bangale8 , U. Barres de Almeida8,9 , J. A. Barrio10 , W. Bednarek11 , E. Bernardini4,12,23 , R. Ch. Berse5 , A. Berti2,22 , W. Bhattacharyya12 , A. Biland1 , O. Blanch13 , G. Bonnoli14 , R. Carosi14 , A. Carosi3 , G. Ceribella8 , A. Chatterjee7 , S. M. Colak13 , P. Colin8 , E. Colombo15 , J. L. Contreras10 , J. Cortina13 , S. Covino3 , P. Cumani13 , P. Da Vela14 , F. Dazzi3 , A. De Angelis4 , B. De Lotto2 , M. Delfino13,25 , J. Delgado13 , F. Di Pierro4 , A. Domínguez10 , D. Dominis Prester6 , D. Dorner16 , M. Doro4 , S. Einecke5 , D. Elsaesser5 , V. Fallah Ramazani17 , A. Fernández-Barral13 , D. Fidalgo10 , M. V. Fonseca10 , L. Font18 , C. Fruck8 , D. Galindo19 , R. J. García López15 , M. Garczarczyk12 , M. Gaug18 , P. Giammaria3 , N. Godinović6 , D. Gora12 , D. Guberman13 , D. Hadasch20 , A. Hahn8 , T. Hassan13 , M. Hayashida20 , J. Herrera15 , J. Hose8 , D. Hrupec6 , K. Ishio8 , Y. Konno20 , H. Kubo20 , J. Kushida20 , D. Kuveždić6 , D. Lelas6 , E. Lindfors17 , S. Lombardi3 , F. Longo2,24 , M. López10 , C. Maggio18 , P. Majumdar7 , M. Makariev21 , G. Maneva21 , M. Manganaro15,? , K. Mannheim16 , L. Maraschi3 , M. Mariotti4 , M. Martínez13 , S. Masuda20 , D. Mazin8,20 , K. Mielke5 , M. Minev21 , J. M. Miranda16 , R. Mirzoyan8 , A. Moralejo13 , V. Moreno18 , E. Moretti8 , T. Nagayoshi20 , V. Neustroev17 , A. Niedzwiecki11 , M. Nievas Rosillo10 , C. Nigro12 , K. Nilsson17 , D. Ninci13 , K. Nishijima20 , K. Noda13 , L. Nogués13 , S. Paiano4 , J. Palacio13 , D. Paneque8 , R. Paoletti14 , J. M. Paredes19 , G. Pedaletti12,? , M. Peresano2 , M. Persic2,26 , P. G. Prada Moroni22 , E. Prandini4 , I. Puljak6 , J. R. Garcia8 , I. Reichardt4 , W. Rhode5 , M. Ribó19 , J. Rico13 , C. Righi3 , A. Rugliancich14 , T. Saito20 , K. Satalecka12 , T. Schweizer8 , J. Sitarek11,20 , I. Šnidarić6 , D. Sobczynska11 , A. Stamerra3 , M. Strzys8 , T. Surić6 , M. Takahashi20 , L. Takalo17 , F. Tavecchio3 , P. Temnikov21 , T. Terzić6 , M. Teshima8,20 , N. Torres-Albà19 , A. Treves2 , S. Tsujimoto20 , G. Vanzo15 , M. Vazquez Acosta15 , I. Vovk8 , J. E. Ward13 , M. Will8 , D. Zarić6 , J. Becerra González15,27, ? , Y. Tanaka28 , R. Ojha27,29,30 , J. Finke31 (for the Fermi-LAT Collaboration), A. Lähteenmäki32,33,34 , E. Järvelä32,33 , M. Tornikoski32 , V. Ramakrishnan32 , T. Hovatta35 , S. G. Jorstad36,37 , A. P. Marscher37 , V. M. Larionov36,38 , G. A. Borman39 , T. S. Grishina36 , E. N. Kopatskaya36 , L. V. Larionova36 , D. A. Morozova36 , S. S. Savchenko36 , Yu. V. Troitskaya36 , I. S. Troitsky36 , A. A. Vasilyev36 , I. Agudo40 , S. N. Molina40 , C. Casadio41,40 , M. Gurwell42 , M. I. Carnerero43 , C. Protasio15,44 , and J. A. Acosta Pulido15,44 (Affiliations can be found after the references) Received 12 January 2018 / Accepted 19 March 2018 ABSTRACT Aims. The very high energy (VHE &100 GeV) γ-ray MAGIC observations of the blazar S4 0954+65, were triggered by an exceptionally high flux state of emission in the optical. This blazar has a disputed redshift of z = 0.368 or z > 0.45 and an uncertain classification among blazar subclasses. The exceptional source state described here makes for an excellent opportunity to understand physical processes in the jet of S4 0954+65 and thus contribute to its classification. Methods. We investigated the multiwavelength (MWL) light curve and spectral energy distribution (SED) of the S4 0954+65 blazar during an enhanced state in February 2015 and have put it in context with possible emission scenarios. We collected photometric data in radio, optical, X-ray, and γ-ray. We studied both the optical polarization and the inner parsec-scale jet behavior with 43 GHz data. Results. Observations with the MAGIC telescopes led to the first detection of S4 0954+65 at VHE. Simultaneous data with Fermi-LAT at high energy γ-ray(HE, 100 MeV < E < 100 GeV) also show a period of increased activity. Imaging at 43 GHz reveals the emergence of a new feature in the radio jet in coincidence with the VHE flare. Simultaneous monitoring of the optical polarization angle reveals a rotation of approximately 100◦ . Conclusions. The high emission state during the flare allows us to compile the simultaneous broadband SED and to characterize it in the scope of blazar jet emission models. The broadband spectrum can be modeled with an emission mechanism commonly invoked for flat spectrum radio quasars (FSRQs), that is, inverse Compton scattering on an external soft photon field from the dust torus, also known as external Compton. The light curve and SED phenomenology is consistent with an interpretation of a blob propagating through a helical structured magnetic field and eventually crossing a standing shock in the jet, a scenario typically applied to FSRQs and low-frequency peaked BL Lac objects (LBL). Key words. gamma rays: galaxies – galaxies: active – BL Lacertae objects: individual: S4 0954+65 ? Corresponding authors: G. Pedaletti (giovanna.pedaletti@desy.de), M. Manganaro (manganaro@iac.es), J. Becerra González (jbecerra@iac.es). Article published by EDP Sciences A30, page 1 of 15 A&A 617, A30 (2018) 1. Introduction Blazars are a subclass of active galactic nuclei (AGN) in which the relativistic jet presents a small viewing angle toward the observer and thus where relativistic effects on the observed emission are more extreme. Conventionally, blazars are subdivided in BL Lac objects and flat spectrum radio quasars (FSRQs) depending on the characteristic of their optical spectrum: while BL Lac objects are dominated by the featureless continuum emission from the jet, FSRQs show wide optical emission lines. The optical spectrum of a fraction of BL Lac objects presents features attributable to the host galaxy. The blazar S4 0954+65 hosts a black hole of mass MBH ∼ 3.3 × 108 M , estimated from the width of the Hα line (Fan & Cao 2004). The detection of the Hα line is not confirmed by Landoni et al. (2015; see the discussion on the redshift determination) so that the mass estimation cannot be confirmed either. This blazar presents strong variability in the optical band, already well studied by Wagner et al. (1990) and by Morozova et al. (2014). Intra night variability has been found both in optical and radio wavelengths (Wagner et al. 1993). The optical high brightness state of February 2015, presented here, is however exceptional for the object, with a brightening of more than three magnitudes in the R-band with respect to the average monitored state1 . This not only spurred many alerts in the community (see Astronomer’s Telegram (ATel) #6996, #7001, #7057, #7083, #7093; Carrasco et al. 2015; Stanek et al. 2015; Spiridonova et al. 2015; Bachev 2015; Ojha et al. 2015), but also the first and only detection of the object at very high energies (VHE, E & 100 GeV), thanks to observations by the MAGIC Telescopes. This detection by MAGIC and the multiwavelength (MWL) data collected alongside it are the focus of the present work. The source GRO J0957+65, detected with the Energetic Gamma Ray Experiment Telescope (EGRET) telescope on board the Compton Gamma-Ray Observatory, has been associated through optical and radio observations with S4 0954+65 by Mukherjee et al. (1995). S4 0954+65 has been afterward always included in the released catalogs of sources detected by the Large Area Telescope (LAT) instrument on board the Fermi satellite (Abdo et al. 2010; Nolan et al. 2012; Ackermann et al. 2013, 2016; Acero et al. 2015; Ajello et al. 2017), with the exclusion of the bright source list released after the first three months of Fermi-LAT data integration. The classification of the object, based on the available literature, is still unclear. In most of the ATels mentioned above S4 0954+65 is referenced as a FSRQ, but in most of the literature this is classified as a BL Lac object due to the small equivalent width of the emission lines in its spectrum (see, e.g., Stickel et al. 1991). Sambruna et al. (1996) classified the spectral energy distribution (SED) of S4 0954+65 as “FSRQ-like”, in a sample limited to the sources with a detection from EGRET data. It indeed presents a flatter spectral index than most BL Lac objects, in both X-ray and γ-ray bands (see Raiteri er al. 1999; and references therein). Among BL Lac objects, a further phenomenological subdivision can be made based on the frequency of the synchrotron peak, ranging from optical to X-ray frequency and identifying the classes of low-, intermediate-, or high-peaked BL Lac object (LBL, IBL, HBL, respectively). Ghisellini et al. (2011) classified this object as a LBL based on the SED. When including the kinematic features from the radio jet in the classification templates, 1 http://users.utu.fi/kani/1m/S4_0954+65.html A30, page 2 of 15 Hervet et al. (2016) classify this as their kinematic class II, mostly composed of FSRQ. S4 0954+65 can thus be interpreted as a transitional object between FSRQ and classical BL Lac objects. The most numerous extragalactic sources detected at VHE from Imaging Air Cherenkov Telescopes (IACTs), presently, belong to the HBL class. Therefore the VHE detection of an object such as S4 0954+65 provides a rare opportunity to study VHE emission conceivably produced in a different kind of environment. Indeed, while emission in HBL can mostly be satisfactorily modeled taking into account only processes in a compact feature in the jet, for FSRQs the inclusion of the interactions of such a feature with the surrounding ambient becomes of greater importance (see e.g., Tavecchio 2016). The structure of the broadband SED collected here will also be put in context with other common characteristics of a FSRQ classification, such as intrinsic brightness, peak of the synchrotron component and Compton dominance. The question of redshift for S4 0954+65 is also still not settled, as claims of line detection in the optical spectrum are not always confirmed. The redshift of the source was first determined at z = 0.368 by the identification of lines by Lawrence et al. (1986, 1996). Stickel et al. (1993) obtained, from different measurements, the same redshift estimate based on line identification. None of these lines were confirmed by the observations reported in Landoni et al. (2015), who instead pose a lower limit of z ≥ 0.45. The latter results were obtained with a superior resolution spectra. At the time of the observation the magnitude in R-band of the object was 15.5, while it is known from variability studies that it could be even two magnitudes lower. In the following we will adopt the redshift z = 0.368. The outline of this paper is as follows. In Sect. 2, we present the MAGIC telescopes and the relative data set on S4 0954+65. Section 3 reviews all the MWL data that were collected during this exceptional burst, whereas Sect. 4 discusses the implication of this burst for the source state and inner jet structure. Additional information on the MAGIC data analysis, the parameters derived from the radio data, and the full dataset for X-ray data will be found in Appendix A, B, and C respectively. 2. MAGIC observations The Major Atmospheric Gamma Imaging Cherenkov Telescopes (MAGIC) telescopes are an array of two IACTs located in the Island of La Palma (Spain) at an altitude of ∼2200 m asl. The system is sensitive down to an energy threshold of E ∼ 50 GeV (Aleksić et al. 2016) for low zenith angle observations. This is of particular relevance for the monitoring of variable sources and of those that tend to exhibit a steep spectrum at VHE. The full data have been analyzed using the standard MAGIC analysis chain and the MAGIC Standard Analysis Software (MARS; Zanin et al. 2013; Aleksić et al. 2016). The MAGIC collaboration supports a program of targets of opportunity (ToO), triggered by MWL monitoring. The ToO program was activated for observations of S4 0954+65 at the end of January 2015 after the first hints of enhanced optical state (triggered by the Tuorla monitoring in R-band, see Sect. 3.3). We observed the source with the MAGIC telescopes for 2 nights (MJD 57049-57050, 2015 January 27 and 28), for a total of 1 h high-quality dark time data, but obtained no detection. We resumed the ToO observations in February after the Tuorla monitoring revealed a very exceptional flux state, later confirmed by other monitoring programs (see Sect. 3.3). We obtained a dN/dE (TeV-1 cm-2 s-1) Nevents MAGIC Collaboration: S4 0954+65 February 2015 flare with MAGIC 1400 Time = 9.48 h Non = 2118; Noff = 1764.2 ± 18.8 1200 Nex = 353.8 Significance (Li&Ma) = 7.41σ 1000 800 600 MAGIC 10−8 MAGIC (EBL deabsorbed for z=0.368) 10−9 400 10−10 200 0 0 0.1 0.2 0.3 detection at a significance of ∼7.4σ from observations during 2015 February 14 (MJD 57067, ATel #7080 Mirzoyan et al. 2015). We continued observing S4 0954+65, barring adverse atmospheric conditions, until full moon days when standard MAGIC observations are not possible due to the elevated level of background light (last day of observation, with already large moonlight contamination, on 2015 March 1, MJD 57082). A detailed breakdown of the observation conditions and relative results can be found in Appendix A. The total excess from the dark-time data is consistent with a point source emission (see Fig. 1). No other significant emission is found in the field of view apart from the one coincident with S4 0954+65 at the center. The SED points presented in Sect. 4 below are derived for the day of the flare (MJD 57067, 2015 February 14), using only data taken in dark conditions (that allow for the lowest threshold and lowest systematic uncertainty, Appendix A). We followed the standard MAGIC unfolding procedure (Albert et al. 2007) to obtain the intrinsic spectrum. The γ-ray emission from sources at high redshift is absorbed via photon–photon pair production on photons from the extragalactic background light (EBL; see e.g., Finke et al. 2010; Domínguez et al. 2011). S4 0954+65 redshift is assumed to be z = 0.368. The spectral shape of the intrinsic emission, that is, after the correction for the EBL absorption, can be fitted with a simple power law !−Γ , Energy (GeV) 2 Fig. 1. Distribution of the squared angular distance (θ2 ) between the reconstructed event direction and the nominal source direction. The filled histogram is the background estimation, obtained from sky regions within the field of view with similar detector acceptance. We show only data taken in dark condition (condition 1, see Appendix A). The standard MAGIC low energy (LE) cuts are applied to the data (see Appendix A and Table A.1). The vertical line corresponds to the optimal cut (θ2 = 0.02 deg2 ) for point source analysis in LE cuts, used to derive significance values. dN E = N0 dE E0 102 0.4 θ [ deg ] 2 (1) with normalization N0 = 13.8 ± 2.1stat ± 1.5sys × 10−10 TeV−1 cm−2 s−1 at E0 = 0.15 TeV and spectral index Γ = 3.98 ± 0.67stat ± 0.15sys . The quoted systematic uncertainties are derived from the standard evaluation in MAGIC data presented by Aleksić et al. (2016). We note that the calculated systematic uncertainty on N0 does not contain the uncertainty on the energy scale, that is about 15%. The unfolded MAGIC spectrum is shown in Fig. 2. The unfolded observed spectrum, that Fig. 2. Spectrum for the VHE MAGIC detection. MAGIC data are for flare night only (2015 February 14, MJD 57067.14). Violet filled circles are for the unfolded observed points, while open circles are deabsorbed for EBL absorption (EBL model by Domínguez et al. 2011). The solid line is the fit for the observed points and the dashed line is the fit for the de-absorbed ones, with details in the text. is, without correcting for the EBL absorption, can be described also by a simple power law with N0 = 9.9 ± 1.5stat ± 1.1sys × −1 −10 −2 −1 10 TeV cm s at E0 = 0.15 TeV and spectral index Γ = 4.58 ± 0.66stat ± 0.15sys . 3. The multiwavelength coverage All the data presented in this section are collected to produce the light curves and SED, whose interpretation is later presented in Sect. 4. 3.1. Fermi-LAT The LAT on board the Fermi satellite scans the entire sky every 3 h. From the data of the first four years of operation, S4 0954+65 was detected with an average significance of 27.2σ in the energy range from 100 MeV to 300 GeV as reported in the Fermi-LAT Third Source Catalog (Acero et al. 2015; 3FGL). A dedicated analysis from MJD 56952 (2014 October 22) to MJD 57208 (2015 July 05) is presented in this work. We selected Pass 8 source class events within a 10◦ circular region centered on the position of S4 0954+65, in the energy range 0.1–500 GeV. The spectral analysis was performed through an unbinned likelihood fit, using the ScienceTools software package version v11-05-00 along with the instrument response functions P8R2_SOURCE_V6. The model of the likelihood fit includes a Galactic diffuse emission model and an isotropic component2 . In addition, we included the sources in the 3FGL catalog within a 20◦ circular region centered on S4 0954+65. The spectral indexes and fluxes of the 3FGL sources located within a region of 10◦ from S4 0954+65 were left free to vary, while the sources in the region from 10◦ to 20◦ were fixed to their catalog values. The results were obtained from two iterations of maximumlikelihood analysis, after the sources with a test statistics (Mattox et al. 1996) T S < 10 were removed. The strongest source located beyond 10◦ from S4 0954+65 is at an angular distance of 10.8◦ . This source has a variability index of 42.4 in the 3FGL catalog, that allows us to treat it as a nonvariable source and thus 2 Model available at https://fermi.gsfc.nasa.gov/ssc/data/ access/lat/BackgroundModels.html. A30, page 3 of 15 A&A 617, A30 (2018) to fix its spectral index and flux to the values reported in the 3FGL catalog. The light curve was calculated in day timescale bins, modeling the source with a single power-law spectrum (as it is also described in the 3FGL). Both the flux and spectral index of S4 0954+65 were left free during the likelihood fits, while the rest of the point sources were fixed and only the diffuse Galactic and isotropic models were allowed to vary. In case of T S < 4, an upper limit on the flux was calculated fixing the spectral index to 2.38 as given in the 3FGL catalog. The results are shown in Fig. 3. The figure also shows the light curve calculated in a 15-day bin as comparison. The light curve was obtained with the same procedure described above for the 1-day binning. During the HE flare in November 2014 (MJD 56976, ATel #6709; Krauss 2014) the LAT spectral index is compatible with its 3FGL value of 2.38 ± 0.04, averaged from four years of data. Moreover, the visibility of the source by MAGIC was at an unfavorable zenith angle of 60◦ (implying a high energy threshold). Therefore, no ToO observation was activated with MAGIC for this flare. MAGIC observations were activated later on during the strong flare on February 2015 when the LAT detected a hardening of the spectrum as shown previously by Tanaka et al. (2016) where the LAT analysis using Pass 7 reprocessed data is presented. The spectral analysis for the MWL SED corresponds to 1-day integration centered in the MAGIC observation (MJD 57067.14, 2015 February 14). From a first likelihood fit we found the best spectral fit was a power-law spectral index of 1.87 ± 0.09 (significantly harder than its average 3FGL value) and was fixed in the model for the spectral points calculation. Moreover, all the sources included in the model except the diffuse Galactic and isotropic models were also fixed. The source was detected during this period with a TS of 379.7. A curved spectral model is not significantly favored in this day (TS for a log parabola fit is T S LP = 380.10 to be compared with a simple power law fit with T S PWL = 379.74). 3.2. Swift dataset The 22 multi epochs event-list obtained by the X-ray Telescope (XRT; Burrows et al. 2004) on board the Neil Gehrels Swift Observatory satellite in the period of 2014 November 17 (MJD 56978.96395) to 2015 March 11 (MJD 57092.26632) with a total exposure time of ∼11.12 h were processed using the procedure described by Fallah Ramazani et al. (2017). All these observations had been performed in photon counting (PC) mode, with an average integration time of 1.8 ks each. The equivalent Galactic hydrogen column density is fixed to the value of nH = 5.17 × 1020 [cm−2 ] (Kalberla et al. 2005). The average integral photon X-ray flux (0.3–10 keV) in this period is 1.64 × 10−11 erg cm−2 s−1 . The X-ray flux is peaking at MJD 57070.76523 with F(0.3−10keV) = 3.18 × 10−11 erg cm−2 s−1 which is a factor of about two higher than the average flux of the analyzed period. The average flux outside the flare period (2006–2015) is F(0.3−10keV) = 4.3 × 10−12 erg cm−2 s−1 , that we derived from a sample of XRT data comprising 25 X-ray exposures in the XRT database, not including the 22 multi epochs event-list described above. This indicates that the source was clearly in its X-ray high state during the VHE γ-ray detection. The X-ray spectral index during the analyzed period varies between 1.15 ± 0.06 ≤ ΓX ≤ 1.82 ± 0.1. It is notable that the softest spectral index was obtained a night prior to the VHE γ-ray flare while the spectra starts to harden after 2015 February 14 and reach its historical hardest spectra ten days after the VHE A30, page 4 of 15 γ-ray flare. The X-ray spectra on the night before and after the VHE γ-ray flare can be well described with a power-law with spectral index of ΓX,Feb.13 = 1.82 ± 0.05 (χ2 /d.o.f. = 1.024/41) and ΓX,Feb.15 = 1.49 ± 0.07 (1.025/24 χ2 /d.o.f.) respectively. The full dataset analysis is given in Appendix C. The Swift satellite hosts an additional instrument, the Ultraviolet/Optical Telescope (UVOT; Poole et al. 2008). The data taken during the period of interest for this work have already been presented by Tanaka et al. (2016). They follow the behavior of the optical light curve that we present below. Therefore, they are not reproduced again nor shown in Fig. 3. The UVOT bands are however important for the SED modeling presented in Sect. 4 and will therefore be included there for MJD 57067 (2015 February 14, day of the VHE detection). The dataset presented by Tanaka et al. (2016) suffers from an incorrect exposure calculation by a factor of two, related to the deadtime correction, and thus a lower reconstructed flux. We, therefore have performed a reanalysis here for the two exposures taken with UVOT on MJD 57066.76. Data reduction has been done on all the available filters (v, b, u, w1, m2, w2), following the standard UVOT data analysis prescriptions3 . We present both exposures separately, due to the high variability in this night (e.g., for the V-band there is a variation of ∼0.3 magnitudes in ∼1.5 h). 3.3. The optical domain Optical data were collected with: 35 cm KVA telescope (La Palma Island, Spain) used in the Tuorla monitoring program; 1.8 m Perkins telescope of Lowell Observatory (Flagstaff, Arizona); 70 cm telescope AZT-8 at the Crimean Astrophysical Observatory (Nauchny, Russia); 40 cm telescope LX-200 of St. Petersburg State University (St. Petersburg, Russia); IAC80/Camelot at the Teide Observatory (Tenerife, Spain). The data analysis from KVA was performed with the semi-automatic pipeline using the standard analysis procedures (Nilsson et al. in prep). The differential photometry was performed using the comparison star magnitudes from Villata et al. (1997). For the Perkins telescope see Jorstad et al. (2010) and references therein. The details of observations and data reductions with AZT-8 and LX-200 are given by Larionov et al. (2008). IAC80/Camelot data were automatically processed by the pipeline Redcam and calibrated astrometrically using XParallax, both available at the telescope. Instrumental magnitudes for IAC80/Camelot data were extracted using Sextractor (Bertin & Arnouts 1996) and calibration of the source magnitude was obtained with respect to the reference stars provided by Raiteri er al. (1999). All the telescopes mentioned above provide R-band photometry. We have applied the calibration of Mead et al. (1990) for all optical measurements to transform magnitudes into flux densities, and dereddened the flux according to the absorption by Schlafly & Finkbeiner (2011). The host galaxy is not detected for this object. From the Perkins, AZT-8+ST7 and LX-200 telescopes we collect also polarization information. In Fig. 3 we show the optical photometry data and time evolution of the fractional linear polarization and the electrical vector position angle (EVPA) in R-band. The EVPA measurements have been arranged such to minimize the impact of the ±180◦ ambiguity, that is, adding or subtracting 180◦ whenever two subsequent measurements differ by more than 90◦ . In the same timeframe of the VHE detection and the optical flare, a substantial change in the optical EVPA can be identified (see Fig. 3). The EVPA rotation starts just before the optical 3 https://swift.gsfc.nasa.gov/analysis/ MAGIC Collaboration: S4 0954+65 February 2015 flare with MAGIC Fig. 3. MWL light curves and polarization evolution of S4 0954+65 ranging from MJD 56970 (2014 November 9) to MJD 57200 (2015 June 27). The energy range of each panel and the corresponding instrument can be found in the legend. Please refer to the text for details on the data taking and reduction for each instrument. and VHE flare and reaches a total change of roughly 100◦ . The optical flare in February 2015 is a factor of about 3 larger in flux than the 2011 flare (see Morozova et al. 2014), that was already exceptional and concurrent with a series of γ-ray flares evident in Fermi-LAT data. During the most extreme flare in 2011, the EVPA rotated by about 300◦ . A30, page 5 of 15 A&A 617, A30 (2018) Fig. 4. Sequence of total (contours) and polarized (segments) intensity images of S4 0954+658 at 43 GHz, convolved with a beam of 0.24 × 0.15 mas2 at PA = –10◦ . The global total intensity peak is 1606 mJy beam−1 and the global polarized intensity peak is 104 mJy beam−1 ; black line segments within each image show the direction of polarization and their length is proportional to the polarized intensity. The black horizontal line indicates the position of the core, A0, and gray, blue, and red circles show the locations of knots K14a,b and K15, respectively. The size of the circles is proportional to the estimated average size in each epoch. The detailed characteristics of the knots can be found in Table 1 and B.1. Table 1. Characteristics of the new radio knots observed from the jet of S4 0954+65. Knot Average flux mJy Maximum flux mJy Average PA deg (◦ ) Average size (FWHM) mas Proper motion mas/yr Apparent speed c Time of ejection MJD K14a K14b K15 120 ± 7 76 ± 25 109 ± 14 286 ± 10 118 ± 6 121 ± 5 −17.6 ± 2.4 −16.2 ± 2.6 −5.9 ± 1.9 0.15 ± 0.07 0.07 ± 0.06 0.05 ± 0.01 0.55 ± 0.04 0.59 ± 0.04 1.11 ± 0.08 12.49 ± 0.91 13.47 ± 0.86 25.27 ± 1.20 56708 ± 26 56891 ± 15 57081 ± 18 Notes. The evolution of parameters with the monitoring snapshots can be found in Appendix B. 3.4. Radio and millimeter ranges The source S4 0954+65 was monitored at 3.5 mm (86 GHz) and 1.3 mm (229 GHz) wavelengths from the IRAM 30 m Millimeter Radiotelescope under the Polarimetric Monitoring of AGN at Millimeter Wavelengths4 (POLAMI) program. The program monitors the four Stokes parameters of a sample of the brightest 40 northern blazars with a cadence better than a month (see Agudo et al. 2018a,b; Thum et al. 2018). Results from the observations are presented in Fig. 3. The data reduction, calibration, and flagging procedures were described in detail by Agudo et al. (2018a), see also Agudo et al. (2010, 2014). Figure 3 includes also the 1.3 mm flux density data that were obtained at the Submillimeter Array (SMA) located in Hawaii. S4 0954+65 is included in an ongoing monitoring program at the SMA to determine the fluxes of compact extragalactic radio sources that can be used as calibrators at millimeter wavelengths (Gurwell et al. 2007). Observations of available potential calibrators are from time to time observed for 3–5 min, and the measured source signal strength calibrated against known standards, typically solar system objects (Titan, Uranus, Neptune, or Callisto). Data from this program are updated regularly and are available at the SMA website5 . The largest flux in the considered period is at MJD 57072-57076, showing an increase of the flux between 1 and 3 mm wavelengths. We note, however, the lack of exactly simultaneous data to the MAGIC peak detection (MJD 57067). S4 0954+65 is monitored monthly by the Boston University (BU) group with the Very Long Baseline Array (VLBA) at 43 GHz within a sample of bright γ-ray blazars through the VLBA-BU-BLAZAR program6 . The VLBA data are calibrated 4 5 6 http://polami.iaa.es/ http://sma1.sma.hawaii.edu/callist/callist.html http://www.bu.edu/blazars/VLBAproject.html A30, page 6 of 15 and imaged in the same manner as discussed by Jorstad et al. (2005, 2017). The VLBA imaging monitoring program allows us to study the kinematics of the inner jet at pc scale. The inner jet has been monitored also for months after the VHE flare (see Fig. 4). In addition to the stable core at mm wavelengths (dubbed A0, see Fig. 4) it was possible to identify the emergence of three new knots whose characteristics are tabulated in Table 1. The nomenclature of the knots follows in sequential order from the beginning of the VLBA monitoring program. Previous knots characteristics can be found in Morozova et al. (2014). Of particular interest is knot K15, which is very compact, with a FWHM average size of 0.05 ± 0.01 mas and presents the largest apparent speed of (25.27 ± 1.20)c, cf. Fig. 5. The zeroepoch separation of this knot is consistent with the VHE flare considering its 18-day uncertainty. The intensity of the core is increasing in the epoch of MJD 57067 observation, but no significant change in the core polarization can be appreciated. The detailed information on the time evolution of the radio knot can be found in Table B.1, while the polarization evolution details are shown in Table B.2. No new knot appears in November 2014, when the source was high in the HE band as observed by Fermi-LAT but without optical enhancement. The zero epochseparation from the core of knots K14a,b are not coincident with the high state in Fermi-LAT data of November 2014, but happen months before. We analyzed Fermi-LAT data for the period included within the error band for K14a,b zero epoch-separation and found no particular enhancements. We also note the position angle of K15 with respect to the core, (PA = −5.9◦ ± 1.9◦ ). This is different than the values reconstructed from previous knots, ranging from roughly PA = −15◦ to PA = −25◦ in Morozova et al. (2014), that are in turn consistent with the values for K14a,b. The mean jet direction is at MAGIC Collaboration: S4 0954+65 February 2015 flare with MAGIC 4. Discussion The coverage of flaring states at VHE is helpful in understanding jet dynamics. We present a discussion of the SED for the day of the flare (2015 February 14). We did not attempt SED modeling for other days, for which the MAGIC data would provide only nonconstraining upper limits to emission at VHE. The day of the VHE detection is instead put in context with a longer time span behavior in the MWL dataset. However, the VHE sampling of the state is too scarce to attempt a numerical correlation study of the light curves. 4.1. Light-curve phenomenology Fig. 5. Apparent distance from the radio core A0 of the new emerging knots, K14a,b and K15, as a function of time. The images from which the apparent distances are calculated can be found in Fig. 4. We note that the K15 knot presents the highest apparent speed. PA ' −20◦ . A difference in PA and in apparent speed could be simply related to a small difference in the angle to the observer. However, the highest apparent speed can be used to estimate the Doppler factor, considering the upper limit to largest possible viewing angle θobs < arcsin(1/βapp ) and ultimately leading to δapp ∼ βapp . Applying this to the above mentioned knots (averaging the apparent speed to βapp ∼ 13c for K14a/b): θobs,K15 < 2.3◦ and δapp,K15 ∼ 25; θobs,K14 < 4.4◦ and δapp,K14 ∼ 13. The 37 GHz observations were made with the 13.7 m diameter telescope at Aalto University Metsähovi Radio Observatory. A detailed description of the data reduction and analysis is given by Teraesranta et al. (1998). The error estimate in the flux density includes contributions from the measurement RMS and the uncertainty of the absolute calibration. The S4 0954+65 observations were done as part of the regular monitoring program and the GASP-WEBT campaign. There are no strictly simultaneous 37 GHz data to the MAGIC detection, however an increase in flux can be seen when comparing observation taken 1 day before (2015 February 13, MJD 57066.15, Fν = 1.27 ± 0.07 Jy) and one day after the MAGIC detection (2015 February 15, MJD 57068.15, Fν = 1.65 ± 0.09 Jy). The Owens Valley Radio Observatory (OVRO) 40 m uses off-axis dual-beam optics and a cryogenic pseudo-correlation receiver with a 15.0 GHz center frequency and 3 GHz bandwidth. Calibration is achieved using a temperature-stable diode noise source to remove receiver gain drifts and the flux density scale is derived from observations of 3C 286 assuming the Baars et al. (1977) value of 3.44 Jy at 15.0 GHz. The systematic uncertainty of about 5% in the flux density scale is not included in the error bars. Complete details of the reduction and calibration procedure are found in Richards et al. (2011). The long-term monitoring program at OVRO monitors the variability of this source at 15 GHz over a longer time than what shown here. While it is obvious that the source was variable also during February 2015, it is not an exceptionally bright flux state of the source in the radio band. From a decade long monitoring, the source shows brighter levels (highest at F15 GHz = 2.53 Jy) and fainter levels (lowest at F15 GHz = 0.85 Jy). Both 15 and 37 GHz data seem to be in agreement with the behavior seen from mm wavelength data. Again we note the lack of strictly simultaneous data to the MAGIC peak detection (MJD 57067). The MWL light curves of the source for all the instruments involved in the present work are reported in Fig. 3, and cover a period of seven months, from MJD 56970 (2014 November 19) to MJD 57200 (2015 June 27). The red region in Fig. 3 indicates the time window where the knot K15 was ejected in the VLBA analysis, as reported in Table 1: a time range of 36 days centered in MJD 57081 (2015 February 28). The VHE detection and the enhanced activity in the other bands are found inside the K15 ejection time window, making this event important for the understanding of the whole scenario. The spectral index at HE as inferred from the Fermi-LAT data is harder than the average spectral index of Γ = 2.38 ± 0.04 from the 3FGL catalog dataspan. In the presented timeframe, the X-ray emission peaks around the observation on MJD 57070.76434 (2015 February 17), with a delay with respect to the detection in VHE. The approximately 3 hours of observations in VHE in the same night did not lead to a detection (see Table A.1). However, during the period of enhanced MWL activity, there is a clear hardening of the X-ray spectrum. Hardening at both X-ray and γ-ray energies points toward the emergence of a new component in the nonthermal spectrum. The optical band is very bright during the VHE detection, reaching peaks of more than 20 mJy of flux density when the average behavior of the source is found around a few mJy (see the optical monitoring from Tuorla observatory). The optical emission is polarized by a fraction of &10% and the polarization angle rotates by ∼100◦ during the flare: Blinov et al. (2015) have shown that from a systematic monitoring (Robopol monitoring) of both γ-ray loud and γ-ray quiet sources, only the former class of object displays polarization angle rotation similar to the one seen here for S4 0954+65. Blinov et al. (2015) studied the change of EVPA as a function of time for smooth changes of >90◦ . Requesting the same smoothness requirements, no smooth rotation of >90◦ can be identified in the dataset presented here, see Fig. 6. A variation of ∆EVPA ' 105◦ that is not smooth can, however, be identified between MJD 57060 and MJD 57075. This variation would imply a change of the EVPA curve slope of ∆EVPA /∆t = 7 deg day−1 , compatible with the bulk of the variations studied by Blinov et al. (2015). The rotations of the polarization angle are often physically linked to high flaring states of the objects in the γ-ray band. While individual occurrences of γ-ray flares and rotations cannot be firmly linked to each other, there is a low probability that all the occurrences are due to chance coincidence (from MonteCarlo simulations in Blinov et al. 2015). This hypothesis is still confirmed from 3 years of Robopol monitoring data in Blinov et al. (2018). Kiehlmann et al. (2017) also study whether a simple stochastic variation can account for the observed rotations in the Robopol monitoring. While their model is failing to recover all the observational characteristics in the monitoring, it also highlights a larger discrepancy from the expectations of stochastic model with A30, page 7 of 15 A&A 617, A30 (2018) Fig. 6. Light curves for R-band polarimetry of S4 0954+65. Please refer to the text for details on the data taking and reduction for each instrument. respect to the occurrence of large variations of EVPA (>90◦ ), however not significant. Smooth variations seem also to be more firmly linked to deterministic processes and not to a random walk effect (Kiehlmann et al. 2016). Robopol monitoring data are also used in Angelakis et al. (2016), to study the difference in the amount of polarization seen on average in γ-ray loud and γ-ray quiet sources. The median fraction variability of the S4 0954+65 dataset presented here is 16.4%. This value can be compared with the average 10% for the γ-ray loud subset of the Robopol monitoring and a value of 17.1% for S4 0954+65 computed for the observations on year 2013 and 2014. According to the interpretation by Angelakis et al. (2016), a higher fractional polarization is also expected in LSP/ISP blazars, due to the fact that in such sources the optical synchrotron emission relates to the peak synchrotron emission. Therefore, the particles associated with this emission are the most energetic, with faster cooling and thus probing a small volume of the emission region near the acceleration region, where it is expected to have a stronger ordered (helical) magnetic field, leading to higher polarization fraction. Images at 43 GHz show the emergence of new knots. In Morozova et al. (2014), a series of optical flares of S4 0954+65 in 2011 are studied, and the emission of knots is found correlated to the simultaneous flaring of the optical and HE bands. The maximum flux in the 2011 state is a factor of 3 lower in optical than the state presented here. The polarization fraction in this 2011 flare was similar to that seen in the present work. In Morozova et al. (2014) the chance coincidence of high optical state and knot emission has very low probability. The phenomenology of the 2015 flare described here agrees very well with the model put forward by Marscher et al. (2008) and applied to the S4 0954+65 dataset of Morozova et al. (2014). In that model, the flare is due to a newly appearing knot accelerating at the base of the jet and propagating through an helical flow streamline. The helical streamline can be expected due to the anchoring of the accelerating flow to the rotating base of the accretion disk or black hole magnetosphere, depending on modeling. The magnetic field topology in the jet is also helical and ordered. Geometrical effects and the propagation through the helical magnetic field account for the rotation of the EVPA. In Zhang et al. (2014), a model is proposed where the EVPA rotation is also related to the propagation through an helical magnetic field, but the streamline of propagation is not necessarily helical itself. In this model the magnitude of the swing can A30, page 8 of 15 depend on the assumptions on the settings for the flare, specifically the magnetic field strength and orientation, the acceleration efficiency and the continuous injection of freshly accelerated particles. The model described in Marscher et al. (2008) allows the emission at radio wavelengths in a flaring state which is not simultaneous with the VHE flare. In this scenario the radio activity could be delayed several days, even months, with respect to the VHE detection. This is expected if synchrotron self absorption is involved, and hence the emission region is located closer to the central engine than the radio core (A0 in Fig. 4). The peak of radio emission is expected to be lagging behind and appear when the disturbance has propagated further down the jet, where the absorption is not an issue. The X-ray emission peak, then, could also be delayed with respect to the optical outburst. As the X-ray emission is probably due to IC of an external soft photon field by electrons in the jet (see above), the X-ray variability traces both the accelerated particle distribution and a change in the soft photon field. This retraces similar interpretation drawn for flares of other sources where the dataset was however richer and more detailed (Marscher et al. 2008, 2010; Aleksić et al. 2014; Ahnen et al. 2017b). 4.2. Emission model for the flare SED The SED of blazars are dominated by their nonthermal emission and can usually be described by two broad components. The low energy nonthermal emission is explained as synchrotron emission, while the high energy emission is most commonly modeled through inverse Compton (IC) emission, where soft photons are upscattered to γ-ray energies by electrons within the jet emitting region. The origin of the soft photon field itself can vary for different blazar subclasses. In particular, for most of the classical BL Lac objects, the VHE emission can be reasonably modeled through synchrotron self-Compton emission (SSC, see e.g., Rees 1967; Maraschi et al. 1992). Instead, for the case of FSRQs, the modeling of the emission usually requires the inclusion of external soft photon fields from, for example, the infrared dusty torus or the optical-ultraviolet emission from the broad line region (BLR) for the IC process (see e.g., Tavecchio 2016). A broadband SED is compiled for 2015 February 14 (MJD 57067). We collect, from the MWL sample described in Sect. 3, the data closest in time to the MAGIC observation. Fermi-LAT data points are obtained from a 1-day integration centered on the MAGIC observation. The specific dates of other wavelength observations are given in the caption of Fig. 7. Tanaka et al. (2016) model the SED of S4 0954+65 during a similar integration time as the 2015 flare studied in this work. The data shown in Fig. 7 include, in addition to what is shown by Tanaka et al. (2016), the VHE data from the MAGIC observation, the AZT-8+ST7 and POLAMI data. Moreover, the Fermi-LAT data are reanalyzed as described in Sect. 3 to be centered at the MAGIC observation time and benefit from the latest Fermi-LAT Pass 8. The Swift-XRT and Swift-UVOT data are also reanalyzed for this work. Tanaka et al. (2016) report that a SSC modeling of the data is challenging, requiring very low magnetic field (B ∼ 1 µG in contrast to the B ∼ 1 G expected in blazar jet components). Alternatively, an External Compton (EC) modeling was able to reproduce the data. In their model, the soft photon field for the EC model was the dusty torus from the source. In Fig. 7, we plot the model from Tanaka et al. (2016). This model reproduces the Fermi-LAT and MAGIC data, although their paper did not MAGIC Collaboration: S4 0954+65 February 2015 flare with MAGIC Fig. 7. Spectral energy distribution for the VHE MAGIC detection. Red symbols are strictly simultaneous to the VHE detection, blue symbols are for data taken during the same day and black symbols are for the closest observations. MAGIC spectral data (red circles) are for flare night only (2015 February 14, MJD 57067.14). Red filled circles are for the unfolded observed data points. The red shaded band shows the region of additional systematic uncertainty. Fermi-LAT data are the Pass 8 data for 2015 February 14 (1-day integration centered on the MAGIC observation, blue squares). Swift-XRT data are for 2015 February 13 (MJD 57066.70992, blue squares). Swift-UVOT data are given for the two separate exposure taken on 2015 February 13 (MJD 57066.76, blue triangles and dark blue triangles). R-band data are for 14th Feb (Tuorla, MJD 57067.16375 and AZT-8+ST7 MJD 57067.1, red diamonds). POLAMI data are for the 18th February (MJD 57071.5, black stars at 100 and 300 GHz). OVRO data are for 2015 February 10 and 19 (black circles at 15 GHz). Metsähovi for 2015 February 13 and 15 (MJD 57066.15, MJD 57068.68, black squares at 37 GHz). The gray data are for NED (light) and SSDC (dark) SED historical data points. The model from Tanaka et al. (2016; gray dashed curve) as well as the model presented here (black solide curve) include an emission component from synchrotron plus inverse Compton on a dusty torus (see text for details and Table 2 for the values of the physical parameters). The effect of the EBL attenuation is included in the modeling using the model by Finke et al. (2010) and a redshift of z = 0.368. include any MAGIC data. However, the model fails to reproduce properly the optical observations. Such underestimation at optical frequencies in the model of Tanaka et al. (2016) is driven by a misreconstruction of the UVOT fluxes, explained in Sect. 3. With the reanalyzed UVOT dataset presented here, we use a new model, using the same code and most of the same assumptions as in Tanaka et al. (2016), including a redshift of z = 0.368. The code is explained in detail in Finke et al. (2008) and Dermer et al. (2009). We note that the presented SED model curves already include the effect of EBL absorption, meaning that the intrinsic emission is absorbed according to the EBL model by Finke et al. (2010). The new EC model provides a good description of the MWL data and is shown in Fig. 7. The parameters of both models are reported in Table 2. The break in the underlying electron population is similar to what expected by classical cooling, with the slope of the electron distribution before of the break (s1 ) and after the break (s2 ) differing by s2 − s1 = 1.2. We also tested a model with a larger break (s2 − s1 = 1.6) and longer variability timescale (105 s) which was better able to reproduce the UV band data. However, the larger s2 − s1 disagrees with expectations from synchro-Compton losses. Further, this model underestimates the MAGIC data and overestimates the emission at 1 mm. We therefore do not present this model here. The use of VHE spectral information is crucial to model the falling part of the high energy peak of the blazars SEDs, which is crucial to constrain the most energetic electrons within the leptonic framework scenario (SSC and EC models). As mentioned in the introduction, the classification of a blazar can be aided by the study of its SED characteristics. According to the SED model presented above, the peak of the synchrotron emission is at νsyn ∼ 8 × 1014 Hz, making it an intermediate synchrotron peaked BL Lac object (Ackermann et al. 2015)7 . The Compton dominance, calculated comparing the luminosity at the peak of the synchrotron emission to that of the IC peak, is LIC /Lsyn ∼ 7. Such Compton dominance value is at least 3.5 times the values obtained by Finke (2013) for long-term blazar studies. 7 Intermediate-synchrotron-peaked blazar (ISP) are defined with restframe synchrotron peak frequencies of 1014 Hz < νsyn < 1015 Hz. A30, page 9 of 15 A&A 617, A30 (2018) Table 2. SED model parameters. Parameter Symbol Redshift Bulk Lorentz factor Doppler factor Variability timescale [s] Comoving radius of blob [cm] Magnetic field [G] Low-energy electron spectral index High-energy electron spectral index Minimum electron Lorentz factor Break electron Lorentz factor Maximum electron Lorentz factor Black hole mass [M ] Disk luminosity [erg s−1 ] Inner disk radius [Rg ] Seed photon source energy density [erg cm−3 ] Seed photon source photon energy [me c2 units] Dust Torus luminosity [erg s−1 ] Dust Torus radius [cm] Dust temperature [K] Jet power in magnetic field [erg s−1 ] Jet power in electrons [erg s−1 ] z Γ δD tv R0b B s1 s2 0 γmin 0 γbrk 0 γmax MBH Ldisk Rin useed seed Ldust Rdust T dust P j,B P j,e 5. Conclusions The census of extragalactic objects that present VHE emission is still limited. We present here the first detection at VHE of the blazar S4 0954+65 obtained through observations with the MAGIC Telescopes. The observations were conducted during an exceptional flare of the source in February 2015, originally identified in the optical band. We collected MWL simultaneous data to better characterize the state of the source. The HE emission is also found in elevated state from the analysis of Fermi-LAT data, which reveal the hardest state of the HE emission to be concurrent with the detection at VHE. The X-ray emission peak is delayed by few days with respect to the VHE detection and shows a trend of spectral hardening during the period presented here. The radio and mm wavelength emission reveal a moderate elevation of the flux, that is however not exceptional in the long term behavior of the source. The source is classified in the literature as a BL Lac, but we have shown here that it presents similarities with the FSRQ class. Results from the monitoring of optical polarization and 43 GHz jet component analysis were compared to archival observation of S4 0954+65 and of statistical behaviour of other sources. Three main measurements were considered: the day of the VHE detection of S4 0954+65 is included in the error box for the zero epoch separation of knot K15; the optical polarization fraction is increasing in the same period; a rotation of optical EVPA of ∼100◦ can be identified, also in the same period, possibly related to the helical structure of the magnetic field in the acceleration region. We discuss how these measurements point to a common behavior with ISP/LSP sources. Both, the best emission model (EC on dust torus) and the MWL light curve behavior show points of contact with other sources that are either clear FSRQ (like PKS 1510-089) or are transitional objects (like BL Lac itself). This is also supported from the moderate A30, page 10 of 15 Model A Tanaka et al. (2016) 0.368 30 30 1.0 × 105 6.6 × 1016 0.6 2.4 4.5 1.0 8.0 × 103 2.0 × 104 3.4 × 108 3.0 × 1043 6.0 2.4 × 10−4 7.5 × 10−7 3.9 × 1042 2.1 × 1017 1500 1.0 × 1046 1.1 × 1045 Model B This work 35 35 4 × 104 3.0 × 1016 0.4 2.4 3.6 1.0 4.0 × 103 4.0 × 104 4.4 × 10−5 5 × 10−7 1.5 × 1042 6.1 × 1017 1000 1.4 × 1045 6.6 × 1045 Compton dominance in the SED model presented here and the fact that the synchtrotron peak show that the source can be classified as ISP source. The work presented here reiterates the importance of VHE γ-ray and detailed MWL studies of blazars during different flux states to test their intrinsic characteristics and shed light on the physical processes taking place within their jets. Acknowledgements. We would like to thank the Instituto de Astrofísica de Canarias for the excellent working conditions at the Observatorio del Roque de los Muchachos in La Palma. The financial support of the German BMBF and MPG, the Italian INFN and INAF, the Swiss National Fund SNF, the ERDF under the Spanish MINECO (FPA2015-69818-P, FPA2012-36668, FPA2015-68378-P, FPA2015-69210-C6-2-R, FPA2015-69210C6-4-R, FPA2015-69210-C6-6-R, AYA2015-71042-P, AYA2016-76012-C3-1-P, ESP2015-71662-C2-2-P, CSD2009-00064), and the Japanese JSPS and MEXT is gratefully acknowledged. This work was also supported by the Spanish Centro de Excelencia “Severo Ochoa” SEV-2012-0234 and SEV-2015-0548, and Unidad de Excelencia “María de Maeztu” MDM-2014-0369, by the Croatian Science Foundation (HrZZ) Project IP-2016-06-9782 and the University of Rijeka Project 13.12.1.3.02, by the DFG Collaborative Research Centers SFB823/C4 and SFB876/C3, the Polish National Research Centre grant UMO-2016/22/M/ST9/00382 and by the Brazilian MCTIC, CNPq, and FAPERJ. The Fermi LAT Collaboration acknowledges generous ongoing support from a number of agencies and institutes that have supported both the development and the operation of the LAT as well as scientific data analysis. These include the National Aeronautics and Space Administration and the Department of Energy in the United States, the Commissariat à l’Energie Atomique and the Centre National de la Recherche Scientifique/Institut National de Physique Nucléaire et de Physique des Particules in France, the Agenzia Spaziale Italiana and the Istituto Nazionale di Fisica Nucleare in Italy, the Ministry of Education, Culture, Sports, Science and Technology (MEXT), High Energy Accelerator Research Organization (KEK), and Japan Aerospace Exploration Agency (JAXA) in Japan, and the K. A. Wallenberg Foundation, the Swedish Research Council and the Swedish National Space Board in Sweden. This work performed in part under DOE Contract DE-AC02-76SF00515. This research has made use of the NASA/IPAC Extragalactic Database (NED), which is operated by the Jet Propulsion Laboratory, California Institute of Technology, under contract with the National Aeronautics and Space Administration. Part of this work MAGIC Collaboration: S4 0954+65 February 2015 flare with MAGIC is based on archival data, software, or online services provided by the Space Science Data Center - ASI. The OVRO 40-m monitoring program is supported in part by NASA grants NNX08AW31G, NNX11A043G, and NNX14AQ89G, and NSF grants AST-0808050 and AST-1109911 The research at Boston University was supported by NASA Fermi Guest Investigator program grant 80NSSC17K0694 and US National Science Foundation grant AST-1615796. The VLBA is an instrument of the Long Baseline Observatory. The Long Baseline Observatory is a facility of the National Science Foundation operated by Associated Universities, Inc. This paper is partly based on observations carried out with the IRAM 30 m Telescope. IRAM is supported by INSU/CNRS (France), MPG (Germany) and IGN (Spain). IA acknowledges support by a Ramón y Cajal grant of the Ministerio de Economía, Industria y Competitividad (MINECO) of Spain. The research at the IAA–CSIC was supported in part by the MINECO through grants AYA2016–80889–P, AYA2013–40825–P, and AYA2010–14844, and by the regional government of Andalucía through grant P09–FQM–4784. St. Petersburg University team acknowledges support from Russian Science Foundation grant 17-12-01029. The Submillimeter Array is a joint project between the Smithsonian Astrophysical Observatory and the Academia Sinica Institute of Astronomy and Astrophysics and is funded by the Smithsonian Institution and the Academia Sinica. References Abdo, A. A., Ackermann, M., Ajello, M., et al. 2010, ApJ, 188, 405 Acero, F., Ackermann, M., Ajello, M., et al. 2015, ApJS, 218, 23 Ackermann, M., Ajello, M., Allafort, A., et al. 2013, ApJS, 209, 34 Ackermann, M., Ajello, M., Atwood, W. 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MAGIC data summary for the observation of S4 0954+65 from 2015 January 27 to 2015 March 1. MJD Observation time [h] Significance σ LE: hadr < 0.28 size > 60phe Significance FR σ FR: hadr < 0.16 size > 300phe F(>150GeV) cm−2 s−1 0.33 0.68 2.05 2.86 0.80 2.53 0.32 2.91 0.97 0.94 0.64 –0.82 7.98 7.26 0.35 3.19 –0.04 2.12 2.41 2.79 0.43 0.19 0.20 –0.09 –0.67 1.16 0.42 1.00 1.62 –0.50 <3.0 × 10−11 <1.4 × 10−11 (3.1 ± 0.6) × 10−11 – <5.0 × 10−11 (1.2 ± 0.5) × 10−11 <5.0 × 10−11 <2.0 × 10−11 <3.5 × 10−11 F(>250GeV) < 2.1 × 10−11 obs condition 57049.176 (1) 57050.164 (1) 57067.139 (1) "(1+3) "(3) 57068.154 (1) 57069.099 (2) 57070.147 (1) 57077.098 (1) 57082.153 (4) Notes. Days of observations are listed along with the data qualification (see text for details), length of observation and significance of detection for different analysis cuts. For detections, also the integrated flux above 150 GeV is given. In the instances of nondetection, we provide a 95% confidence level upper limit. The MAGIC telescopes are supported by an extensive weather monitor program. Atmospheric transmission at different heights within the MAGIC field of view is obtained with the use of a LIDAR (for details on this see Fruck & Gaug 2015). For data quality selection we consider the transmission measured at a height of 9 km, with T 9km = 1 representing a perfectly clear sky and T 9km = 0 a complete opacity. MAGIC can carry out observations also during partial moonlight, with the drawback of having a higher energy threshold and larger systematic errors due to a higher contamination from the elevated night sky background (NSB), see Ahnen et al. (2017a). The brightness of the NSB can be monitored from the average current in the camera (DC). S4 0954+65 was observed in a zenith range ranging from 35◦ to 50◦ , for a total of 12.5 h of data, of which approximtely one hour was lost due to bad weather. In the following we refer to the different observation conditions of our data set as follows: – good dark data: data taken with dark sky (DC < 1.5 µA) and good atmospheric condition (T 9km > 0.85), used for detection and spectral reconstruction; – dark data needing atmospheric correction: data taken with dark sky (DC < 1.5 µA) but under nonoptimal weather conditions (0.55 < T 9km < 0.85), used for detection and spectral reconstruction after atmospheric correction; – good low moon data: data taken with elevated NSB due to moonlight (1.5 µ < DC < 4 µA) and good atmospheric condition (T 9km > 0.85), used only for detection in this particular dataset; – good moon data: data taken during high NSB due to moonlight (DC > 4 µA) and good atmospheric condition (T 9km > 0.85), used only for detection. The subsample of dataset selected with condition (1) (9.48 h of good quality data) has been analyzed with the standard MAGIC analysis chain (Zanin et al. 2013). The subsample of dataset selected with condition (2) (0.32 h of data) follows the same analysis chain until the estimation of the energy for the events and evaluation of the flux. For this last step, the estimated energy and the effective area are corrected taking into account the enhanced atmospheric absorption (for validation of the procedure see Fruck & Gaug 2015). The subsample of dataset selected with condition (3) is applicable only at the day of 14th February, with the first VHE detection. The detection can be claimed from dark data alone (i.e., selected with condition 1), but an extra 0.81 h of data were taken under low moonlight. The data are presented here for completeness, but are not used for spectral reconstruction so as not to increase the systematic error and energy threshold. The subsample of dataset selected with condition (4) (0.94 h of data) requires a special analysis that takes care of the effect of moonlight on data taking, reconstruction and analysis. Details of the procedure can be found in Ahnen et al. (2017a). The detailed breakdown of significances and estimated VHE fluxes is given in Table A.1. Numbers are presented for the socalled low energy (LE) and full range (FR) cuts. The LE cuts are optimized for an energy range of E & 100 GeV and are particularly appropriate for steep spectrum sources, while FR cuts are optimized for an energy range of E & 250 GeV. The cuts are applied on 2 parameters: the “size” parameter, integrated charge (in photoelectrons) in the cleaned shower image; the “hadronness” parameter, computed from the gamma-hadron separation random forest (RF), with a value ranging from zero for the most gamma-like images to one for the most hadronlike images. Indeed the standard MAGIC analysis chain relies on RF techniques to discriminate among gamma and hadronic shower and to better reconstruct the event directions. Lookup tables are used for energy estimation. This is achieved starting from a parametrization of the shower images in the detector. The significance of signal is then calculated with Eq. (17) from Li & Ma (1983) and using five regions of equal size and distance to the center of camera as the signal region for background estimation. Fluxes are calculated above an energy threshold of 150 GeV, which corresponds to the peak of the differential energy distribution of the excess events as a function of estimated event energy. The high energy threshold is due to the high zenith angle of the observation. We note that for data of condition 4, strong moon, we apply an additional minimum cut in the “size” parameter (“size” > 150 phe) of the reconstructed Cherenkov image as prescribed by the moonlight-adapted analysis. This increases the energy threshold to a value of ∼250 GeV. In case of nondetection, we provide 95% confidence level upper limits to the flux, calculated following Rolke et al. (2005), considering a systematic error on flux estimation of 30% (Aleksić et al. 2016). A30, page 13 of 15 A&A 617, A30 (2018) Appendix B: Additional VLBA derived parameters Table B.1. Time evolution of characteristics of the new radio knots observed from the jet of S4 0954+65. Epoch 23 Sep 2014 2014.7288 2014.7288 2014.7288 2014.7288 15 Nov 2014 2014.8740 2014.8740 2014.8740 2014.8740 5 Dec 2014 2014.9288 2014.9288 2014.9288 2014.9288 29 Dec 2014 2014.9945 2014.9945 2014.9945 2014.9945 14 Feb 2015 2015.1233 2015.1233 2015.1233 2015.1233 11 Apr 2015 2015.2767 2015.2767 2015.2767 2015.2767 11 May 2015 2015.3589 2015.3589 2015.3589 2015.3589 9 Jun 2015 2015.4385 2015.4385 2015.4385 2015.4385 2 Jul 2015 2015.5014 2015.5014 2015.5014 2015.5014 MJD Flux(Jy) x y R(mas) PA(deg) Size(mas) Knot 56924 56924 56924 56924 0.558 0.118 0.160 0.071 0.000 –0.018 –0.077 –0.246 0.000 0.084 0.289 0.533 0.000 0.086 0.300 0.587 0.0 –12.1 –14.9 –24.8 0.016 0.058 0.066 0.269 A0 K14b K14a K13 56977 56977 56977 56977 0.613 0.057 0.089 0.025 0.000 –0.040 –0.094 –0.328 0.000 0.147 0.336 0.576 0.000 0.152 0.349 0.663 0.0 –15.4 –15.7 –29.7 0.024 0.060 0.077 0.226 A0 K14b K14a K13 56997 56997 56997 56997 0.655 0.092 0.105 0.046 0.000 –0.025 –0.094 –0.333 0.000 0.109 0.319 0.636 0.000 0.112 0.332 0.717 0.0 –12.9 –16.4 –27.6 0.025 0.065 0.069 0.366 A0 K14b K14a K13 57021 57021 57021 57021 0.664 0.079 0.114 0.038 0.000 –0.084 –0.124 –0.456 0.000 0.267 0.419 0.688 0.000 0.280 0.437 0.826 0.0 –17.5 –16.5 –33.5 0.026 0.105 0.115 0.587 A0 K14b K14a K13 57067 57067 57067 57067 0.899 0.070 0.286 0.031 0.000 –0.090 –0.158 –0.426 0.000 0.302 0.463 0.759 0.000 0.315 0.489 0.870 0.0 –16.5 –18.9 –29.3 0.021 0.123 0.196 0.420 A0 K14a K14b K13 57123 57123 57123 57123 0.679 0.119 0.111 0.084 0.000 –0.008 –0.099 –0.156 0.000 0.120 0.368 0.533 0.000 0.121 0.381 0.555 0.0 –3.9 –15.0 –16.3 0.018 0.048 0.110 0.137 A0 K15 K14b K14a 57153 57153 57153 57153 0.354 0.103 0.052 0.084 0.000 –0.017 –0.121 –0.177 0.000 0.204 0.388 0.568 0.000 0.205 0.407 0.595 0.0 –4.7 –17.4 –17.3 0.028 0.040 0.101 0.195 A0 K15 K14b K14a 57182 57182 57182 57182 0.440 0.121 0.050 0.097 0.000 –0.037 –0.166 –0.232 0.000 0.302 0.458 0.634 0.000 0.304 0.487 0.675 0.0 –6.9 –19.9 –20.1 0.016 0.049 0.112 0.253 A0 K15 K14b K14a 57205 57205 57205 57205 0.469 0.092 0.059 0.060 0.000 –0.050 –0.178 –0.269 0.000 0.360 0.514 0.651 0.000 0.363 0.544 0.704 0.0 –8.0 –19.1 –22.4 0.014 0.051 0.176 0.238 A0 K15 K14b K14a Notes. For each identified component and for each epoch (i.e., observation), we present: flux, position with respect to core AO, projected size, and position angle. The detailed information on the time evolution of the radio knot can be found in Table B.1, while the polatization evolution details are shown in Table B.2. A30, page 14 of 15 Table B.2. Time evolution of polarization parameters (percentage and angle) for the core A0 observed from the jet of S4 0954+65. MJD P ± dP(%) EVPA ± dE(deg) 56924 56977 56997 57021 57067 57123 57153 57182 57205 5.22 ± 0.77 6.99 ± 0.80 7.74 ± 0.72 8.15 ± 0.69 9.78 ± 0.94 8.52 ± 0.41 2.38 ± 0.83 3.19 ± 0.63 1.06 ± 0.56 5.25 ± 4.23 16.86 ± 3.28 –16.57 ± 2.64 –7.33 ± 2.43 0.31 ± 2.74 –7.03 ± 1.37 –7.00 ± 9.93 –9.34 ± 5.66 –51.79 ± 15.3 MAGIC Collaboration: S4 0954+65 February 2015 flare with MAGIC Appendix C: Swift-XRT full dataset Table C.1. S4 0954+65 Swift-XRT exposures. F(2–10 keV) [10−12 ] [erg cm−2 s−1 ] F(0.3–10 keV) [10−12 ] [erg cm−2 s−1 ] Index χ2RED DOF 8620.6 2.76+0.22 −0.19 4.08+0.20 −0.21 1.62 ± 0.06 0.69 30 00035381001 54187.38 3578.6 2.00+0.34 −0.31 3.12+0.42 −0.33 1.72 ± 0.12 1.16 9 00036326001 54475.05 3748.5 1.61+0.23 −0.23 2.68+0.29 −0.26 1.82 ± 0.11 1.11 10 00036326002 2.47+0.47 −0.41 3.71+1.68 −1.14 1.08+0.12 −0.12 1.81+0.42 −0.27 1.40+0.51 −0.36 3.72+0.39 −0.38 3.98+0.25 −0.25 3.47+0.22 −0.27 2.95+1.14 −0.75 1.67+0.29 −0.35 2.16+0.43 −0.48 5.36+0.74 −0.65 1.17+0.51 −0.32 12.08+0.95 −0.80 3.74+0.35 −0.29 3.89+0.49 −0.45 11.15+0.84 −0.82 10.35+0.91 −0.84 16.22+1.06 −1.18 21.32+1.42 −1.47 14.92+1.17 −0.94 13.59+1.23 −1.43 10.89+1.38 −0.90 15.54+1.07 −1.34 14.28+1.21 −1.05 12.62+1.59 −1.31 22.37+1.40 −1.70 12.77+0.83 −0.72 16.54+1.55 −1.13 7.93+0.83 −0.83 9.30+1.19 −1.18 8.69+1.30 −1.82 8.47+0.99 −0.95 7.19+1.42 −1.11 8.66+0.83 −0.90 5.18+1.88 −1.28 3.78+0.78 −0.51 2.82+0.56 −0.44 4.07+1.25 −1.09 4.14+1.16 −1.01 4.19+0.74 −0.55 6.49+1.57 −1.51 3.03+0.55 −0.46 3.11+0.57 −0.61 3.61+0.47 −0.39 1.60 ± 0.12 0.25 8 00036326003 4.84+1.53 −0.96 1.34 ± 0.23 0.89 3 00036326004 1.67+0.13 −0.15 1.70 ± 0.08 1.32 14 00036326005 2.48+0.37 −0.38 1.46 ± 0.16 0.17 4 00036326006 Date-Time MJD EXP 2006-07-04T00:49:40 53920.04 2007-03-28T09:06:11 2008-01-10T01:09:39 [s] 2008-01-11T01:20:01 54476.06 2891.9 2008-01-15T16:10:28 54480.67 1513.4 2009-01-09T10:57:37 54840.46 10524.0 2009-11-01T22:49:53 55136.95 2784.5 2009-11-05T08:26:28 55140.35 2906.9 2009-12-12T18:45:25 55177.78 3848.3 2010-01-23T14:26:34 55219.60 8873.9 2010-03-12T05:57:53 55267.25 7980.6 2011-10-13T04:06:03 55847.17 1563.3 2011-10-14T13:19:43 55848.56 3074.2 2014-04-28T14:10:59 56775.59 1540.8 2014-05-28T20:08:46 56805.84 1920.4 2014-06-25T20:09:39 56833.84 1670.7 2014-11-17T23:06:57 56978.96 3262.1 2014-11-22T13:31:43 56983.56 4108.0 2015-01-27T19:19:19 57049.81 1942.9 2015-02-13T17:01:10 57066.71 1962.9 2015-02-15T07:15:48 57068.30 1893.0 2015-02-16T13:17:09 57069.55 1905.4 2015-02-17T18:20:49 57070.77 1775.6 2015-02-18T10:00:55 57071.42 2092.7 2015-02-19T08:22:52 57072.35 1071.3 2015-02-20T16:19:13 57073.68 983.9 2015-02-21T19:29:42 57074.81 1735.6 2015-02-22T14:41:28 57075.61 1937.9 2015-02-23T03:29:19 57076.15 994.0 2015-02-24T05:07:19 57077.21 1371.0 2015-03-04T19:34:43 57085.82 2205.1 2015-03-05T06:44:02 57086.28 1578.3 2015-03-06T11:26:12 57087.48 1875.5 2015-03-07T10:04:13 57088.42 1311.1 2015-03-08T14:43:10 57089.61 1210.7 2015-03-09T16:02:02 57090.67 1838.0 2015-03-10T06:27:07 57091.27 1098.8 2015-03-11T06:22:22 57092.27 1863.0 2015-06-21T17:29:18 57194.73 1465.9 2015-06-22T20:25:32 57195.85 1965.4 2015-06-24T04:33:29 57197.19 2202.6 2015-06-25T02:44:56 57198.12 1635.7 2015-06-26T01:06:48 57199.05 986.4 2015-06-27T04:36:46 57200.19 1808.0 2015-06-28T00:54:48 57201.04 1773.1 2015-06-29T05:54:31 57202.25 1748.1 2015-06-30T09:00:28 57203.38 1962.9 OBSID 2.06+0.42 −0.40 1.60 ± 0.21 1.31 2 00036326007 4.95+0.35 −0.33 1.39 ± 0.08 0.89 15 00036326008 5.63+0.28 −0.23 1.52 ± 0.04 1.18 48 00090100001 4.89+0.19 −0.24 1.52 ± 0.05 1.69 38 00090100003 3.95+1.07 −0.76 1.40 ± 0.23 1.56 2 00036326009 2.49+0.38 −0.31 1.64 ± 0.14 1.76 6 00036326010 3.06+0.52 −0.43 1.53 ± 0.16 1.81 3 00091892001 7.40+0.64 −0.58 1.48 ± 0.10 1.47 12 00091892002 2.05+0.50 −0.34 1.90 ± 0.23 0.32 2 00091892003 15.07+0.74 −0.81 1.20 ± 0.06 1.21 32 00033530001 5.59+0.38 −0.33 1.64 ± 0.07 1.65 23 00033530002 6.02+0.59 −0.56 1.70 ± 0.10 1.31 10 00033530003 18.45+0.92 −0.77 1.81 ± 0.05 1.02 41 00033530004 14.38+0.84 −0.84 1.49 ± 0.07 1.02 24 00033530008 22.73+1.12 −1.10 1.51 ± 0.05 0.70 39 00033530009 31.82+1.67 −1.31 1.64 ± 0.05 1.03 36 00033530010 20.95+0.97 −1.12 1.51 ± 0.05 1.25 36 00033530011 19.35+1.41 −1.30 1.54 ± 0.08 0.81 18 00033530012 15.92+1.59 −1.16 1.60 ± 0.09 0.41 14 00033530013 20.09+1.26 −1.16 1.31 ± 0.06 1.03 28 00033530014 18.66+1.20 −0.95 1.34 ± 0.05 1.37 30 00033530015 16.81+1.65 −1.47 1.39 ± 0.09 0.93 13 00033530017 27.47+2.16 −1.78 1.15 ± 0.06 1.05 27 00033530018 18.42+0.86 −0.85 1.57 ± 0.05 1.13 40 00033530019 20.93+1.53 −1.23 1.25 ± 0.06 1.03 24 00033530020 10.88+0.85 −1.01 1.46 ± 0.08 0.96 18 00033530021 14.04+1.22 −1.00 1.66 ± 0.10 0.99 13 00033530022 11.41+1.46 −1.42 1.35 ± 0.13 1.13 6 00033530023 10.76+0.96 −0.78 1.26 ± 0.08 0.67 14 00033530024 9.27+1.41 −1.19 1.30 ± 0.13 1.00 6 00033530025 11.49+0.83 −0.83 1.38 ± 0.07 0.48 16 00033530026 6.38+1.99 −1.33 1.16 ± 0.24 0.70 1 00033829001 5.12+0.58 −0.55 1.43 ± 0.13 0.80 6 00033829002 00033829004 4.06+0.61 −0.49 1.57 ± 0.15 0.72 6 5.30+1.26 −1.01 1.33 ± 0.19 0.54 4 00033829005 5.83+1.49 −0.97 1.52 ± 0.20 1.04 3 00033829006 5.70+0.86 −0.72 1.44 ± 0.12 1.10 7 00033829007 7.62+1.78 −1.57 0.99 ± 0.19 0.34 4 00033829008 4.40+0.59 −0.59 1.58 ± 0.13 1.20 6 00033829009 4.21+0.76 −0.56 1.43 ± 0.15 0.76 5 00033829010 Notes. For each observation, identified by its date and Swift observation identifier (OBSID), we present: the duration of the exposure (EXP), the integrated energy flux in two energy bands, the best-fit spectral index, the χ2 and degrees of freedom of the fit (DOF). Table C.1 collects all the analyzed exposures for the Swift-XRT dataset described in Sect. 3. Fluxeshave been extracted from a 20 pixel circular aperture. A different aperture was used on 2015 February 17 (MJD 57070.76), due to pile-up effects. A30, page 15 of 15
https://openalex.org/W4310335255	https://wwwnc.cdc.gov/eid/article/28/12/pdfs/22-1144.pdf	English	null	Laboratory Features of Trichinellosis and Eosinophilia Threshold for Testing, Nunavik, Quebec, Canada, 2009–2019	Emerging infectious diseases	2,022	cc-by	2,835	RESEARCH LETTERS T richinella nativa infection is associated with inges tion of parasitized sylvatic animals and periodic outbreaks among residents of northern Canada (1–3). In the Arctic region of Nunavik in Quebec, outbreaks associated with polar bear and walrus consumption have prompted public health interventions, includ ing a highly successful community-led active surveil lance system that examines hunted meat for evidence of Trichinella encystment (4,5). We report a 10-year case series of Trichinella infection in Nunavik and de scribe the laboratory features. Eosinophilia is a well- characterized feature of infection that is readily avail able for most cases. We performed receiver operating characteristic (ROC) analysis to define an optimal threshold of eosinophilia to prompt reflex Trichinella antibody testing and rapid reporting to public health authorities for timely outbreak investigation (1–3). Tt p g j 8. Martín-Carrillo N, Feliu C, Abreu-Acosta N, Izquierdo-Rodriguez E, Dorta-Guerra R, Miquel J, et al. A peculiar distribution of the emerging nematode Angiostrongylus cantonensis in the Canary Islands (Spain): recent introduction or isolation effect? Animals (Basel). 2021;11:1267. https://doi.org/10.3390/ani11051267 p g j 8. Martín-Carrillo N, Feliu C, Abreu-Acosta N, Izquierdo-Rodriguez E, Dorta-Guerra R, Miquel J, et al. A peculiar distribution of the emerging nematode Angiostrongylus cantonensis in the Canary Islands (Spain): recent introduction or isolation effect? Animals (Basel). 2021;11:1267. https://doi.org/10.3390/ani11051267 p // g/ / 9. Paredes-Esquivel C, Sola J, Delgado-Serra S, Puig Riera M, Negre N, Miranda MA, et al. Angiostrongylus cantonensis in North African hedgehogs as vertebrate hosts, Mallorca, Spain, October 2018. Euro Surveill. 2019;24:1900489. https://doi.org/10.2807/1560-7917.ES.2019.24.33.1900489 p g 9. Paredes-Esquivel C, Sola J, Delgado-Serra S, Puig Riera M, Negre N, Miranda MA, et al. Angiostrongylus cantonensis in North African hedgehogs as vertebrate hosts, Mallorca, Spain, October 2018. Euro Surveill. 2019;24:1900489. https://doi.org/10.2807/1560-7917.ES.2019.24.33.1900489 p g 9. Paredes-Esquivel C, Sola J, Delgado-Serra S, Puig Riera M, Negre N, Miranda MA, et al. Angiostrongylus cantonensis in North African hedgehogs as vertebrate hosts, Mallorca, Spain, October 2018. Euro Surveill. 2019;24:1900489. https://doi.org/10.2807/1560-7917.ES.2019.24.33.1900489 p g 10 Nguyen Y, Rossi B, Argy N, Baker C, Nickel B, Marti H, et al. Autochthonous case of eosinophilic meningitis caused by Angiostrongylus cantonensis, France, 2016. Emerg Infect Dis. 2017;23:1045–6. https://doi.org/10.3201/eid2306.161999 g 10 Nguyen Y, Rossi B, Argy N, Baker C, Nickel B, Marti H, et al. Autochthonous case of eosinophilic meningitis caused by Angiostrongylus cantonensis, France, 2016. Emerg Infect Dis. 2017;23:1045–6. RESEARCH LETTERS https://doi.org/10.3201/eid2306.161999 In a retrospective test-negative case–control study, we reviewed laboratory and public health records to identify cases of trichinellosis in Nunavik that occurred from 2009 through 2019. Our study was approved by the Research Institute of the McGill University Health Centre Research and Ethics Board (REB #2020-5312). ( ) We first reviewed all requests for Trichinella se rologic testing sent from Quebec to the National Ref erence Centre for Parasitology, the only testing site for Quebec, during 2009–2019 (Appendix, https:// wwwnc.cdc.gov/EID/article/28/12/22-1144-App1. pdf). To define an initial set of cases (with positive Trichinella serologic results), we selected specimens originating from Nunavik. One author (L.B.H.) re viewed the charts and confirmed cases if the clinical evolution was compatible with the positive serologic results. Because trichinellosis is notifiable by provin cial law, we cross-referenced cases with the public health database to identify other cases determined epidemiologically and reviewed those charts. We de fined a set of region-matched controls as those with negative Trichinella serologic results. Those controls are therefore persons from the general population, from the same region who had clinical manifestations that prompted testing for trichinellosis. Although serologic results early in the disease course could be negative, chart review of controls did not yield ad ditional suspected cases on the basis of clinical evo lution. We extracted available clinical and laboratory data by chart review at the McGill University Health Centre and at regional health centers in Nunavik. We calculated summary statistics and tests (t-test and χ2), comparing cases and controls by using R (6), and gen erated ROC curves by using the pROC R package (7). W id tifi d 43 f t i hi ll i d t Address for correspondence: María Teresa Galán-Puchades, Department of Pharmacy, Pharmaceutical Technology and Parasitology, Faculty of Pharmacy, University of Valencia Av. Vicent Andrés Estellés s/n, 46100 Burjassot-Valencia, Spain; email: mteresa.galan@uv.es RESEARCH LETTERS RESEARCH LETTERS RESEARCH LETTERS Laboratory Features of Trichinellosis and Eosinophilia Threshold for Testing, Nunavik, Quebec, Canada, 2009–2019 Luke B. Harrison, Michael D. Libman, Chelsea Caya, Momar Ndao, Cedric P. Yansouni Author affiliations: McGill University Health Centre, Montreal, Quebec, Canada (L.B. Harrison, M.D. Libman, C. Caya, C.P. Yansouni); National Reference Centre for Parasitology, Montreal (M. Ndao) Luke B. Harrison, Michael D. Libman, Chelsea Caya, Momar Ndao, Cedric P. Yansouni Author affiliations: McGill University Health Centre, Montreal, Quebec, Canada (L.B. Harrison, M.D. Libman, C. Caya, C.P. Yansouni); National Reference Centre for Parasitology, Montreal (M. Ndao) Luke B. Harrison, Michael D. Libman, Chelsea Caya, Momar Ndao, Cedric P. Yansouni Luke B. Harrison, Michael D. Libman, Chelsea Caya, Momar Ndao, Cedric P. Yansouni Luke B. Harrison, Michael D. Libman, Chelsea Caya, Momar Ndao, Cedric P. Yansouni Author affiliations: McGill University Health Centre, Montreal, Quebec, Canada (L.B. Harrison, M.D. Libman, C. Caya, C.P. Yansouni); National Reference Centre for Parasitology, Montreal (M. Ndao) Author affiliations: McGill University Health Centre, Montreal, Quebec, Canada (L.B. Harrison, M.D. Libman, C. Caya, C.P. Yansouni); National Reference Centre for Parasitology, Montreal (M. Ndao) DOI: https://doi.org/10.3201/eid2812.221144 Prolonged eosinophilia is characteristic of trichinellosis. To determine the optimal eosinophil threshold for reflex Trichinella testing, we examined all 43 cases in Nunavik, Quebec, Canada, during 2009–2019. Using receiver operating characteristic analysis, we determined that eosinophil counts >0.8 × 109 cells/L should prompt con sideration of trichinellosis and testing to rapidly identify potential outbreaks. y g g We identified 43 cases of trichinellosis and a set of 31 region-matched controls (Table). We excluded 4 2567 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 28, No. 12, December 2022 RESEARCH LETTERS Table. Characteristics of case-patients and controls in study of Trichinella infections in Nunavik, Quebec, Canada, 2009–2019* Characteristic Case-patients, n = 43 Controls, n = 31 Difference in means (95% CI) Test statistic† p value Demographics Age, y 39.1 (5–75, 16.1) 45.8 (0–80, 22.4) –6.63 (–15.59 to 2.32) t = –1.48 0.144 Female 30 (69.8) 16 (51.6) χ2 = 3.77 0.052 Male 13 (30.2) 15 (48.4) Level of care received, no. with available information/total no. ‡Optical density >0.3. §Four cases were determined epidemiologically, and initially negative serologic testing was not repeated. ‡Optical density 0.3. §Four cases were determined epidemiologically, and initially negative serologic testing was not repeated. that differed most between cases and controls was peak absolute eosinophilia (5.35 vs. 0.80 × 109 cells/L; p<0.001). Among case-patients, peak eosinophilia that differed most between cases and controls was peak absolute eosinophilia (5.35 vs. 0.80 × 109 cells/L; p<0.001). Among case-patients, peak eosinophilia possible case-patients with weakly positive serologic results but ambiguous clinical manifestations con sistent with past infection. Information on signs and symptoms was available for only 19/43 case-patients, but demographic, laboratory, and clinical outcomes were well documented. gov/eid • Vol 28 No 12 December 2022 Figure. Receiver operating characteristic curve comparing performance of thresholds of absolute and relative eosinophilia to trigger automatic reporting of possible trichinellosis. Sn and Sp for the thresholds of absolute eosinophilia examined were 0.5 × 109 (Sn = 1.0, Sp = 0.65), 0.8 × 109 (Sn = 1.0, Sp = 0.71), 1.0 × 109 (Sn = 0.91, Sp = 0.71), 2.0 × 109 (Sn = 0.63, Sp = 0.94) and for relative eosinophilia were 5% (Sn = 1.0, Sp = 0.65), 10% (Sn = 0.88, Sp = 0.71), 15% (Sn = 0.85, Sp = 0.76), and 30% (Sn = 0.36, Sp = 1.0). Case-patients had a median age of 40 years and were mostly female (30/43, 69.8%), which may result from chance (p = 0.052), differential exposure to para sitized meat, food sharing, food preparation practices, or selection bias (8). When available, clinical features were similar to those previously described for trichi nellosis (i.e., fever, rash and myalgia) (9). No patients died, and 9/27 (33%) patients with documented ill ness required hospitalization. Epidemiologic inves tigations revealed sporadic cases and 2 suspected point-source outbreaks (8). In 1 outbreak, seals were suspected as the source of infection, which could rep resent a change in epidemiology from previous out breaks associated with polar bear and walrus meat and might reflect the surveillance program targeting game meat from the latter animals but not seals. Laboratory information was available for 41/43 case-patients, a larger series of findings in Trichi nella infection in Nunavik than previously reported. Features of Trichinella infection in Nunavik, presump tively caused by T. nativa, are similar to those report ed for T. spiralis infection (9), including elevated cre atinine kinase and eosinophilia (Table). The variable Figure. Laboratory Features of Trichinellosis and Eosinophilia Threshold for Testing, Nunavik, Quebec, Canada, 2009–2019 (%) Outpatient 18/27 (67) 15/24 (63) NA NA Inpatient 9/27 (33) 9/24 (38) NA NA Critical care 0/27 0/24 NA NA Evacuated to southern Quebec 7/27 (26) 8/24 (33) NA NA Unknown 16/43 (37) 7/31 (23) NA NA Positive Trichinella serologic result 39 (91)§ NA NA NA Biochemical features during illness Eosinophils,  109 cells/L 5.35 (0.80–17.40, 3.81) 0.80 (0–4.5, 1.00) 4.55 (3.14 to 5.95) t = 6.47 <0.001 Platelets,  109/L 545 (294–977, 169) 479 (208–1,009, 210) 62.23 (–24.43 to 154.90) t = 1.45 0.151 Creatinine kinase, U/L 1562 (103–8,081, 1511) 956 (28–6,470, 1,730) 606.49 (–245.92 to 1,458.90) t = 1.42 0.160 C-reactive protein, mg/L 66.5 (8–191.7, 42.1) 66.6 (0.5–253.0, 82.9) –0.61 (–38.05 to 36.83) t = –0.03 0.974 Alanine aminotransferase, U/L 140.2 (21.0–541.0, 128.1) 85.1 (16.0–334.0, 75.6) 55.17 (–5.56 to 115.91) t = 1.82 0.074 *Values are no. (%) or mean (range, SD) except as indicated. NA, not applicable; t, Student t statistic. †Expressed as t value (95% CI). ‡Optical density >0.3. §F d t i d id i l i ll d i iti ll ti l i t ti t t d RESEARCH LETTERS was noted early and declined over months; among controls, counts were frequently elevated but stable over time (Appendix Figure). Using ROC analysis, we identified an absolute eosinophilia threshold of >0.8 × 109 cells/L, which identified all cases in this series with a specificity of 71% (Figure). We assessed the potential effect on resource use of this threshold by examining the region’s whole-population distribu tion of absolute eosinophil counts. Among 8,562 per sons who submitted a specimen for complete blood count for any reason from January 2019 through April 2022, a total of 287 had eosinophil counts that exceed ed our threshold (86 [1.2%] specimens/year).ll 4. Proulx JF, MacLean JD, Gyorkos TW, Leclair D, Richter AK, Serhir B, et al. Novel prevention program for trichinellosis in Inuit communities. Clin Infect Dis. 2002;34:1508–14. https://doi.org/10.1086/340342 4. Proulx JF, MacLean JD, Gyorkos TW, Leclair D, Richter AK, Serhir B, et al. Novel prevention program for trichinellosis in Inuit communities. Clin Infect Dis. 2002;34:1508–14. https://doi.org/10.1086/340342 p // g/ / 5 .Larrat S, Simard M, Lair S, Bélanger D, Proulx J-F. From science to action and from action to science: the Nunavik Trichinellosis Prevention Program. Int J Circumpolar Health. 2012;71:18595. https://doi.org/10.3402/ijch.v71i0.18595 p g 5 .Larrat S, Simard M, Lair S, Bélanger D, Proulx J-F. From science to action and from action to science: the Nunavik Trichinellosis Prevention Program. Int J Circumpolar Health. 2012;71:18595. https://doi.org/10.3402/ijch.v71i0.18595 6 R Core Team. R: a language and environment for statistical computing. Version 4.1.3 [cited 2022 Apr 1]. https://www.R-project.org 6 R Core Team. R: a language and environment for statistical computing. Version 4.1.3 [cited 2022 Apr 1]. https://www.R-project.org p p j g 7 Robin X, Turck N, Hainard A, Tiberti N, Lisacek F, Sanchez J-C, et al. pROC: an open-source package for R and S+ to analyze and compare ROC curves. BMC Bioinformatics. 2011;12:77. https://doi.org/10.1186/1471-2105-12-77 j g 7 Robin X, Turck N, Hainard A, Tiberti N, Lisacek F, Sanchez J-C, et al. pROC: an open-source package for R and S+ to analyze and compare ROC curves. BMC Bioinformatics. 2011;12:77. https://doi.org/10.1186/1471-2105-12-77 8 Ducrocq J, Proulx J-F, Simard M, Lévesque B, Iqaluk M, Elijassiapik L, et al. The unique contribution of a local response group in the field investigation and management of a trichinellosis outbreak in Nunavik (Québec, Canada). Can J Public Heal_th. 2020;111:31–9. Acknowledgments We appreciate the collaboration of Nunavik Public Health, the National Reference Centre for Parasitology, and the staff of Inuulitsivik Health Centre, including members of medical records department. This work was supported by a FRQ-S Clinician-Researcher Career Junior 2 award to C.P.Y. About the Author Dr. Harrison is chief resident in Infectious Diseases and Medical Microbiology at the McGill University Health Centre. His research interests include evolutionary biology. Centre. His research interests include evolutionary biology. RESEARCH LETTERS https://doi.org/10.17269/ s41997-019-00255-8 ( p y ) Automated flags and reflex testing in Nunavik now incorporate the threshold identified in our analy sis. In the absence of a defined alternative diagnosis, eosinophil counts of >0.80 ×109 cells/L should prompt clinical consideration of trichinellosis and further in vestigation. Early identification of outbreaks is critical in this region—where hunted meat is shared widely within and among communities—to limit exposures and enable delivery of postexposure prophylactic an thelmintic therapy, which has evidence of effectiveness in this serious illness (1,4,10). The cost–benefit ratio of this threshold will require ongoing assessment. 9 .Gottstein B, Pozio E, Nöckler K. Epidemiology, diagnosis, treatment, and control of trichinellosis. Clin Microbiol Rev. 2009;22:127–45. https://doi.org/10.1128/CMR.00026-08 9 .Gottstein B, Pozio E, Nöckler K. Epidemiology, diagnosis, treatment, and control of trichinellosis. Clin Microbiol Rev. 2009;22:127–45. https://doi.org/10.1128/CMR.00026-08 9 .Gottstein B, Pozio E, Nöckler K. Epidemiology, diagnosis, treatment, and control of trichinellosis. Clin Microbiol Rev. 2009;22:127–45. https://doi.org/10.1128/CMR.00026-08 10 Faber M, Schink S, Mayer-Scholl S, Ziesch S, Schönfelder R, Wichmann-Schauer H, et al. Outbreak of trichinellosis due to wild boar meat and evaluation of the effectiveness of post exposure prophylaxis, Germany, 2013. Clin Infect Dis. 2015;60:e98-104. p // g/ / 10 Faber M, Schink S, Mayer-Scholl S, Ziesch S, Schönfelder R, Wichmann-Schauer H, et al. Outbreak of trichinellosis due to wild boar meat and evaluation of the effectiveness of post exposure prophylaxis, Germany, 2013. Clin Infect Dis. 2015;60:e98-104. p g 10 Faber M, Schink S, Mayer-Scholl S, Ziesch S, Schönfelder R, Wichmann-Schauer H, et al. Outbreak of trichinellosis due to wild boar meat and evaluation of the effectiveness of post exposure prophylaxis, Germany, 2013. Clin Infect Dis. 2015;60:e98-104. ‡Optical density >0.3. §Four cases were determined epidemiologically, and initially negative serologic testing was not repeated. Receiver operating characteristic curve comparing performance of thresholds of absolute and relative eosinophilia to trigger automatic reporting of possible trichinellosis. Sn and Sp for the thresholds of absolute eosinophilia examined were 0.5 × 109 (Sn = 1.0, Sp = 0.65), 0.8 × 109 (Sn = 1.0, Sp = 0.71), 1.0 × 109 (Sn = 0.91, Sp = 0.71), 2.0 × 109 (Sn = 0.63, Sp = 0.94) and for relative eosinophilia were 5% (Sn = 1.0, Sp = 0.65), 10% (Sn = 0.88, Sp = 0.71), 15% (Sn = 0.85, Sp = 0.76), and 30% (Sn = 0.36, Sp = 1.0). Figure. Receiver operating characteristic curve comparing performance of thresholds of absolute and relative eosinophilia to trigger automatic reporting of possible trichinellosis. Sn and Sp for the thresholds of absolute eosinophilia examined were 0.5 × 109 (Sn = 1.0, Sp = 0.65), 0.8 × 109 (Sn = 1.0, Sp = 0.71), 1.0 × 109 (Sn = 0.91, Sp = 0.71), 2.0 × 109 (Sn = 0.63, Sp = 0.94) and for relative eosinophilia were 5% (Sn = 1.0, Sp = 0.65), 10% (Sn = 0.88, Sp = 0.71), 15% (Sn = 0.85, Sp = 0.76), and 30% (Sn = 0.36, Sp = 1.0). 2568 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 28, No. 12, December 2022 RESEARCH LETTERS Address for correspondence: Luke B. Harrison, McGill University Health Centre, Rm E5.1815, 1001 Blvd Decarie, Montreal, QC H4A 3J1, Canada; email: luke.harrison@mail.mcgill.ca References 1. Schellenberg RS, Tan BJK, Irvine JD, Stockdale DR, Gajadhar AA, Serhir B, et al. An outbreak of trichinellosis due to consumption of bear meat infected with Trichinella nativa, in 2 northern Saskatchewan communities. J Infect Dis. 2003;188:835–43. https://doi.org/10.1086/378094 Author affiliations: Wythenshawe Hospital, Manchester, UK (S. Ugolini); University of Manchester NHS Foundation Trust, Manchester (S. Ugolini); IRCCS Azienda Ospedaliero– Universitaria di Bologna, Bologna, Italy (M. Lima, M. Maffi, M. Vastano, T. Gargano, S. Varani); Ospedali Riuniti di Ancona, Ancona, Italy (F. Pierangeli); Alma Mater Studiorum–University of Bologna, Bologna (M. Lima, T. Gargano, S. Varani, A. Gustinelli, M. Caffara, M.L. Fioravanti) p // g/ / 2. Dalcin D, Zarlenga DS, Larter NC, Hoberg E, Boucher DA, Merrifield S, et al. Trichinella nativa outbreak with rare thrombotic complications associated with meat from a black bear hunted in northern Ontario. Clin Infect Dis. 2017;64:1367–73. https://doi.org/10.1093/cid/cix165 p g 3. MacLean JD, Viallet J, Law C, Staudt M. Trichinosis in the Canadian Arctic: report of five outbreaks and a new clinical syndrome. J Infect Dis. 1989;160:513–20. https://doi.org/10.1093/infdis/160.3.513 DOI: https://doi.org/10.3201/eid2812.220424 DOI: https://doi.org/10.3201/eid2812.220424 2569 merging Infectious Diseases • www.cdc.gov/eid • Vol. 28, No. 12, December 2022 2569 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 28, No. 12, December 2022
https://openalex.org/W4306750487	https://www.researchsquare.com/article/rs-2013176/latest.pdf	English	null	Clustering of Charged Colloidal Particles in Microgravity Environment of Space	Research Square (Research Square)	2,022	cc-by	8,541	Clustering of Charged Colloidal Particles in Microgravity Environment of Space Junpei Yamanaka (  yamanaka@phar.nagoya-cu.ac.jp ) Graduate School of Nagoya City University Hiroyuki Miki Graduate School of Nagoya City University Teruyoshi Ishigami Graduate School of Nagoya City University Tohru Okuzono Graduate School of Nagoya City University Akiko Toyotama Nagoya City University Jitendra Mata Australian Centre for Neutron Scattering https://orcid.org/0000-0001-9225-7900 Honoka Komazawa Nagoya City University Yushi Takeda Nagoya City University Madoka Minami Nagoya City University Minori Fujita Graduate School of Nagoya City University Maho Doi Nagoya City University Tsunehiko Higuchi Nagoya City University Hiroshi Takase Graduate School of Medical Sciences, Nagoya City University Satoshi Adachi Japan Aerospace Exploration Agency Tetsuya Sakashita J A E l ti A Clustering of Charged Colloidal Particles i Microgravity Environment of Space Junpei Yamanaka (  yamanaka@phar.nagoya-cu.ac.jp ) Graduate School of Nagoya City University Hiroyuki Miki Graduate School of Nagoya City University Teruyoshi Ishigami Graduate School of Nagoya City University Tohru Okuzono Graduate School of Nagoya City University Akiko Toyotama Nagoya City University Jitendra Mata Australian Centre for Neutron Scattering https://orcid.org/0000-0001-9225-7900 Honoka Komazawa Nagoya City University Yushi Takeda Nagoya City University Madoka Minami Nagoya City University Minori Fujita Graduate School of Nagoya City University Maho Doi Nagoya City University Tsunehiko Higuchi Nagoya City University Hiroshi Takase Graduate School of Medical Sciences, Nagoya City University Satoshi Adachi Japan Aerospace Exploration Agency Tetsuya Sakashita Japan Aerospace Exploration Agency Taro Shimaoka Clustering of Charged Colloidal Particles in Microgravity Environment of Space Junpei Yamanaka ( yamanaka@phar nagoya-cu ac jp ) Akiko Toyotama Nagoya City University Keywords: License:   This work is licensed under a Creative Commons Attribution 4.0 International License. License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Clustering of Charged Colloidal Particles in Microgravity Environment of Space Hiroyuki Miki1, Teruyoshi Ishigami1, Junpei Yamanaka1, Tohru Okuzono1, Akiko Toyotama1, Jitendra Mata2, Honoka Komazawa3, Yushi Takeda3, Madoka Minami3, Minori Fujita1, Maho Doi3, Tsunehiko Higuchi1, Hiroshi Takase4, Satoshi Adachi5, Tetsuya Sakashita5, Taro Shimaoka6, Masae Nagai6, Yuki Watanabe7, and Seijiro Fukuyama ( ) g 3. Faculty of Pharmaceutical Sciences, Nagoya City University, Japan y g y y y p 4. Core Laboratory, Graduate School of Medical Sciences, Nagoya City University, Japan y 5. Japan Aerospace Exploration Agency (JAXA), Japan 7. Advanced Engineering Services (AES) Co., Ltd., Japan 1. Graduate School of Pharmaceutical Sciences, Nagoya City University, Japan 7. Advanced Engineering Services (AES) Co., Ltd., Japan Abstract We conducted a charge-charge clustering experiment of positively and negatively charged colloidal particles in aqueous media under a microgravity environment at the International Space Station. A special setup was used to mix the colloid particles in microgravity and then immobilize these structures in gel cured using UV light. The samples returned to the ground were observed by optical microscopy. The space sample of polystyrene particles with a specific gravity d (=1.05) close to the medium had an average association number of approximately 50% larger than the ground control and better structural symmetry. The effect of electrostatic interactions on the clustering was also confirmed for titania particles (d~3), whose association structures were only possible in the microgravity environment without any sedimentation they generally suffer on the ground. This study suggests that even slight sedimentation and convection on the ground significantly affect the structure formation of colloids. Knowledge from this study will help us to develop a model which will be used to design photonic materials and better drugs. 6. Japan Space Forum (JSF), Japan y 5. Japan Aerospace Exploration Agency (JAXA), Japan Introduction Self-assembly of submicron to micron-sized particles has been observed in various phenomena in nature, including aggregation of unstable colloidal dispersions1-4 and association of proteins in living cells5. The formation of colloidal clusters, composed of a small number of colloidal particles, has been reported in numerous studies 6-11. In particular, tetrahedral clusters are the building blocks of diamond lattices, which are photonic crystals capable of confining light12-15, and the conditions for their formation have been explored16-21. The colloidal self-assembly enables the construction of novel materials that are useful in the fields of photonics, optoelectronics and sensing, and even clinical diagnostics. In this study, we examined the association of positively and negatively charged colloidal particles (hereafter referred to as p and n particles) by electrostatic attraction (Fig.1). A schematic diagram of various clusters consisting of one p-particle and n-particles with an association number m (=1,2,3,...). Tetrahedral clusters (m=4) are of interest as building blocks of the diamond lattice, a photonic crystal with a perfect photonic band gap. Fig. 1 Schematic of clustering and clusters of positively and negatively charged colloidal particles. m is the association number. + → negatively charged(n) particles positively charged(p) particles clusters aggregation m = 1 m = 2 m = 3 m = 4 clusters Fig. 1 Schematic of clustering and clusters of positively and negatively charged colloidal particles. m is the association number. To construct materials with excellent optical properties, colloidal particles with high refractive indexes (nr) are often required. For example, to achieve a perfect photonic band that reflects all incident light from all directions, a diamond lattice structure with particles with a refractive index nr > 2 is required. Generally, materials with high refractive indexes have, at the same time, high specific gravity. For example, a titania particle with nr = 2.4 has a specific gravity of ~4. On the ground, the structure formation of such heavy particles is often difficult due to gravitational settling effects. Assuming gravitational sedimentation with Stokes resistance F = 6πhav (h: viscosity of the medium, a: radius of the particle, v: settling velocity), v for 1 μm-sized titania particles at a temperature = 25 °C is 1.2 μm/sec, respectively. This implies that the settling distance in one day is 2 5 cm. Microgravity environments, where sedimentation and convection are negligible, are ideal for experiments on the structure formation of micron-sized colloidal particles22,23. Introduction Numbers of microgravity experiments have been conducted on colloidal crystallization in rigid sphere dispersions. Here, colloidal crystallization is a phenomenon in which particles are regularly arranged in a liquid, and particles in a crystalline structure are not in contact with each other. Microgravity experiments using the space shuttle revealed that particle sedimentation significantly affects nucleation and growth processes23. It has also been reported that dendritic growth is the dominant crystal growth mode under microgravity24. These have led to a review of the kinetics of crystallization of hard spheres, and models have been devised that incorporate the interaction of diffusion fields in crystals25,26. We have also reported that the nucleation rate also decreases in the crystallization of charged colloids under microgravity obtained by parabolic flight27. However, the formation of clusters, in which particles are in contact with each other, has not been reported so far. We conducted a clustering experiment of positively and negatively charged colloidal particles in aqueous media on the Japanese Experiment Module (JEM) "Kibo" of the International Space Station (ISS)28 in 2020. Details of the space experiments are described in Supplementary Text 1. p- and n- particles of about 1 µm were mixed in the microgravity environment of the ISS to form clusters. The main driving forces for clustering were electrostatic interactions and van der Waals (vdW) attraction between particles. Polystyrene (PS) particles, which have almost the same specific gravity d (= 1.05) as water, and titania particles with high refractive index and high specific gravity (d~3) were used as colloidal particles. In this paper, we report the results of the analysis of colloidal clusters generated in space. Overview of our space experiments Here, we briefly describe our space experiment on the ISS. Additional information is provided in Supplementary Text 1. The properties of the colloid samples are shown in Table 1(a). Negatively charged fluorescent PS particles were obtained from Thermo Scientific (Massachusetts, U.S.A.). Other particles were synthesized by methods described in Supplementary Text 2. The titania particles were coated with a silica layer on their surfaces, and a polyelectrolyte was introduced to the silica surface to provide them with electric charges. p- and n-particles were stained with red (rhodamine B isothiocyanate) and green fluorescent (fluorescein isothiocyanate) dyes for identification. Samples were prepared with various concentrations of sodium chloride NaCl, [NaCl], to adjust the magnitude of the electrostatic interaction. The composition of the space samples is compiled in Table 1(b). The ground-based studies have shown that for obtaining isolated clusters, (i) the concentration of p- and n-particles must be 3 sufficiently dilute (less than about 0.1 vol% in total), and (ii) the composition must be highly asymmetric, with a number concentration ratio of twenty or higher. The experimental conditions were chosen accordingly. UV curable gelation reagents29,30 had been dissolved in the colloidal samples. They were composed 0.1M dimethylacrylamide (gel monomer), 10 mM methylene-bis- acrylamide (cross-linker), and 10 mg/mL VA-086 (photo-induced polymerization initiator). They undergo radical polymerization upon UV irradiation, forming a three-dimensional network structure in water, and the medium becomes a polymer gel. able 1 (a) Properties of the particles used in the space experiments (b) Composition of the samples used the space experiments. in the space experiments. (a) (b) sample No. p n C p C n [NaCl] [HCl] (10-3 vol%) (10-3 vol%) (mM) (mM) 1 PS(+) PS(-) 2 10 0 10 2 PS(+) PS(-) 2 10 50 10 3 PS(+) PS(-) 2 10 100 10 4 PS(+) PS(-) 2 10 200 10 9 TiO2(+) TiO2(-)1 48 2 0 0 14 TiO2(+) TiO2(-)2 48 2 0 0 19 TiO2(+) TiO2(-)3 48 2 0 0 23 TiO2(+) TiO2(-)4 48 2 0 0 24 TiO2(+) TiO2(-)4 48 2 50 0 25 TiO2(+) TiO2(-)4 48 2 100 0 26 TiO2(+) TiO2(-)4 48 2 200 0 particle d ζ (nm) (mV) PS(+) 789±22 38.7 PS(-) 1025±36 -50.1 TiO2(+) 957±33 48.7 TiO2(-)1 907±35 -56.2 TiO2(-)2 1016±38 -50.4 TiO2(-)3 1004±32 -44.5 TiO2(-)4 1016±38 -36.1 (b) (a) 4 4 Fig. 2 Experimental set-up and samples used in the space experiment. Overview of our space experiments (a) Sample bag, consisting of two tetra-pack compartments connected via a breakable separator. (b) The solution in the two chambers is mixed by squeezing the bag. (c) An example of a sample used in a space experiment. The sample is tied with string for proper handling in microgravity. (d) Sample bag in a shipping container (e) UV irradiation system. The bag is placed in the apparatus and the sample is immobilized by UV irradiation. The sample is dissolved in a UV-curing gelator. (f) Samples (Titania #23) returned to the ground. The sample was removed from the bag. (c) Cross section of the gel-fixed space sample and the ground sample. The space sample is uniformly white, while the particles in the ground sample are localized near the gel surface. Fig. 2 Experimental set-up and samples used in the space experiment. (a) Sample bag, consisting of two tetra-pack compartments connected via a breakable separator. (b) The solution in the two chambers is mixed by squeezing the bag. (c) An example of a sample used in a space experiment. The sample is tied with string for proper handling in microgravity. (d) Sample bag in a shipping container (e) UV irradiation system. The bag is placed in the apparatus and the sample is immobilized by UV irradiation. The sample is dissolved in a UV-curing gelator. (f) Samples (Titania #23) returned to the ground. The sample was removed from the bag. (c) Cross section of the gel-fixed space sample and the ground sample. The space sample is uniformly white, while the particles in the ground sample are localized near the gel surface. Fig. 2(a) is an overview of the experimental apparatus we used. Two polymer bags of tetra- pack shapes with a volume of 3 mL are connected by a breakable seal. The connection is broken by crushing the bag to mix the liquids in the bags [Fig. 2(b)]. The two bags were filled with dilute aqueous dispersions of p- and n-particles, respectively. Fig. 1(c) shows an example of the sample used in the space experiment; the bags are connected by a string to prevent the sample from scattering under microgravity in the ISS. Fig. 2(d) is the sample bag in the shipping container, which was transported to the ISS using this container. (e) is a metal UV irradiation device. Overview of our space experiments Sample bags (30 pieces) were fixed inside the apparatus, and the samples were gelatinized by UV irradiation with a UV-LED (365 nm, 9.0 W) installed behind the box. The space experiment was carried out approximately eight months after the sample preparation. The space crew mixed the two liquids at ISS by pushing and squashing alternately 100 times to have homogeneity within the samples. Preliminary studies on the ground showed that it takes approximately two days to cluster the particles to reach equilibrium. In the space experiment, 5 the sample was left in microgravity for two days and gelled by UV irradiation. More details are given in Supplementary Text 3. Fig. 2(f) shows the appearance of the space sample (TiO2 # 23) taken out of the bag after returning to the ground. All of the space samples were successfully immobilized by the gel. The elastic modulus of gel E was approximately 25 kPa. This value was about 70% of the value when the samples were gelled immediately after preparations (E ~ 35 kPa), but it was large enough for the sample handling. The samples were then cut with a surgical scalpel, and the cross sections were observed by optical microscopy. Fig. 2(g) compares the cross sections of the space samples and ground control samples prepared under a pseudo-microgravity obtained by the clinostat. No distinct supernatant was observed in the space sample, though the ground control sample had significant particle precipitation and the center of the sample was transparent. A portion of the gelled space experimental sample was also sent to the Australian Nuclear Science and Technology Organisation (ANSTO) for small- and ultra-small-angle neutron scattering experiments to obtain information on the number of associations and aggregation structure. Association number of PS clusters Association number of PS clusters Association number of PS clusters Fig. 3 (a) Fluorescence micrographs of polystyrene (PS) clusters from the space experiment and magnified images of m = 1 to m = 4 clusters. (b) Distribution of the number of clusters m at various NaCl concentrations in the (b) space experiment and (c) ground experiment. (d) Plot of average cluster number <m> vs. [NaCl]. (e) Plot of average radius of inertia <Rg> vs. [NaCl] for clusters obtained by neutron scattering. Fig. 3 (a) Fluorescence micrographs of polystyrene (PS) clusters from the space experiment and magnified images of m = 1 to m = 4 clusters. (b) Distribution of the number of clusters m at various NaCl concentrations in the (b) space experiment and (c) ground experiment. (d) Plot of average cluster number <m> vs. [NaCl]. (e) Plot of average radius of inertia <Rg> vs. [NaCl] for clusters obtained by neutron scattering. Fig. 3(a) shows a micrograph of sample #2 ([NaCl] = 50 μM). Because the PS samples have a large excess of n-particles, their isolated clusters are centered on p-particles and surrounded by n- particles. As shown in Fig. 3(a), clusters with various association numbers m were identified in the space sample. Fig. 3(b) shows the distribution of m obtained from the results of about 1000 clusters in samples #1-#4 ([NaCl] = 0, 50, 100, and 200 μM), respectively. With increasing [NaCl], the electrostatic shielding effect becomes stronger, and the Coulomb force between particles becomes weaker. Note that a small number of clusters containing two or more p-particles were also present (< 6% of the entire p-particles); they were classified as "aggregates." Fig. 3(c) shows the distribution of m for the ground control. Because the sample at [NaCl] = 0 μM was used for particle characterization without gelation, clustering experiments were carried out for samples at [NaCl] = 50, 100, and 200 μM. Fig. 3(d) shows a plot of the average association number <m> versus [NaCl]. In both the space and ground experiments, <m> increased on increasing [NaCl], reaching a maximum at [NaCl] = 100 μM. As seen from equation (1), the electrostatic interaction between the particles is shielded by ions and decreases on increasing Cs (µ k2). Fig. 3(e) shows the relationship between the [NaCl] and mean radius of gyration <Rg> of the clusters obtained by the Guinier plot from ultra-small angle neutron scattering. Ground control experiments On the sample preparation, two samples having the same composition were made. One was used for the space experiment and the other as a ground control sample. The ground control samples were stored at 4 °C in JAXA Space Center (Tsukuba, Japan) for approximately eight months, as was the case of the space experiment. The control experiments were carried out in August 2020. To examine changes in characteristics of samples during the storage, some of the bags of PS and titania samples were opened without gelation and analyzed. For the remaining samples, clustering experiments were conducted. On the ground, the sedimentation of the particles in two days is significant, particularly for titania particles. The settling distances estimated from the Stokes law in two days are 2.6 mm and 10 cm for PS and titania, comparable and much greater than the inner dimension of the sample bag. On the ground, after the p- and n-particles were mixed by squashing the bags in the same manner in the space experiment and by rotating them gently, they were placed under pseudo-microgravity for two days. For PS and titania samples, we used a rotator (3 rpm) and a clinostat device (10 rpm). The samples were then immediately gelled by UV irradiation. 6 Association number of PS clusters The scattering curves are given in Fig. 4. The trends of the Cs dependence of <Rg> and <m> are in good agreement. Thus, 7 the statistical scattering measurements corroborated the results obtained from microscopic observations. Fig. 4 SANS-USANS scattering profiles (left), and Guinier plot (right) for PS clusters in space sample for four values of [NaCl]. The curves are vertically shifted and superimposed for clarity. In the Guinier region, scattering intensity I(q) is proportional to exp(-Rg・q2), where Rg is the radius of gyration of the clusters, and q is the scattering vector. Rg is obtained as the slope of the plot of log I(q) versus q2. 0 1x10-7 2x10-7 3x10-7 4x10-7 5x10-7 0 50 100 200 I(q) q (Å-2) 2 [NaCl] (µM) 106 107 103 104 105 10 2 108 0 1x10-7 2x10-7 3x10-7 4x10-7 5x10-7 0 50 100 200 I(q) q (Å-2) 2 [NaCl] (µM) 106 107 103 104 105 10 2 108 Fig. 4 SANS-USANS scattering profiles (left), and Guinier plot (right) for PS clusters in space sample for four values of [NaCl]. The curves are vertically shifted and superimposed for clarity. In the Guinier region, scattering intensity I(q) is proportional to exp(-Rg・q2), where Rg is the radius of gyration of the clusters, and q is the scattering vector. Rg is obtained as the slope of the plot of log I(q) versus q2. Structural symmetry of PS clusters To examine the effects of microgravity in more detail, we then examined the structural symmetry of tetrahedral (m=4) PS clusters. The relative positions of each particle in the tetrahedral clusters were determined by confocal microscopy. The structural symmetry of the tetrahedral clusters can be quantified by the bond orientational order parameter qtetra31,32, defined as 𝑞!"!#$ = 1 −3 8 ' ' (cos 𝜃%& + 1 3. ' ( &)%+ , %)+ , 0１1 0１1 where θjk is the angle between the bonds of particles j and k (illustrated in Fig. 5). For a perfect tetrahedron, qtetra = 1. Fig. 5(a) is the mean value of qtetra, <qtetra>, for tetrahedrons of the PS particles at various values of [NaCl] in the space and ground experiments. Fig. 5(b)-5(d) depicts the distribution of qtetra for the space and ground experiments at the three values of [NaCl]. In the entire [NaCl] regions, the samples obtained from the space experiment had larger <qtetra>, that is, better symmetry, than the ground samples. 8 Fig. 5 Comparison of the bond orientational order parameter qtetra for PS particle tetrahedra produced by the space and ground experiments. (a) plot of mean <qtetra> versus [NaCl], (b)-(d) distribution of qtetra at [NaCl] = 50 μM, 100 μM, and 200 μM. Fig. 5 Comparison of the bond orientational order parameter qtetra for PS particle tetrahedra produced by the space and ground experiments. (a) plot of mean <qtetra> versus [NaCl], (b)-(d) distribution of qtetra at [NaCl] = 50 μM, 100 μM, and 200 μM. Clusters of titania particles Cluster formation of titania particle systems, which is difficult to study on the ground due to gravitational sedimentation, was investigated by analyzing space samples. In the titania sample, the central particle of the isolated cluster is an n-particle because p-particles are present in significant excess. The gel-immobilized space samples were uniform and no sedimentation effects were observed (Fig. 2(e)). However, two unexpected things happened in the titania space experiment. One was the formation of numerous aggregates of more than 1000 particles in the sample. The other was discoloration of the fluorescent dye; p-particles were stained with a red fluorescent pigment, but many of them turned red-green in color. Both of these issues were probably due to the degradation of the particles over time. Details of the degradation are described in the Supplementary Discussion. On the other hand, various isolated clusters were also observed, as shown in Fig. 6(a). Because the n-particles were stained green and did not exhibit red coloration, the p- and n- particles were distinguished by the presence of a red-colored component. Images of red fluorescence (left) and white light (right) are shown for each cluster in Fig. 6(a). p-particles are observed in both pictures, while n-particles are observed only in white light. Fig. 6(b) shows distributions of m at various values of [NaCl]. The population of m=1 clusters was more significant at lower [NaCl], in accordance with the stronger electrostatic attraction between n- and p-particles. Fig. 6(c) shows the dependence on charge number Z, confirming that the number of m=1 clusters is more significant with increasing Z. Note that m=4 clusters were observed at [NaCl]=50 μM. In Fig. 6(d) and 6(e), we also present the plot of the population of various clusters 9 plotted against [NaCl] and Z, respectively. Thus, the space experiment confirmed the effect of electrostatic interaction on the clusters of titania particles, which is difficult to verify on the ground. Fig. 6 (a) Magnified images of titania-based qtetra clusters from m = 1 to m = 4 (left and right images with red fluorescence only detected and white light source, respectively). Distribution of the number of clusters m in the titania-based sample. (b) [NaCl]-dependence and (c) Z-dependence. (d) [NaCl] dependence and (e) Z-dependence of the proportion of m = 0 to m = 4 clusters. Fig. Clusters of titania particles 6 (a) Magnified images of titania-based qtetra clusters from m = 1 to m = 4 (left and right images with red fluorescence only detected and white light source, respectively). Distribution of the number of clusters m in the titania-based sample. (b) [NaCl]-dependence and (c) Z-dependence. (d) [NaCl] dependence and (e) Z-dependence of the proportion of m = 0 to m = 4 clusters. Aggregation structure of titania particles Aggregation structure of titania particles Fig. 7 Microscopic images of aggregates formed in the titania-based space experimental samples. (a-1), (a-2) inverted microscope images, and (a-3) confocal laser microscope image (b) Fractal dimension Df of the aggregates obtained for samples having various values of [NaCl]. Red and blue symbols represent results for space and ground control samples, respectively. Fig. 7(a-1)~7(a-3) show microscopic images of large aggregates of titania particles. These aggregates are made almost exclusively of p-particles. (a-1) (a-2) (a-3) (b) (a-1) (a-2) (b) (a-3) (b) (a-2) Fig. 7 Microscopic images of aggregates formed in the titania-based space experimental samples. (a-1), (a-2) inverted microscope images, and (a-3) confocal laser microscope image (b) Fractal dimension Df of the aggregates obtained for samples having various values of [NaCl]. Red and blue symbols represent results for space and ground control samples, respectively. Fig. 7(a-1)~7(a-3) show microscopic images of large aggregates of titania particles. These aggregates are made almost exclusively of p-particles. Fig. 7(a-1)~7(a-3) show microscopic images of large aggregates of titania particles. These aggregates are made almost exclusively of p-particles. 10 The fractal dimension of the aggregates was determined from the microscopic images33,34,35,36,37. Fig. 5(b) shows the fractal dimension Df determined for samples of various values of [NaCl] (red symbols). Df was largely independent of [NaCl] and ranged between reaction-limited aggregation (~2.1) and diffusion-limited aggregation (~1.7). This suggests that the aggregates are mainly formed by vdW forces, with negligible Cs dependence. We note that the titania particles form dense aggregates close to Df ~ 3 on the ground due to significant sedimentation (shown by blue symbols). The bulky aggregates with Df = 2 observed in a microgravity environment again indicate the effect of microgravity on the assembly of titania particles. Thus, the significant influence of microgravity on the formation of aggregates of high specific gravity particles was verified. Clustering of charged colloids , (6) (6) 𝐴7;8 = 3ℎ𝜈< 8√2 (𝑛+ ' −𝑛, ')(𝑛' ' −𝑛, ') (𝑛+ ' + 𝑛, ') + '(𝑛' ' + 𝑛, ') + ' P(𝑛+ ' + 𝑛, ') + '+(𝑛' ' + 𝑛, ') + 'Q . (7) (7) Here eri and nri are the particle’s relative permittivity and refractive index (i = 1, 2) and the medium (i = 3). f'(r) is determined by the size of the two particles, while the A value depends on the difference between the permittivity and the refractive index of the medium and particles. For example, the A between two polystyrene (PS) particles (nr =1.59, er=2.5) interacting across a water gap is 4.80 kBT, and 49.4 kBT for titania (nr ~3, er =48) particles. Computer simulations use the two- body potential to study the relationship between the number of associations and the charge number of particles. Here eri and nri are the particle’s relative permittivity and refractive index (i = 1, 2) and the medium (i = 3). f'(r) is determined by the size of the two particles, while the A value depends on the difference between the permittivity and the refractive index of the medium and particles. For example, the A between two polystyrene (PS) particles (nr =1.59, er=2.5) interacting across a water gap is 4.80 kBT, and 49.4 kBT for titania (nr ~3, er =48) particles. Computer simulations use the two- body potential to study the relationship between the number of associations and the charge number of particles. The attraction between p- and n-particles is stronger at lower Cs. The smaller <m> at lower Cs is attributable to stronger electrostatic repulsion between n particles, which prevents additional attachments of n particles to the clusters. As Cs increase, reduction of the electrostatic repulsion between n particles causes increases in <m>. However, on further increase in Cs, the attraction between the n- and p- particles becomes weaker, resulting in the reduction of <m>, as observed in the experiment. This trend is consistent with the results of the Monte Carlo simulation. In the polystyrene colloids, the number of associations and symmetry of clusters were superior to the space experiments. These results suggest that slight sedimentation and convection effect on the ground may affect the association of micrometer-order colloidal particles. Clustering of charged colloids Here we discuss the clustering of the oppositely charged colloids in terms of interparticle interaction. The main driving forces for the clustering of charged colloidal particles are electrostatic interactions and vdW attraction between particles. The former is often discussed using the Yukawa- type two-body interaction potential UYukawa(r), as, 𝑈Yukawa(𝑟) = exp (𝜅𝑎1) 1 + 𝜅𝑎1 exp (𝜅𝑎2) 1 + 𝜅𝑎2 𝑍1𝑍2𝑒2 4𝜋𝜀 exp (−𝜅𝑟) 𝑟 , (2) (2) where a1 and a2 are the radius of the two particles, Z1 and Z2 are the signed charge numbers, e is the dielectric constant of the medium, and e is the elementary charge. k is the Debye parameter, where i is the type of the i-th ion in the medium, and ci and zi are their concentrations and valences are given by where a1 and a2 are the radius of the two particles, Z1 and Z2 are the signed charge numbers, e is the dielectric constant of the medium, and e is the elementary charge. k is the Debye parameter, where i is the type of the i-th ion in the medium, and ci and zi are their concentrations and valences are given by by 𝜅! = 𝑒! 𝜀𝑘B𝑇* 𝑐"𝑧" ! . (3) 𝜅! = 𝑒! 𝜀𝑘B𝑇* 𝑐"𝑧" ! . (3) (3) It can be seen that the higher the ion concentration, the more pronounced the electrostatic interaction is shielded. The vdW potential UvdW(r) acting between the two particles is given by It can be seen that the higher the ion concentration, the more pronounced the electrostatic interaction is shielded. The vdW potential UvdW(r) acting between the two particles is given by 𝑈456(𝑟) = 𝐴𝑓 (𝑟), (4) (4) 𝑓(𝑟) = −1 6 D 2𝑎+𝑎' 𝑟' −(𝑎++𝑎')' + 2𝑎+𝑎' 𝑟' −(𝑎+−𝑎')' + ln 𝑟' −(𝑎++𝑎')' 𝑟' −(𝑎+−𝑎')'H , (5) (5) 11 where kB is the Boltzmann constant, T is temperature, h is the Planck constant, and νe is the principal absorption frequency of the material (~ 3 × 1015 /sec). A is the Hamaker constant, which according to Lifshitz theory, is given as A =Aν=0 + Aν>0 (ν is the frequency of electromagnetic waves), and 𝐴7)8 = 3 4 𝑘9𝑇(𝜀:+ −𝜀:, 𝜀:+ + 𝜀:, . (𝜀:' −𝜀:, 𝜀:' + 𝜀:, . Electrostatic interaction To investigate the formation mechanism of macroscopic aggregates observed for titania samples, we calculated electrostatic and vdW interactions for positively charged TiO2-SiO2 core- shell particles (TiO2 (+)). The silica layer's thickness and the medium's salt concentration were parameters. From SEM observations, the size of the TiO2 core of the sample is estimated to be 860 nm, and the thickness of the silica layer is 50 nm when the sample is filled. Henry's equation determined the zeta potential from the measured electrophoretic mobility of the ground control particles without gel fixation, and the surface charge number Z(P-B) of the particles was calculated using the Poisson- Boltzmann equation Z(P-B) depending on the particle size, which in turn depends on the shell thickness. The calculation results are shown in Fig. 8. Fig. 8 Number of particle charges calculated assuming various SiO2 shell layer thicknesses Fig. 8 Number of particle charges calculated assuming various SiO2 shell layer thicknesses Yukawa potentials were calculated for various salt concentrations according to equation (1). The results are described in (4) and (5). Aggregation structure of titania Here we discuss the mechanism of large aggregate formation in titania colloids. The titania particles we used were covered with a shell layer of silica. The determination of silica concentration in the medium of the space sample showed that approximately 25% of the silicates in the shell are dissolved in the medium. The dissolution of the silica shell may have two effects on the interparticle interactions. (i) The electrostatic repulsion is reduced because the polyelectrolyte on the silica surface is also removed, resulting in reduced Z values. (ii) The refractive index of the titania layer is about twice that of silica, and the thinner the silica layer, the stronger the vdW attraction between particles. Concerning (i), the Z value of the ground control sample was reduced by about 28% from 12 the initial values. In addition, since the silica surface is negatively charged, partial desorption of the positive macromolecules of the p-particles could have resulted in a negatively charged patch region of some of the particles, which could have agglomerated due to Coulomb attraction between the positively charged portions of the other particles. We assume that the large aggregates observed were produced for these reasons. The detailed discussion will be described in the followings. vdW interaction Viravathana and Marr38 calculated the vdW potential acting between two TiO2-SiO2 core-shell particles by splitting the potential between regions, such as between cores and shells. 13 In calculating the Hamaker constant A by Lifshitz theory (Eqs. (6) and (7)), we used refractive indices of 2.4, 1.43, and 1.33 for TiO2, SiO2, and water, respectively. The refractive index of TiO2 was determined by analyzing the interference of a thin film of synthesized TiO2 core particles adsorbed as two-dimensional colloidal crystals on a glass substrate. Other values are from literature. For dielectric constants, anatase type literature values of e = 48 were used for TiO2, and 3.8 and 78.3 for SiO2 and water. A = 49.4 kBT, 1.27 kBT, and 5.77 kBT between TiO2 and TiO2, SiO2 and SiO2, and SiO2 and TiO2, respectively, in pure water at 25 °C. A between TiO2 -TiO2 was about 40 times the value between SiO2 - SiO2, and the vdW force became stronger the thinner the shell. Interaction potential The interaction pair potential was calculated as the sum of the Yukawa and vdW potentials. An example of a potential curve is shown in Fig. 9(a). When the contribution of the electrostatic repulsive force expressed by the Yukawa potential is sufficiently significant, the total potential has a maximum. The value of the potential maximum (Umax) at the maximum was calculated for various shell thicknesses. The results are shown in Fig. 9(b). Fig. 9 (a)Interaction potential between TiO2-SiO2 core-shell particles (Shell thickness 20 nm, Cs = 200 mM). (b) Maximum values of the interaction potential between TiO2-SiO2 core-shell particles (From top, shell thickness = 50,40,30,20,10, and 0 nm). (b) (a) (b) (a) (b) Fig. 9 (a)Interaction potential between TiO2-SiO2 core-shell particles (Shell thickness 20 nm, Cs = 200 mM). (b) Maximum values of the interaction potential between TiO2-SiO2 core-shell particles (From top, shell thickness = 50,40,30,20,10, and 0 nm). The higher the salt concentration, the weaker the electrostatic interaction, so the potential maximum is lower. Also, the thinner the shell thickness, the stronger the vdW attraction, so the potential maximum is also lower. Thus, it is shown that the dissolution and desorption of the silica shell layer can cause particle aggregation due to the vdW force. Note that from the analysis of the rate of aggregation based on the Smoluchowski theory, the concentration of free particles, which are not involved in aggregation, can be calculated as a function of time. However, it is assumed that the particles irreversibly aggregate when they come into contact. Under the space experiment conditions 14 (particle diameter 1 μm, particle concentration 0.1 vol%), the half-life t1/2 of the free particle concentration is t1/2 = 85 seconds when there is no interaction between particles, and 11 hours and 69 days for Umax/kBT = 10 and 15, respectively. Fig. 9(b) shows the conditions for Umax/kBT =10 with red lines; the storage period at ISS is about 250 days, and aggregation is expected within the storage period when the shell layer is thin, and the salt concentration is sufficiently high. However, the ionic strength expected from the conductivity measurements was about 100 µM, insufficient for most particles to aggregate. Although the eluted silica layer was slight and the overall charge of the particles remained positive, it is possible that the silica layer eluted non-uniformly, resulting in non- uniform charge and aggregation. Interaction potential The space experiment confirmed the fractal aggregation, which is not produced on the ground. The findings of this study will be helpful in the development of models used in the design of photonic materials using particles with high refractive indices and so large specific gravities. Colloidal particles and characterization Details of the synthesis of PS and titania particles are given in Supplementary Text 2. We summarized the characteristics of the particles used in this study in Table 1. Particle size was measured by scanning electron microscopy (JCM-6000, JEOL, Tokyo, Japan). The electrophoretic mobility of the particles was measured by a microscopic electrophoresis apparatus (type Zeecom ZC-3000U, Microtec Co., Ltd., Chiba, Japan) with more than 100 particles at [NaCl] = ten μM. ζ potential of the particles was calculated from the mobility using Henry's formula. Methods Colloidal particles and characterization Microscope observations An inverted fluorescence microscope (ECLIPSE, Ti-S, Nikon, Co., Ltd.) with an oil immersion objective (100×, Plan Flour, Nikon) was used. Movies of the sample were taken while moving the viewing field, and the number of clusters having various m values was counted. Approximately 1000 clusters were counted in the total for PS samples. For the titania sample, more than 50 clusters were counted because most of the particles formed large aggregates. Large aggregates of titania samples were observed using a confocal laser scanning microscope (type C2, Nikon) and an all-in-one microscope (BZ-X800, KEYENCE, Osaka, Japan) equipped with an optical sectioning module (BZ-H4XF, KEYENCE, Osaka, Japan). The particles' surface structure and elemental analysis were performed using a transmission electron microscope (type S-4800, Hitachi, Tokyo) and a high-speed, high-sensitivity EDS/EDX detector (ULTIM MAX65, Oxford Instruments, Tokyo, Japan) at the Analysis Center, Nagoya City University. Ground control experiment For sample PS#1 ([NaCl] = 0 μM), samples were taken out of the bag. The electrical conductivity of the positively and negatively charged particle dispersions and the zeta potential z of the particles were measured to determine how the samples changed over time. For the positive and negative particle dispersion systems, s = 3.90 μS/cm and 5.46 μS/cm, respectively; for the five μM HCl solution, s = 2.7 μS/cm, indicating that s increased by about 1~3 μS/cm. This value is likely due to contamination by ionic impurities in storage, corresponding to 10~30 μM for [NaCl]. Also, z = +23.9 mV and -47.3 mV for positively and negatively charged particles, respectively (measured at [NaCl] =10 μM). At the time of sample preparation (November 2019), z values were +38.7 mV and -50.1 mV, respectively: the z of positively and negatively charged particles were reduced by about 38% and 5%, respectively: the surface charge numbers calculated from the z using the Poisson-Boltzmann equation were +6450 (when filled: +3690) and -13100 (when filled: -14200), respectively. The 15 significant rate of decrease in positive charge may be since the charge of the amino group giving the positive charge is affected by even a slight change in pH. For sample PS#2 , the two solutions were mixed as in the space experiment. Then the bags were put under pseudo microgravity by using a rotator (type M T R-107, As-One, Tokyo, Japan, rotation radius = 6 cm) for two days at 3 rpm. Gelation was performed using a UV-LED lamp. Cross sections of the gels were observed with an optical microscope, and the elastic modulus was measured with a rheometer. The elastic modulus was E₀ = 24.5 ± 4.2 kPa. Samples PS#3 and #4 were prepared similarly. The titania sample #23 ([NaCl] =0 μM) was taken out of the bag and the z of the particles were measured. The p- and n- titania particles had z = +37.7±6.5 mV and -30.4±4.8 mV, respectively. The initial values were z = +48.7 mV and -36.1 mV, respectively. The other samples were rotated using a clinostat (Portable Microgravity Simulator PMS-VIII, AES Co., Ltd.) at 10 rpm for two days and then immediately gelled using a UV-LED lamp. Monte Carlo simulation We calculated the equilibrium configurations of various clusters using the Monte Carlo (MC) method39,40 with experimental parameters for PS samples to see if the abovementioned experimental results are theoretically valid. Details of the calculations are given in Supplementary Text 4. Since the vdW force is sufficiently smaller than the electrostatic interaction in the PS sample, we solely used Yukawa-type potentials, Eq. (1), for the calculation. In Fig. 2(d), a plot of <m> versus [NaCl] obtained from the MC simulation is presented. The distribution of m at each Cs is shown in Supplementary Fig.3. Although the simulation gave <m> 16 values several times larger than in the experiment, <m> increased on increasing [NaCl] and reached a constant value. This trend was qualitatively consistent with the experimental results in the low [NaCl] region. Although we assume that the particles are perfectly smooth spheres in the MC simulation, actual particle surfaces are more or less rough. Consequently, when two particles come into contact, frictional forces are generated, preventing the particles' most stable configuration. The observed difference in <m> between simulation and experiment appears, at least partly, to be due to the presence of the frictional forces. The size distribution of actual particles (coefficient of variation = 4%) may also affect the <m> value. However, in MC simulations for particles with a size polydispersity of 4%, the m values at [NaCl] = 2 µM-100 µM agreed within 2% for the monodisperse case. Results are presented in Supplementary Fig.4. Data Availability The datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request. Acknowledgments We thank the MSPR engineering and integration staff for enterprising research (MEISTER) and the on-orbit crew for their precise operations that made the experiment a success. This work was partly supported by a JSPS KAKENHI grant (17K04990). We sincerely thank Misato Suko, Hajime Hattori, Yoko Kondo, and Kazushi Yamamoto, Faculty of Pharmaceutical Sciences, Nagoya City University, for their participation in the preparatory study to determine the conditions of the space experiment. Sincere thanks are due to Yui Nakata, AES Co., Ltd., for her help in preparing the space experiments. We thank Yumika Uenishi, Yuri Nakamura, and Natsumi Masuda for their help in the data analysis of the space sample aggregates. We acknowledge the support of the Australian Centre for Neutron Scattering, ANSTO, and the Australian Government through the National Collaborative Research Infrastructure Strategy in providing the neutron research infrastructure used in this work via ACNS proposals 7063 & 9971. This work benefited from the use of the SasView application, originally developed under NSF award DMR-0520547. SasView contains code developed with funding from the European Union’s Horizon 2020 research and innovation program under the SINE2020 project, grant agreement No 654000. We thank Mr. Yutaka Hattori, KEYENCE Co., Ltd., for the discussion. We acknowledge the assistance of the Research Equipment Sharing Center at Nagoya City University for the SEM observation. This research was supported by Japan Science and Technology Agency, 'Establishment of University Fellowships towards the Creation of Science Technology Innovation'. Neutron scattering experiments The neutron scattering experiments were performed by using a small-angle neutron scattering instrument (Quokka) and an ultra-small-angle neutron scattering instrument equipped with a Bonse- Hart camera (Kookaburra) at the Australian Centre for Neutron Scattering, Australian Nuclear Science and Technology Organisation (ANSTO). Combining these instruments evaluated microstructures from 1 nm to 10 µm. The medium of the experimental space samples was replaced from water with heavy water, and measurements were performed by increasing the difference in scattering length density between the polystyrene and titania particles and the medium. Gel matrix itself has negligible scattering (data are not included here) and was subtracted from overall scattering as background. Particle association in gel matrix was studied using the Quokka small-angle neutron scattering (SANS) (ANSTO, Sydney, Australia)41 and Kookaburra ultra-small-angle neutron scattering (USANS) (ANSTO, Sydney, Australia)42 instrument at ANSTO. The gels were socked in D2O and then were loaded in the demountable sample holders (20 mm diameter for SANS and 40 mm diameter for USANS) with a 1 mm path length filled with D2O as a background medium. The scattering vector q defined as q = 4πsinθ/λ, where λ is the neutron wavelength and 2θ is the scattering angle. The SANS data were collected in the q range of 0.0007–0.1 Å−1, with source aperture to sample aperture distances of 1.3, 12, and 20 m, and a neutron wavelength (λ) of 5.0 Å−1 and 8.1 Å−1 (for lens optics), respectively. The USANS data were collected in the q range of 0.00004–0.001 Å−1, with λ of 4.74 Å−1. 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Colloid Interf. Sci. 221, 301-307 (2000). 39. Allen, M. P. and Tildesley, D. J. Computer Simulation of Liquids (Clarendon, Oxford, 1987). 20 40. Frenkel, D. & Smit, B. Understanding Molecular Simulation - From Algorithms to Applications, 2nd Ed. (Academic, San Diego, 2002). 41. Wood, K., et al. QUOKKA, the pinhole small-angle neutron scattering instrument at the OPAL Research Reactor, Australia: design, performance, operation and scientific highlights. J Appl Crystallogr 51, 294-314 (2018). 41. Wood, K., et al. QUOKKA, the pinhole small-angle neutron scattering instrument at the OPAL Research Reactor, Australia: design, performance, operation and scientific highlights. J Appl Crystallogr 51, 294-314 (2018). 42. Rehm, C. et al. Design and performance of the variable-wavelength Bonse–Hart ultra-small- angle neutron scattering diffractometer KOOKABURRA at ANSTO. J Appl Crystallogr 51, 1-8 (2018). 42. Rehm, C. et al. Design and performance of the variable-wavelength Bonse–Hart ultra-small- angle neutron scattering diffractometer KOOKABURRA at ANSTO. J Appl Crystallogr 51, 1-8 (2018). Author Contributions J.Y., the principal investigator of the Colloidal Clusters Project, and T.O., A.T., T.H., and J.M., the chief investigators of the same project, designed the basic research. H.M., T.I., H.K., Y.T., M.M., and M.F. synthesized the colloidal samples and determined the space experimental conditions. S.A., T.Sakashita, T.Shimaoka, M.N., Y.W., and S.F. developed the experimental apparatus and planned space experiments on the ISS. H.M., T.I., and M.D. analyzed the structure of space samples using optical microscopy. J.M. performed neutron scattering. H.T. performed high-resolution electron microscopy. The paper was written with input from all authors, with J.Y., H.M., and J.M. writing the part on experimental studies and T.O. and H.M. writing the computer simulation part. Competing interests All the authors declare no competing interests. Competing interests All the authors declare no competing interests. Additional information Correspondence and requests for materials should be addressed to J. Yamanaka Additional information Correspondence and requests for materials should be addressed to J. Yamanaka 21 Supplementary Files This is a list of supplementary ¦les associated with this preprint. Click to download. SupplementaryInformation1007.pdf
https://openalex.org/W3045523281	http://www.helmholtz-berlin.de/pubbin/oai_publication?VT=1&ID=102677	English	null	Implementation of Flexible Embedded Nanowire Electrodes in Organic Light‐Emitting Diodes	Physica status solidi. Rapid research letters	2,020	cc-by	5,980	Implementation of Flexible Embedded Nanowire Electrodes in Organic Light-Emitting Diodes Lukas Kinner, Felix Hermerschmidt,* Theodoros Dimopoulos,* and Emil J. W. List-Kratochvil are easily achieved on glass substrates.[1] Yet, the trend in industry is moving toward production on poly(ethylene terephthalate) (PET) substrates, because roll-to-roll (R2R) production offers faster production speeds than traditional bulk production of inor- ganic semiconductor devices, such as silicon solar cells.[7,8] In consumer electronics, the trend goes toward ﬂexible devices, like fold- able smart phones or rollable screens.[9] The implementation of silver nanowires (NWs) as ﬂexible transparent electrodes (FTEs) in solution-processed organic light-emitting diodes (OLEDs) still faces two major challenges: the high roughness of NW ﬁlms and heat sensitivity of the most commonly used transparent substrate poly(ethylene terephthalate) (PET). A solution-based, roll-to-roll, and sheet-to-sheet scalable process to create indium tin oxide (ITO)-free FTEs is reported. This FTE is realized by spraying NWs on PET, without the necessity of postdeposition heat treatment. To overcome the roughness limitation, NW ﬁlms are reverse transfer embedded on another PET substrate. As a result, the FTE shows a low roughness, as well as high mechanical, thermal, organic-solvent, and plasma stability. This developed FTE shows comparable transmittance with ITO but lower sheet resistance and higher mechanical stability. The FTE is implemented in a solution-processed OLED with PDY-132 (Super Yellow) as the emissive layer. In contrast to many other works in this ﬁeld, a ZnO-nanoparticle electron-injection layer is used on the NWs instead of poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) or other organic hole injectors. The use of ZnO nanoparticles instead of organic layers yields many advantages in terms of process and device stability. The resulting devices show greater ﬂexibility, conductivity, and luminance than PET/ITO reference devices, while having the same power efﬁcacy. However, ITO fails to deliver a low sheet resistance on PET due to the lower possible substrate temperature during the sputter deposition process and the high deposition rates necessary to achieve high conductiv- ity.[1] Typical PET/ITO foils show Rsh of 60 Ω sq1. Further, ITO has the drawback of being brittle, which deteriorates its conductivity when subjected to mechanical strain.[10] This fact compromises its use in ﬂexible applications (e.g., bendable devices).[11] Many different approaches for solution- processed TEs were brought forward in recent years to tackle the aforementioned drawbacks of ITO. www.pss-rapid.com www.pss-rapid.com RAPID RESEARCH LETTER Implementation of Flexible Embedded Nanowire Electrodes in Organic Light-Emitting Diodes The use of inkjet-printed metal grids offers the possibility of producing already structured electrodes in an industrial-scale process.[2,12–14] Other important TE mate- rials include conductive polymers,[15] ultrathin metallic layers,[16] dielectric/metal/dielectric layers,[8] carbon nanotubes,[17] and metal nanowires (NWs).[5,18] Modern optoelectronics, such as organic light-emitting diodes (OLEDs) and thin-ﬁlm photovoltaics, rely on transparent electro- des (TEs).[1–5] TEs assure that light can leave or reach the active materials (emitters or absorbers), while simultaneously serving to inject or extract the charge carriers. Indium tin oxide (ITO) is the industrial standard for TEs, due to its high optical transmittance in the visible spectral range (>80%), together with its low sheet resistance (Rsh) of <15 Ω sq1.[6] These key features Modern optoelectronics, such as organic light-emitting diodes (OLEDs) and thin-ﬁlm photovoltaics, rely on transparent electro- des (TEs).[1–5] TEs assure that light can leave or reach the active materials (emitters or absorbers), while simultaneously serving to inject or extract the charge carriers. Indium tin oxide (ITO) is the industrial standard for TEs, due to its high optical transmittance in the visible spectral range (>80%), together with its low sheet resistance (Rsh) of <15 Ω sq1.[6] These key features The most widely used metal for NWs is silver. Silver NWs feature high transparency (90–96%), as well as low Rsh (9–70 Ω sq1).[19] In addition to these optical and electrical L. Kinner, Dr. F. Hermerschmidt, Prof. E. J. W. List-Kratochvil Institut für Physik Institut für Chemie & IRIS Adlershof Humboldt-Universität zu Berlin Brook-Taylor-Straße 6, 12489 Berlin, Germany E-mail: felix.hermerschmidt@hu-berlin.de Prof. E. J. W. List-Kratochvil Helmholtz-Zentrum Berlin für Materialien und Energie GmbH Brook-Taylor-Straße 6, 12489 Berlin, Germany The ORCID identiﬁcation number(s) for the author(s) of this article can be found under https://doi.org/10.1002/pssr.202000305. © 2020 The Authors. Published by Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution- NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. © 2020 The Authors. Published by Wiley-VCH GmbH L. Kinner, Dr. T. Dimopoulos Center for Energy, Photovoltaic Systems AIT Austrian Institute of Technology Gieﬁnggasse 6, 1210 Vienna, Austria E-mail: theodoros.dimopoulos@ait.ac.at Prof. E. J. W. List-Kratochvil Helmholtz-Zentrum Berlin für Materialien und Energie GmbH Brook-Taylor-Straße 6, 12489 Berlin, Germany www.advancedsciencenews.com properties, NWs show excellent mechanical stability, which makes them a good candidate for ﬂexible and R2R-processed electrodes.[20] For R2R processing, different deposition methods for NWs have been presented, such as electrostatic spraying,[21] wire bar coating,[22] inkjet printing,[23] air brush spraying,[24] and spray coating.[18] evaporated hole-injection layers on the NWs to manufacture OLEDs. In the presented work a mixture of ZnO nanoparticles and polyethyleneimine (PEI) is used as electron-injection layer on NWs, creating an inverted OLED architecture. g A PEDOT:PSS-free OLED is less prone to humidity and has an additional encapsulating effect by ZnO. Furthermore, it is not necessary to apply many thin organic interlayers, which are dif- ﬁcult to control during processing and the inverted architecture offers better applicability in display production.[30] Independent of the deposition technique, NW ﬁlms feature two major drawbacks, namely their high roughness and their need for postdeposition treatments. High roughness is a bottleneck for thin-ﬁlm device fabrication, as spikes from the one electrode may reach the opposing electrode and hence short circuit the device. Postdeposition treatments are necessary to remove the organic capping layer around NWs and assure silver–silver contact between the NWs. Both steps are necessary to reduce contact resistance between individual wires.[25,26] We prove the applicability of this embedded electrode on PET in solution- processed OLED devices. The presented inverted OLEDs on embedded NWs show the same power efﬁcacy as ITO-based devices but a luminance which surpasses devices on standard PET/ITO substrates. For comparison, we initially produced OLEDs with nonem- bedded sprayed NW ﬁlms on PET. All devices were short circuited, due to a NW ﬁlm roughness of 46 nm. Hence a plana- rization process, based on the embedding of the NWs into the Ormocomp layer, was used.[18] The schematic of the process is shown in Figure 1a. NWs are sprayed on PET at 110 C (step I). Elevated substrate temperature during spraying allows faster drying of the spray solution droplets. This in turn prevents coalescence to larger droplets and thereby increases homogeneous ﬁlm morphology.[31] For the case of PET, Scardaci et al. showed that a substrate temperature of 110 C shows optimum condi- tions.[29] At this temperature no deformation of the PET substrate was observed, as the continuous air and solution ﬂow cools the PET substrate during spraying. Following the spraying, NWs are drop coated with Ormocomp (step II). www.advancedsciencenews.com On top of this arrange- ment the ﬁnal substrate [PET, coated with Ormoprime 08 (“Ormoprime” is a trademark of micro resist technology)] is placed and then illuminated with UV light (step III). Then a lift-off is con- ducted, yielding NWs embedded in Ormocomp on PET (step IV). To overcome the roughness issue, different planarization and embedding processes have been put forward. Lian et al. proposed an embedding process for NWs, by pressing the spin- coated NWs into a poly(vinyl alcohol) (PVA) ﬁlm, which was spin coated on a poly(ethylene naphthalate) substrate.[25] Although the processing is straightforward and does not require vacuum proc- essing, it is unclear if the PVA ﬁlm resists solution processing, as the electrodes have been tested for evaporated OLEDs only. A more stable approach for solution-processed embedded NWs was presented by Yu et al.[27] NWs were drop cast on a host glass substrate, cured, and covered with a UV-curable poly (acrylate), which is peeled off of the sacriﬁcial glass substrate, containing the NW ﬁlm. The embedded NWs feature a low root-mean-square (RMS) roughness, as the new surface is the initial glass/NW interface. On the embedded electrode, a solution- processed OLED with the following architecture, poly(3,4-ethylene- dioxylthiophene):polystyrene sulfonate (PEDOT:PSS)/Super Yellow/CsF/Al, was tested. The presented devices feature a similar current efﬁcacy performance to ITO, but lower electrical currents and lower luminance, compared with ITO. , y g p ( p ) To characterize the optical properties of the obtained electrode, optical transmittance measurements were carried out. In Figure 1b transmittance spectra of the NW ﬁlms on PET at various process steps are shown. The dashed orange line shows the spectrum of the sprayed ﬁlm after step I. Due to spray- ing of the NWs at 110 C substrate temperature, the organic capping is removed during spraying and the resulting ﬁlms have an Rsh of 13 Ω sq1. Spraying NWs without substrate heating yielded ﬁlms with Rsh > 200 kΩ sq1. Spectra of NW ﬁlms embedded in Ormocomp show an additional dip in the transmit- tance spectrum of the electrode at 400 nm (light blue line), due to the light absorption from the Ormocomp layer. Overcoating the embedded NWs with the electron-injecting ZnO:PEI mixture does not change the transmittance signiﬁcantly (dark blue line). It was observed that most NW device samples initially shorted or showed high dark currents, although the NWs were embedded. Phys. Status Solidi RRL 2020, 14, 2000305 DOI: 10.1002/pssr.202000305 2000305 (1 of 6) Phys. Status Solidi RRL 2020, 14, 2000305 Phys. Status Solidi RRL 2020, 14, 2000305 www.pss-rapid.com www.advancedsciencenews.com www.advancedsciencenews.com A more detailed (12 12 μm2) SFM image of the same sample is shown in Figure 2b, indicating an RMS roughness of 25 nm. The high roughness value is mainly caused by holes in the Ormocomp from the plasma treatment and not by the sharp spikes which may cause shunts.[18] By overcoating the embedded NW with ZnO:PEI (Figure 2c), an even smoother sur- face (RMS ¼ 8.1 nm) is formed, which is close to the ITO/ZnO: PEI roughness of 5 nm (Figure S2, Supporting Information). As described earlier, devices with nonembedded NW electro- des showed short circuits due to RMS roughness of 46 nm. Scanning force microscopy (SFM) measurements were carried out to quantify the roughness decrease caused by embedding. The results are shown in Figure 1c. The height proﬁle of the nonembedded NW ﬁlm (orange line) shows signiﬁcantly higher spikes, indicating higher roughness than the embedded NW ﬁlms (light blue). The embedded electrodes show however some holes in the embedding polymer, caused by plasma treatment. Figure S1 in the Supporting Information shows an SFM image of the embedded NW electrode before plasma treatment, with no holes visible. Further details on the process and the effects of the plasma treatment can be found in a previous study.[18] These holes are partially ﬁlled by the spin-coated ZnO:PEI layer (Figure 1d, dark blue). For comparison, also the proﬁle of the ITO/ZnO:PEI electrode (black) is shown. To get a bigger picture of the roughness of the NW electrodes than just line proﬁles, con- focal laser microscopy and SFM images are shown in Figure 2. One method to do so is by conducting bending tests on the electrode and investigating the measured resistance as a function of bending strain and bending cycles. The electrode resistance R was measured initially (R0) and then after each iteration of bending cycles n. The tensile strain ε during bending is given by the formula: ε ¼ d(2r)1, where r is the bending radius and d the substrate thickness.[32–35] For these experiments, r ¼ 4 mm and the substrate thickness d (Ormocomp þ PET) equals 480 μm, which yields 6% tensile ﬁlm strain. In addition, to tensile bending, compressive bending (ﬁlm inside the curvature) was conducted. Figure 2d shows the mechanical stability of the embedded electrode in comparison with ITO. www.advancedsciencenews.com Hence, the embedded NW ﬁlms were plasma treated to improve the wetting and reduce remaining NW spikes, before spin coating them with ZnO:PEI. Plasma treatment improved the number of working devices and individual device performance. PET/ITO samples were also plasma treated prior to ZnO:PEI coating to enhance the wetting and the work function. In the visible spectral wavelength range NW electrodes coated with ZnO:PEI show a slightly lower transmittance than PET/ITO electrodes coated with ZnO:PEI, but a far lower sheet resistance, as the ITO-based electrode has an Rsh of 60 Ω sq1. Finally, Jung et al. presented an R2R process to create embed- ded and transferred NW electrodes.[28] The NWs were wire-bar- coated on the host substrate (thermally stable polyimide [PI]) and thermally cured with an infrared dryer. It was shown that areas up to 45 15 cm2 had a homogeneous sheet resistance distribu- tion. Then the NWs were embedded in a UV-curable polymer, while simultaneously transferring them to a new PET substrate (ﬁnal substrate). It was shown that an evaporated OLED on this electrode achieved the same luminance but higher efﬁcacy than a glass/ITO-based reference. Electrode performance in this case was largely inﬂuenced by the use of high-aspect-ratio NWs (length of 27 μm and width of 32 nm). ( g ) Recently, we have shown that sprayed and embedded NW elec- trodes in the UV-curable polymer Ormocomp (“Ormocomp” is a trademark of micro resist technology) can be obtained using PET as both host and target substrate.[18] This approach avoids the use of an expensive PI host substrate and yields a conductive substrate that is compatible to solution processing of organic devices. Further, it is compatible with R2R and sheet-to-sheet processing. Sprayed NW electrodes have already been shown for sizes up to 10 10 cm2, only limited by the hot plate size used in the study.[29] In the work presented here, we prove the applicability of this embedded electrode on PET in solution-processed OLED devices. The previously presented devices use either PEDOT:PSS or 2000305 (2 of 6) © 2020 The Authors. Published by Wiley-VCH GmbH www.pss-rapid.com Figure 1. a) Schematic of the production process of the embedded NW electrodes: spraying (Step I), overcoating with Ormocomp (Step II), UV curing with Ormoprime 08 (green layer)-coated ﬁnal substrate (Step III), ﬁnal electrode (Step IV). www.advancedsciencenews.com b) Transmittance spectra of the different electrodes showing a transmittance of the developed electrode close to ITO; the inset shows the developed electrode. c) SFM line proﬁle of nonembedded and embedded NWs, showing the reduction of large spikes. d) SFM proﬁle of NW and ITO electrodes with ZnO:PEI, showing a further reduction of large spikes, which may occur on PET/ITO substrates. www.advancedsciencenews.com www.pss-rapid.com www.pss-rapid.com www.advancedsciencenews.com www.pss-rapid.com Figure 1. a) Schematic of the production process of the embedded NW electrodes: spraying (Step I), overcoating with Ormocomp (Step II), UV curing with Ormoprime 08 (green layer)-coated ﬁnal substrate (Step III), ﬁnal electrode (Step IV). b) Transmittance spectra of the different electrodes showing a transmittance of the developed electrode close to ITO; the inset shows the developed electrode. c) SFM line proﬁle of nonembedded and embedded NWs, showing the reduction of large spikes. d) SFM proﬁle of NW and ITO electrodes with ZnO:PEI, showing a further reduction of large spikes, which may occur on PET/ITO substrates. Having now conﬁrmed that the developed electrode surface roughness is almost as low as the roughness of ITO and hence low enough for device manufacturing, the mechanical behavior was investigated. As described earlier, devices with nonembedded NW electro- des showed short circuits due to RMS roughness of 46 nm. Scanning force microscopy (SFM) measurements were carried out to quantify the roughness decrease caused by embedding. The results are shown in Figure 1c. The height proﬁle of the nonembedded NW ﬁlm (orange line) shows signiﬁcantly higher spikes, indicating higher roughness than the embedded NW ﬁlms (light blue). The embedded electrodes show however some holes in the embedding polymer, caused by plasma treatment. Figure S1 in the Supporting Information shows an SFM image of the embedded NW electrode before plasma treatment, with no holes visible. Further details on the process and the effects of the plasma treatment can be found in a previous study.[18] These holes are partially ﬁlled by the spin-coated ZnO:PEI layer (Figure 1d, dark blue). For comparison, also the proﬁle of the ITO/ZnO:PEI electrode (black) is shown. To get a bigger picture of the roughness of the NW electrodes than just line proﬁles, con- focal laser microscopy and SFM images are shown in Figure 2. From confocal laser microscopy (Figure 2a) the RMS rough- ness of a 330 330 μm2 area of the embedded NW electrode is shown to be 30 nm. Phys. Status Solidi RRL 2020, 14, 2000305 © 2020 The Authors. Published by Wiley-VCH GmbH www.advancedsciencenews.com Rsh of ITO increases 30 times within 70 compressive bending cycles, whereas the NW electrode showed no change in Rsh for compres- sive bending at Rbend ¼ 4 mm for 5000 cycles. At the same bending radius in tensile bending, Rsh increased only three times over 5000 circles, which is ten times less than ITO. Rsh changes strongly in tensile bending compared with compressive bending because tensile bending is more strenuous for the ﬁlm.[32,33] The SEM images of the ITO and NW electrodes before and after bending (Figure S3) show the strong contrast in morphology, explaining the observed difference in Rsh. While ITO ﬁlms contain visible cracks after bending, the NW electrode shows no such damage. py g g From confocal laser microscopy (Figure 2a) the RMS rough- ness of a 330 330 μm2 area of the embedded NW electrode is shown to be 30 nm. A more detailed (12 12 μm2) SFM image of the same sample is shown in Figure 2b, indicating an RMS roughness of 25 nm. The high roughness value is mainly caused by holes in the Ormocomp from the plasma treatment and not by the sharp spikes which may cause shunts.[18] By overcoating the embedded NW with ZnO:PEI (Figure 2c), an even smoother sur- face (RMS ¼ 8.1 nm) is formed, which is close to the ITO/ZnO: PEI roughness of 5 nm (Figure S2, Supporting Information). 2000305 (3 of 6) © 2020 The Authors. Published by Wiley-VCH GmbH Phys. Status Solidi RRL 2020, 14, 2000305 www.pss-rapid.com www.advancedsciencenews.com Figure 2. a) Large-area surface morphology of the electrode, b) SFM image of embedded NW electrode showing holes in the electrode due to plasma treatment, c) SFM image of embedded NW electrode, coated with ZnO:PEI, showing lower roughness, and d) bending tests of the NW electrode and commercial ITO substrates showing the much higher mechanical stability of the NW electrode. Figure 2. a) Large-area surface morphology of the electrode, b) SFM image of embedded NW electrode showing holes in the electrode due to plasma treatment, c) SFM image of embedded NW electrode, coated with ZnO:PEI, showing lower roughness, and d) bending tests of the NW electrode and commercial ITO substrates showing the much higher mechanical stability of the NW electrode. Utilizing both ITO and NW-based electrodes, OLEDs were fabricated, and their performance was investigated. www.advancedsciencenews.com To do so, current density J and luminance L were measured as a function of applied voltage V to determine the so-called J–V–L curves. Figure 3 shows the performance data of OLEDs manufactured on embedded NW FTEs, as well as on PET/ITO reference sub- strates. In Figure 3a the corresponding J–V characteristics of the devices are shown. The NW-based devices show up to two orders of magnitude higher dark currents, caused by remaining shunts through the light-emitting layer. In the range from 2 to 4 V, devices based on NW electrodes show similar electrical behavior to the ITO reference devices. Starting at 4 V, the NW-based devices show a higher current increase as the ITO reference devices. The sheet resistance of the NW electrode is 5 times lower than the ITO electrode. This results in a faster current increase. Similar behavior of current increase in OLEDs on NWs, compared with ITO devices, has been shown in literature for a comparable device architecture.[36] It is important to point out that the saturation current of the NW devices is higher than that of ITO devices. This behavior also results from the ﬁve times lower Rsh of the NW electrode, compared with the ITO reference electrode. a maximum luminance of 5000 cd m2; both values were achieved at 7 V. At 7 V the current density of the NW device is a factor 7 higher than the current density of the ITO device, correlating with the ﬁve times lower sheet resistance of the NW electrode. This results in the seven times higher luminance value. Power efﬁcacy (lm W1) of the devices was determined by calculating the electrical power consumed by the OLED (P ¼ IU) and calculating lm from cd according to Forrest et al. by assum- ing a Lambertian light source: 1 lm ¼ π (1 cd).[37] The resulting power efﬁcacy over luminance graph is shown in Figure 3 c. At lower luminance the ITO reference device shows a higher efﬁcacy. This results from the fact that at lower current densities ITO-based devices show higher luminance values than the NW-based devices. The highest power efﬁcacy is achieved by the ITO reference device with 2.2 lm W1 at 5000 cd m2, whereas the highest efﬁcacy of embedded NW devices is also 2.2 lm W1 but at a considerably higher luminance value of 34 000 cd m2. Phys. Status Solidi RRL 2020, 14, 2000305 © 2020 The Authors. Published by Wiley-VCH GmbH Experimental Section and ohmic losses in the ITO-based devices become more impor- tant than the initial leakage losses in the NW-based devices. Fabrication of NW Electrodes: The fabrication of the embedded NW elec- trodes was realized following the process described in a previous study.[18] Clogging of the spraying nozzle by the NWs was not observed, because the nozzle diameter (3 mm) was far larger than the average NW length (10 μm), and in contrast to inkjet printing, spraying is a continuous process, which prevents the drying of the solution at any place in the tub- ing system. Further, the shaping air was set to its highest value, which removed almost all NW residuals from the nozzle surface. The ﬁlm was kept uniform by choosing the spraying pattern over the substrate in a zig–zag form and having a small offset at the start of every new spray- ing circle. The amount of wires on the substrate and therefore the “thick- ness” was set by the number of spraying circles. Further details are found in a previous study.[18] To match the contacts of the OLED measurement setup, the devices were patterned during spraying by covering the sides with Kapton tape to leave a 5 mm-wide middle strip of the conducting NW ﬁlm; then, Cr/Au ﬁngers were evaporated. Finally, to analyze the color of the light source depending on the electrode type, electroluminescence (EL) spectra were taken of both device types. Independently of the electrode, both device types show the same EL spectra (Figure 3d), with the Commission Internationale de l’Éclairage (CIE) coordinates: x ¼ 0.44 and y ¼ 0.54 for the NW electrode and x ¼ 0.45 and y ¼ 0.54 for the ITO reference. This indicates the same light out-coupling properties of both electrode types, as well as the same chemical stability for both types because no color changing degradation of the light-emitting layer is observed. In conclusion we have demonstrated a NW-based ITO-free FTE, implemented in a solution-processed OLED. Our work shows a vacuum-free way to process NW ﬁlms on PET to obtain FTEs with a sheet resistance ﬁve times lower than that of ITO, while offering similar transmittance. In terms of ﬂexibility the developed electrode shows no resistance change in compres- sive bending and only three times increase over 5000 tensile bending cycles with 6% strain, whereas the comparison ITO elec- trode shows large resistance increases after 70 bending cycles. www.advancedsciencenews.com This same efﬁcacy of the NW electrode device results from the higher luminance at higher current densities, which is achieved due to its lower Rsh. This indicates that our OLED architecture is in general able to achieve these efﬁcacies and efﬁciently convert higher currents to higher luminance values, as previously shown. Similar behavior has been shown in OLEDs with a comparable architecture in the literature.[36] In Figure 3b the luminance characteristics corresponding to the J–V characteristics in Figure 3a are shown. At 4.5 V, the luminance values of the NW devices exceed the ITO reference devices. One can conclude that the higher luminance starting at 4.5 V is caused by the higher currents starting at 4 V (Figure 3a). As stated earlier, this is derived from the lower Rsh of the NW elec- trode. The maximum luminance of embedded NW electrode devi- ces was 34 000 cd m2, whereas the ITO reference device reached One can conclude that the ITO-based devices have a higher power efﬁcacy at lower current densities and resulting lower luminance due to lower leakage. However, this advantage over the NW-based electrode is lost at higher current densities and the resulting higher luminance, when Rsh plays a larger role 2000305 (4 of 6) © 2020 The Authors. Published by Wiley-VCH GmbH www.pss-rapid.com d d i www.advancedsciencenews.com www.pss-rapid.com Figure 3. Characteristics of OLED devices using the embedded NW electrodes on PET and a PET/ITO reference. a) Current density versus bias voltage, b) luminance versus bias voltage, c) power efﬁcacy versus luminance, and d) electroluminescence spectra of OLEDs; the insets show the working devices on ITO and NW electrodes, respectively. Figure 3. Characteristics of OLED devices using the embedded NW electrodes on PET and a PET/ITO reference. a) Current density versus bias voltage, b) luminance versus bias voltage, c) power efﬁcacy versus luminance, and d) electroluminescence spectra of OLEDs; the insets show the working devices on ITO and NW electrodes, respectively. Phys. Status Solidi RRL 2020, 14, 2000305 © 2020 The Authors. Published by Wiley-VCH GmbH www.advancedsciencenews.com [10] J. Li, Y. Tao, S. Chen, H. Li, P. Chen, M.-z. Wei, H. Wang, K. Li, M. Mazzeo, Y. Duan, Sci. Rep. 2017, 7, 16468. [11] R. P. Xu, Y. Q. Li, J. X. Tang, J. Mater. Chem. C 2016, 4, 9116. [12] E. Georgiou, S. A. Choulis, F. Hermerschmidt, S. M. Pozov, I. Burgués-Ceballos, C. Christodoulou, G. Schider, S. Kreissl, R. Ward, E. J. W. List-Kratochvil, C. Boeffel, Sol. RRL 2018, 1700192. [13] S. M. Pozov, G. Schider, S. Voigt, F. Ebert, K. Popovic, F. Hermerschmidt, E. Georgiou, I. Burgués-Ceballos, L. Kinner, D. Nees, B. Stadlober, C. Rapley, R. Ward, S. A. Choulis, E. J. W. List-Kratochvil, C. Boeffel, Flex. Print. Electron. 2019, 4, 025004. [14] L. Kinner, S. Nau, K. Popovic, S. Sax, I. Burgués-Ceballos, F. Hermerschmidt, A. Lange, C. Boeffel, S. A. Choulis, E. J. W. List-Kratochvil, Appl. Phys. Lett. 2017, 110, 101107. [15] Y. H. Kim, J. Lee, S. Hofmann, M. C. Gather, L. 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During bending tests, the resistance was measured from one edge of the sample to the other, perpendicular to the strain direction. [10] J. Li, Y. Tao, S. Chen, H. Li, P. Chen, M.-z. Wei, H. Wang, K. Li, M. Mazzeo, Y. Duan, Sci. Rep. 2017, 7, 16468. [11] R. P. Xu, Y. Q. Li, J. X. Tang, J. Mater. Chem. C 2016, 4, 9116. Keywords [28] E. Jung, C. Kim, M. Kim, H. Chae, J. H. Cho, S. M. Cho, Org. Electron. Phys. Mater. Appl. 2017, 41, 190. ﬂexible materials, indium tin oxide free, organic light-emitting diodes, poly(ethylene terephthalate), transparent electrodes [29] V. Scardaci, R. Coull, P. E. Lyons, D. Rickard, J. N. Coleman, Small 2011, 7, 2621. Received: June 17, 2020 Revised: July 16, 2020 Published online: August 11, 2020 [30] R. Kacar, S. P. Mucur, F. Yildiz, S. Dabak, E. Tekin, Nanotechnology 2017, 28, 245204. [31] M. Majumder, C. Rendall, M. Li, N. Behabtu, J. A. Eukel, R. H. Hauge, H. K. Schmidt, M. Pasquali, Chem. Eng. Sci. 2010, 65, 2000. [32] S. Aura, V. Jokinen, M. 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Mater. 2012, 24, 3326. Current density/voltage/luminance device characterization was conducted with a Keithley 2450 source measurement unit, together with a Konica Minolta LS-160 luminance meter in a purpose-built setup. The EL spectra were measured with an Ocean Optics CS2000 spectrometer and recorded using Ocean View software. [14] L. Kinner, S. Nau, K. Popovic, S. Sax, I. Burgués-Ceballos, F. Hermerschmidt, A. Lange, C. Boeffel, S. A. Choulis, E. J. W. List-Kratochvil, Appl. Phys. Lett. 2017, 110, 101107. E. J. W. List-Kratochvil, Appl. Phys. Lett. 2017, 110, 101107. [15] Y. H. Kim, J. Lee, S. Hofmann, M. C. Gather, L. Müller-Meskamp, [15] Y. H. Kim, J. Lee, S. Hofmann, M. C. Gather, L. Müller-Meskamp, K. Leo, Adv. Funct. Mater. 2013, 23, 3763. K. Leo, Adv. Funct. Mater. 2013, 23, 3763. [16] K. Ellmer, Nat. Photonics 2012, 6, 808. Acknowledgements [20] Y. Jin, D. Deng, Y. Cheng, L. Kong, F. Xiao, Nanoscale 2014, 6, 4812. [21] T. Kim, A. Canlier, G. H. Kim, J. Choi, M. Park, S. M. Han, ACS Appl. Mater. Interfaces 2013, 5, 788. This work was partially funded by the Austrian Climate Fund project ﬂex!PV_2.0 (project number 853603). This work was conducted in the framework of the Joint Lab GEN_FAB and was supported by the HySPRINT innovation lab at Helmholtz-Zentrum Berlin. Open access funding enabled and organized by Projekt DEAL. [22] N. de Guzman, J. Lopez, M. Vasquez Jr., M. D. Balela, Mater. Sci. Forum 2017, 890, 89. [23] P. Maisch, K. C. Tam, L. Lucera, H. J. Egelhaaf, H. Scheiber, E. Maier, [23] P. Maisch, K. C. Tam, L. Lucera, H. J. Egelhaaf, H. Scheiber, E. C. J. Brabec, Org. Electron. Phys. Mater. Appl. 2016, 38, 139. C. J. Brabec, Org. Electron. Phys. Mater. Appl. 2016, 38, 139. [24] T. Akter, W. S. Kim, ACS Appl. Mater. Interfaces 2012, 4, 1855. Conﬂict of Interest [25] L. Lian, D. Dong, D. Feng, G. He, Org. Electron. 2017, 49, 9. [26] S. Bai, H. Wang, H. Yang, H. Zhang, X. Guo, Mater. Res. Express 2018, 5, 026406 The authors declare no conﬂict of interest. [27] Z. Yu, Q. Zhang, L. Li, Q. Chen, X. Niu, J. Liu, Q. Pei, Adv. Mater. 2011, 23, 664. [1] A. I. Hofmann, E. Cloutet, G. Hadziioannou, Adv. Electron. Mater. 2018, 4, 1700412. Experimental Section OLED Manufacturing: To reduce the remaining spikes of NWs and optimize wetting behavior, all samples were plasma treated in a Femto Diener Plasma tool. Ambient air was used as plasma gas at a pressure of 0.3 mbar and 100 W power for 4 min. Following the plasma treatment, the samples were spin coated with a ZnO:PEI (2:1 by volume) in isopropyl alcohol (IPA) solution with 2500 rpm for 60 s and heated for 10 min at 110 C. The ZnO nanoparticle solution was purchased from Genesink, whereas PEI was purchased from Sigma Aldrich, and the PEI solution pre- pared according to the recipe of Zhou et al.[38] The emitting layer PDY-132 (Super Yellow, Merck) was spun at 2500 rpm for 60 s from a 5 mg mL1 toluene solution, with no heating applied. Following the spin-coating steps, 10 nm MoO3 and 200 nm Ag were evaporated. All processing and measuring except the vacuum steps were conducted in ambient air. During device studies it was shown that the presented elec- trode works well with a solution-processed ZnO:PEI electron- injection layer to realize an efﬁcient inverted OLED architecture. It was further shown that plasma treatment can reduce possible shunts in NW-based electrodes. In device tests our electrode showed similar power efﬁcacy performance as commercial ITO PET substrates, but almost a seven times higher maximum luminance values. Overall, a ﬂexible and solution-processed alter- native to ITO was presented. Characterization Methods: The sample surfaces were characterized with a scanning force microscope (Molecular Imaging, Pico Plus) in tap- ping mode, using tapping mode cantilevers (PPP-NCHR) from 2000305 (5 of 6) Phys. Status Solidi RRL 2020, 14, 2000305 www.advancedsciencenews.com www.pss-rapid.com © 2020 The Authors. Published by Wiley-VCH GmbH Keywords Interfaces 2018, 10, 24099. [38] Y. Zhou, C. Fuentes-Hernandez, J. Shim, J. Meyer, A. J. Giordano, H. Li, P. Winget, T. Papadopoulos, H. Cheun, J. Kim, M. Fenoll, A. Dindar, W. Haske, E. Najafabadi, T. M. Khan, H. Sojoudi, S. Barlow, S. Graham, J. Brédas, S. R. Marder, A. Kahn, B. Kippelen, Science 2012, 873, 327. C. G. Granqvist, Sol. Energy Mater. Sol. Cells 2012, 99, 1. [8] L. Kinner, M. Bauch, R. A. Wibowo, G. Ligorio, E. J. W. List-Kratochvil, T. Dimopoulos, Mater. Des. 2019, 168, 107663. [8] L. Kinner, M. Bauch, R. A. Wibowo, G. Ligorio, E. J. W. List-Kratochvil, [9] M. Luo, Y. Liu, W. Huang, W. Qiao, Y. Zhou, Y. Ye, L.-S. Chen, Micromachines 2017, 8, 12. 2000305 (6 of 6) © 2020 The Authors. Published by Wiley-VCH GmbH Phys. Status Solidi RRL 2020, 14, 2000305 © 2020 The Authors. Published by Wiley-VCH GmbH
https://openalex.org/W3167469052	https://www.frontiersin.org/articles/10.3389/fimmu.2021.666543/pdf	English	null	Innate Immune Responses to Wildtype and Attenuated Sheeppox Virus Mediated Through RIG-1 Sensing in PBMC In-Vitro	Frontiers in immunology	2,021	cc-by	7,637	Innate Immune Responses to Wildtype and Attenuated Sheeppox Virus Mediated Through RIG-1 Sensing in PBMC In-Vitro Tesfaye Rufael Chibssa 1,2,3, Richard Thiga Kangethe 1, Francisco J. Berguido 1, Tirumala Bharani K. Settypalli 1, Yang Liu 4, Reingard Grabherr 2, Angelika Loitsch 5, Elena Lucia Sassu 1,6, Rudolf Pichler 1, Giovanni Cattoli 1, Adama Diallo 7,8, Viskam Wijewardana 1 and Charles Euloge Lamien 1 1 Animal Production and Health Laboratory, Joint FAO/IAEA Agricultural and Biotechnology Laboratory, Division of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna, Austria, 2 Institute of Biotechnology, University of Natural Resources and Life Sciences (BOKU), Vienna, Austria, 3 National Animal Health Diagnostic and Investigation Center (NAHDIC), Sebeta, Ethiopia, 4 China National Clinical Research Center for Neurological Diseases, Beijing Tiantan Hospital, Capital Medical University, Beijing, China, 5 Austrian Agency for Health and Food Safety (AGES), Vienna, Austria, 6 Department for Farm Animals and Veterinary Public Health, University Clinic for Swine, University of Veterinary Medicine, Vienna, Austria, 7 Laboratoire National d’Elevage et de Recherches Ve´ te´ rinaires, Institut Se´ ne´ galais de Recherches Agricoles (ISRA), Dakar, Se´ ne´ gal, 8 UMR CIRAD INRA, Animal, Sante´ , Territoires, Risques et Ecosystèmes (ASTRE), Montpellier, France ORIGINAL RESEARCH published: 15 June 2021 doi: 10.3389/fimmu.2021.666543 Reviewed by: Reviewed by: Shawn Babiuk, National Centre for Foreign Animal Disease (NCFAD), Canada Amit Kumar, Indian Veterinary Research Institute (IVRI), India Gnanavel Venkatesan, ICAR-Indian Veterinary Research Institute, India Sheeppox (SPP) is a highly contagious disease of small ruminants caused by sheeppox virus (SPPV) and predominantly occurs in Asia and Africa with signiﬁcant economic losses. SPPV is genetically and immunologically closely related to goatpox virus (GTPV) and lumpy skin disease virus (LSDV), which infect goats and cattle respectively. SPPV live attenuated vaccines (LAVs) are used for vaccination against SPP and goatpox (GTP). Mechanisms related to innate immunity elicited by SPPV are unknown. Although adaptive immunity is responsible for long-term immunity, it is the innate responses that prevent viral invasion and replication before LAVs generate speciﬁc long-term protection. We analyzed the relative expression of thirteen selected genes that included pattern recognition receptors (PRRs), Nuclear factor-kb p65 (NF-kb), and cytokines to understand better the interaction between SPPV and its host. The transcripts of targeted genes in sheep PBMC incubated with either wild type (WT) or LAV SPPV were analyzed using quantitative PCR. Among PRRs, we observed a signiﬁcantly higher expression of RIG-1 in PBMC incubated with both WT and LAV, with the former producing the highest expression level. However, there was high inter-individual variability in cytokine transcripts levels among different donors, with the expression of TNFa, IL-15, and IL-10 all signiﬁcantly higher in both PBMC infected with either WT or LAV compared to control PBMC. Correlation studies revealed a strong signiﬁcant correlation between RIG-1 and IL-10, between TLR4, TNFa, and NF-kb, between IL-18 and IL-15, and between NF-kb and IL-10. There was also a signiﬁcant negative correlation between RIG-1 and IFNg, between TLR3 and IL-1 b, and between TLR4 and IL-15 (P< 0.05). This study identiﬁed RIG-1 as an important PRR in the signaling pathway of innate immune activation during SPPV infection, possibly Correspondence: Richard Thiga Kangethe R.T.Kangethe@iaea.org Specialty section: This article was submitted to Comparative Immunology, a section of the journal Frontiers in Immunology Received: 10 February 2021 Accepted: 27 May 2021 Published: 15 June 2021 Received: 10 February 2021 Accepted: 27 May 2021 Edited by: Edited by: Stephanie DeWitte-Orr, Wilfrid Laurier University, Canada Viruses The Wild type isolate SPPV Algeria/93 Djelfa, obtained from the Institut National de la Médecine Vétérinaire, Algiers, Algeria, and Romanian SPPV vaccine, obtained from BioPharma, Morocco, were used for this study. Both viruses were propagated on embryonic skin cell lines from sheep (ESH-L cells) in Hank’s Minimum Essential Medium (MEM) supplemented with 10% fetal calf serum and 1% antibiotics (17). The viral suspensions were titrated (18) and stored at −80°C until further use. All procedures were performed in the Bio-Safety Level-3 laboratory facilities at AGES, Austria. ( ) Most SPPV LAVs were developed by serial passages of virulent or low virulent SPPVs in cell culture. Immune responses and host interactions with viruses, including poxviruses, are widely studied in vitro using peripheral blood mononuclear cells (PBMC) (13). In addition to ﬁbroblasts, PBMC also includes target cells for SPPV, such as monocytes and macrophages (14). The cytokine expression of PBMC collected from animals vaccinated with SPPV (Romanian vaccine strains), and goatpox virus (Gorgan strain) has previously been studied (15). However, PBMC used in the 2017 study were stimulated with inactivated viruses for the evaluation of immune responses to vaccines using qPCR to measure IL-4 and IFNg. SPPV infection of sheep PBMC induces an innate antiviral response that leads to the recruitment of antigen-presenting cells which in turn initiate adaptive immunity. Innate immune responses are responsible for recognizing pathogens at the initial encounter through pattern- recognition receptors (PRRs) such as Toll-like receptors (TLRs) and RIG-1-like receptors (RLRs). The recognition of viral pathogens activates a cascade of events leading to the induction of downstream signaling molecules and transcription factors, consequently inducing inﬂammatory cytokines and chemokines (16). Therefore, the investigation of innate immune responses provides vital information about virus-host Citation: Chibssa TR, Kangethe RT, Berguido FJ, Settypalli TBK, Liu Y, Grabherr R, Loitsch A, Sassu EL, Pichler R, Cattoli G, Diallo A, Wijewardana V and Lamien CE (2021) Innate Immune Responses to Wildtype and Attenuated Sheeppox Virus Mediated Through RIG-1 Sensing in PBMC In-Vitro. Front. Immunol. 12:666543. doi: 10.3389/fimmu.2021.666543 Chibssa TR, Kangethe RT, Berguido FJ, Settypalli TBK, Liu Y, Grabherr R, Loitsch A, Sassu EL, Pichler R, Cattoli G, Diallo A, Wijewardana V and Lamien CE (2021) Innate Immune Responses to Wildtype and Attenuated Sheeppox Virus Mediated Through RIG-1 Sensing in PBMC In-Vitro. Front. Immunol. 12:666543. doi: 10.3389/fimmu.2021.666543 June 2021 \| Volume 12 \| Article 666543 Frontiers in Immunology \| www.frontiersin.org Chibssa et al. SPPV Increases Sheep RIG-1 Expression In-Vitro through intermediate viral dsRNA. The role of immunomodulatory molecules produced by SPPV capable of inhibiting downstream signaling activation following RIG-1 upregulation is discussed. These ﬁndings advance our knowledge of the induction of immune responses by SPPV and will help develop safer and more potent vaccines against SPP and GTP. INTRODUCTION interactions. However, little is known on the innate immune response mechanisms elicited by wild-type (WT) and LAV SPPVs. Interrogation of immune responses by directly comparing WT versus LAV SPPVs could provide information on the escape of host immunity by WT. Additionally, any immune markers that LAV induces in the host could also be used for vaccine design. Sheeppox (SPP) is a highly contagious viral disease of domestic and wild small ruminants, causing signiﬁcant economic losses in sheep and goat productivity (1). Sheeppox virus (SPPV), the causative agent of the disease, and two other viruses, goatpox virus (GTPV) and lumpy skin disease virus (LSDV), are the three members of the genus Capripoxvirus within the family Poxviridae (2). Commonly, SPPV and GTPV cause SPP and GTP in sheep and goats, respectively, while LSDV is restricted to cattle. However, host speciﬁcity of SPPV and GTPV is not strict as cross-infection between goat and sheep by those two viruses have been noted (3–5). The two viruses are closely related genetically and immunologically, with some cross-protection observed (6). Given their economic relevance and severity, SPP, GTP, and LSD are listed as notiﬁable diseases by the world organization for animal health (7). Live attenuated vaccines (LAV) derived from various SPPV strains are used to control SPP and GTP (8–10). Several reports have recognized LAVs as effective vaccines against CaPV infections; however, ﬁeld data on vaccination suggest some efﬁcacy and safety concerns. There are some vaccination failure cases and adverse reactions (11, 12). This study analyzed the innate immune responses to SPPV infections in sheep PBMC. The expression levels of thirteen targeted genes, including pattern recognition receptors (PRRs), Nuclear factor-kb p65 (NF-kb), and cytokines in sheep PBMC, were compared, following in vitro infections using SPPV WT or LAV viruses. Frontiers in Immunology \| www.frontiersin.org Keywords: gene expression, PRRs, cytokine, RIG-1, LAV, SPPV RNA Extraction, cDNA Synthesis, and qPCR Surface and Intracellular Cytokine Staining After 4 days of incubation of PBMC with SPPV (WT or LAV) or PBS, protein transport in cells was blocked by adding BrefeldinA (GolgiStop, BD Biosciences, USA) overnight. Positive control for cytokine production was generated by adding a cell stimulation cocktail (Leukocyte Activation Cocktail with GolgiStop, BD Biosciences) to a third well and incubated overnight. On day 5, PBMC were harvested, washed in PBS, transferred to microcentrifuge tubes, and stained for ﬂow cytometry. Surface staining master mix containing mouse anti-sheep CD4 antibody (clone 44.38: Bio-Rad) and mouse anti-bovine CD8 (clone CC63, cross-reacts with sheep: Bio-Rad) were added to each tube, and samples were incubated at 4°C for 30 min. Following surface staining, dead cell exclusion staining was done with a ﬁxable viability stain (BD Biosciences) before cell ﬁxation. The cells were washed, ﬁxed, and permeabilized with a saponin-based buffer (Fixation/Permeabilization Solution Kit, BD Biosciences) according to the manufacturer’s protocol. Intracellular cytokine staining was done with mouse anti-bovine interferon-gamma antibody (clone CC302, cross-reacts with sheep, Bio-Rad) for 30 min at room temperature in the dark. Cells were washed twice in 1x Perm/Wash buffer and resuspended in FACS buffer (PBS containing 2% FBS and 2mM ethylenediaminetetraacetic acid). Flow cytometry data were acquired using the Gallios ﬂow cytometer (Becton Dickinson Beckman Coulter, USA) and analyzed with Kaluza software (Becton Dickinson, Beckman Coulter). Cell populations were gated by forward and side-light- scatter parameters, as shown in Figure 1. Intracellular cytokine expression was calculated as a percentage of the parent population. q After an incubation period of 2 days, PBMC and CD14- cells were harvested, and total RNA extracted from PBMC incubated either with WT or LAV and from control cells using the RNeasy mini kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions with on-column DNAse treatment. The total RNA was resuspended in 30µl of RNase-free water and quantiﬁed using a Nanodrop ND-1000 spectrophotometer. Total RNA was converted into cDNA using the SuperScript™III First- Strand Synthesis System (Invitrogen, USA) as per the manufacturer’s instructions. Approximately one µg of each RNA sample was used with random hexamers according to the manufacturer’s instructions. The resulting cDNA was stored at - 20°C until further use. For quantitative analysis, a cDNA dilution of 1: 100 was used as a template for real-time PCR using speciﬁc primers presented in Supplementary Table 1 (20), using GAPDH as a housekeeping gene. Quantiﬁcation of Viral Growth in PBMC The quantiﬁcation of the active virus using cDNA from PBMC and CD14- cells incubated with WT or LAV was carried out with primers and probes targeting the RPO30 gene as previously described (21). Brieﬂy, RNA extracted with on-column DNAse treatment that destroys inoculating SPPV was used as the template for one-step RT-PCR. Absolute quantiﬁcation of replicating virus was calculated based on the ampliﬁcation of serially diluted RPO30 plasmid with target copies from 108 to 102 per 2 mL. RNA without reverse transcription was used as a control. PBMC Isolation, CD14- Cell Isolation and Viral Infection Blood samples were collected from healthy local Austrian sheep via jugular vein using heparinized vacutainer tubes and needles. A certiﬁed veterinary service collected the samples according to the Austrian Agency for Health and Food Safety (AGES) local guidelines and international guidelines by OIE. PBMC were separated by density-gradient centrifugation using Ficoll-Paque (density, 1.077 g/ml; GE health care, Sweden) and washed twice with RPMI 1640 medium (Gibco, Carlsbad, CA, USA). The resultant pellet was resuspended in complete media (RPMI 1640 media containing 10% FBS and 100 IU/mL penicillin and 100 µg/mL streptomycin) and cell numbers were determined using a hemocytometer. CD14+ monocytes were captured from PBMC using magnetic beads conjugated to a bovine cross- reacting anti-human CD14 monoclonal antibody (human CD14 microbeads, Miltenyi Biotech, Germany) and the CD14- ﬂow-through cells collected as previously described (19). June 2021 \| Volume 12 \| Article 666543 Frontiers in Immunology \| www.frontiersin.org 2 SPPV Increases Sheep RIG-1 Expression In-Vitro Chibssa et al. PBMC derived from the donor sheep (n=5) were suspended in complete media and placed in either 24-well plates containing 5 x 106 cells in 1 ml per well (for ﬂow cytometry) or 6-well plates containing 20 x 106 cells in 5 ml per well (for real-time PCR analysis). PBMC were then cultured either with 10µl (ﬂow cytometry) or 100µl (real-time PCR analysis) of WT or LAV strain of SPPV at a concentration of 1x106 TCID50/ml. CD14- cells were also cultured for real-time PCR analysis at 20 x 106 cells in 5 ml with WT or LAV strain of SPPV. Uninfected controls consisting of 10µl or 100µl of PBS added to PBMC or CD14- cell suspensions were also prepared. The plates were incubated at 37°C in a humidiﬁed atmosphere containing 5% CO2 for 2 days (for real-time PCR analysis) or 4 days (for ﬂow cytometry). The incubation period and the MOI were determined by conducting a series of experiments with variable time points for harvesting and using different MOI. relative quantities (GAPDH). Cq values of the ampliﬁed templates in sheep PBMC cultured with either WT or LAV were used for the calculation of gene expression. Differences in expression levels were given as fold-change (FC) using the sheep GAPDH gene for normalization and compared with PBMC control. RNA Extraction, cDNA Synthesis, and qPCR Real-time PCR was performed in a total reaction volume of 10 ml containing 2 ml of diluted cDNA as template, 1 ml of forward and reverse primers (Supplementary Table 1), 5 ml of iQ SYBR Green Supermix 2X (Bio-Rad, USA), and 1 µl of water. All samples were tested in triplicate. Real-time PCR was performed in a CFX96 real-time PCR Detection System (Bio-Rad, Hercules, USA). The cycling conditions and quantiﬁcation of targets were performed as previously described (20). Frontiers in Immunology \| www.frontiersin.org Quantitative Expression Analysis p y The relative fold-change for the thirteen selected genes (Supplementary Table 1) in PBMC infected either with WT or LAV compared to PBMC control were analyzed by real-time PCR. These genes were chosen as representative markers from various immune system components, enabling them to interrogate diverse pathways. A panel of speciﬁc primers compatible with the same thermal cycling program was used. The speciﬁcity of each primer pair was evaluated by melting curve analysis. All ampliﬁed PCR products showed distinct and unique Tms that were compatible with the ampliﬁcation of ovine targets, as previously reported (20). The relative quantities of the target genes were normalized against the internal standard’s Data Analysis and Presentation Data Analysis and Presentation In the present study, gene expression was analyzed using the efﬁciency-corrected calculation models, based on multiple samples, to estimate the relative changes in the gene expressions (normalized with the GAPDH housekeeping genes) (22). Boxplots of the log2 (FC) values and the parent population percentage for ﬂow cytometry data were generated using ggplot2 package in R (23). One sample t-test and two-sample June 2021 \| Volume 12 \| Article 666543 Frontiers in Immunology \| www.frontiersin.org 3 SPPV Increases Sheep RIG-1 Expression In-Vitro Chibssa et al. A B C GURE 1 \| Gate represents gating strategy for Flow cytometry. Analytic gating of ﬂow cytometry showed a representative graph of the change in the mean uorescence intensity of IFNg in the PBMC subpopulation. (A) Single cells were selected in the forward scatter-height (FSC-HA) versus forwarding scatter-width SC-WA) plot, then, live/dead was gated to identify live cells. Next, monocytes and lymphocytes were gated from the live cells on FSC-A versus SSC-A plot. Gating lymphocyte subset was performed as CD4+, CD8+, and CD4/CD8 double-negative (DN). (B) Expression of IFNg by PBMC, monocytes, and lymphocytes: IFNg ositive populations were gated using PBMC cultured with PBS (negative control) and PBMC cultured with cell stimulation cocktail (positive control). (C) Expression IFNg by CD4+, CD8+, and DN: IFNg positive populations were gated using PBMC cultured with PBS (negative control) and PBMC cultured with cell stimulation ocktail (positive control). A A B B B C C FIGURE 1 \| Gate represents gating strategy for Flow cytometry. Analytic gating of ﬂow cytometry showed a representative graph of the change in the mean ﬂuorescence intensity of IFNg in the PBMC subpopulation. (A) Single cells were selected in the forward scatter-height (FSC-HA) versus forwarding scatter-width (FSC-WA) plot, then, live/dead was gated to identify live cells. Next, monocytes and lymphocytes were gated from the live cells on FSC-A versus SSC-A plot. Gating of lymphocyte subset was performed as CD4+, CD8+, and CD4/CD8 double-negative (DN). (B) Expression of IFNg by PBMC, monocytes, and lymphocytes: IFNg positive populations were gated using PBMC cultured with PBS (negative control) and PBMC cultured with cell stimulation cocktail (positive control). (C) Expression of IFNg by CD4+, CD8+, and DN: IFNg positive populations were gated using PBMC cultured with PBS (negative control) and PBMC cultured with cell stimulation cocktail (positive control). Frontiers in Immunology \| www.frontiersin.org Expression Analysis p y The expression level of the transcripts for the PRRs, including TLR3, TLR4, TLR8, and RIG-1, following the infection of sheep PBMC with WT SPPV or LAV SPPV, were converted to log2 fold change and presented in Figure 2. At 48 hours post- infection, there was a highly signiﬁcant expression of RIG-1 in both WT (100-FC) and LAV (60 FC) in infected PBMC cultures (P < 0.001). Moreover, RIG-1 expression in PBMC that were cultured with WT was signiﬁcantly higher than in those cultured with LAV (P < 0.05). NF-kb p65 was clearly upregulated in two donors for both LAV and WT; however, the mean fold changes for PBMC infected with either WT (4.3) or LAV (3.7) were not statistically different from that of the control PBMC (P > 0.05; Figure 2). The expression level of transcripts for IFNa, IFNg, TNFa, IL-1b, IL-6, IL-10, IL-15, and IL-18 showed high variability in the cytokine transcripts levels between different donors: 3.1-fold for IL-18 to 28.14-fold for IL- 1b for PBMC treated with LAV; and 4.22-fold for IL-18 to 27.26-fold for IL-1b for those treated with WT. The mean fold changes for TNFa (WT = 5.3, LAV = 3.5), IL-15 (WT = 5.6, LAV = 3.6), and IL-10 (WT = 9.3, LAV = 4.1) were signiﬁcantly higher in both PBMC infected with either WT or LAV as compared control PBMC (P < 0.05, Figure 2). The expression levels of TNFa, IL-15, and IL-10 were higher in PBMC incubated with WT than LAV, though the difference was not signiﬁcant (P > 0.05). IFNg, IFNa, IL-6, and p g Since many immune markers followed a similar expression pattern between the two (WT and LAV) treatments, we next investigated which markers correlated in their expression. Indeed, heatmap analysis revealed relationships between receptors, transcription factors, and cytokines (Figure 3). Our results showed a strong signiﬁcant correlation between the expression of RIG-1, which recognizes dsRNA, and IL-10 (P< 0.01), and a signiﬁcant negative correlation between RIG-1 and IFNg (P< 0.05) in PBMC. TLR3, another PRR that can recognize dsRNA, is moderately correlated with IL-6, IL-15, and IL-18. However, these correlations were not statistically signiﬁcant. We also observed a highly signiﬁcant negative correlation between the expression of TLR3 and IL-1 b (P< 0.01) in PBMC. Bioinformatic Analysis paired t-test were performed in R to compare gene expression differences between the infected and control groups. Infection of PBMC between WT, LAV, PBMC, and CD14- groups were analyzed by ﬁtting a mixed-effects model ANOVA using GraphPad Prism 9. The heatmap.2 function of R gplots package was used to create both expression and correlation heatmaps. The Pearson correlations were computed in R. Hierarchical clustering was performed using the complete linkage method to compute the distance between clusters. Based on the previous knowledge that the vaccinia virus E3 and N1L proteins can inhibit the signaling to NF-kb (24–26), we analyzed their SPPV orthologs in comparison to vaccinia virus and other poxviruses. Sequence alignment of the E3 and the N1L amino acid sequences were done using clustalW implemented in BioEdit version 7.2.6. The ortholog sequence of N1L from SPPV Djelfa was analyzed using i-Tasser model prediction software (Yang Frontiers in Immunology \| www.frontiersin.org June 2021 \| Volume 12 \| Article 666543 SPPV Increases Sheep RIG-1 Expression In-Vitro Chibssa et al. Zhang Laboratory – University of Michigan, Ann Arbor, MI, USA). LOMETS meta-threading software (RCSB Protein Databank) determined the structural similarities based on z- scores. Pymol (Schroedinger, Inc – New York, NY USA) was used to model the structures. IL-18 each presented various patterns of regulation depending on the donor. In general, none of the cytokines were differentially expressed between WT and LAV treatments. These data collectively reveal a greater innate immune gene expression in WT infected sheep PBMC cultures than LAV infections, likely due to increased levels of viral dsRNA within the cytoplasm of infected PBMC. Additionally, inter-individual variability was more pronounced with cytokine transcripts levels. Expression Analysis Similarly, there was a signiﬁcant positive correlation between the expression levels of TLR4, TNFa, and NF-kb (P< 0.05) and a signiﬁcant negative correlation between TLR4 and IL-15 (P< 0.05) in PBMC (Figure 3). There was a strong and signiﬁcant (WT = 9.3, LAV = 4.1) were signiﬁcantly higher in both PBMC infected with either WT or LAV as compared control PBMC (P < 0.05, Figure 2). The expression levels of TNFa, IL-15, and IL-10 were higher in PBMC incubated with WT than LAV, though the difference was not signiﬁcant (P > 0.05). IFNg, IFNa, IL-6, and FIGURE 2 \| Box plots representing the differential expression of mRNA of thirteen genes. The expression of RIG-1, IL-10, IL-15 and TNFa were signiﬁcantly upregulated. Note that the expression RIG-1 was signiﬁcantly higher in WT as compared to LAV. Data represent the Log2 FC of ﬁve independent experiments. Signiﬁcance level was set at P value (p<0.05, p<0.001, *p<0.0001). FIGURE 3 \| Heat map for correlations of target genes expression FC using qPCR. The correlation heat map describes the combined Pearson correlation coefﬁcient based on the distance between the two gene expression value against all target groups. This shows that the heatmap will cluster together, genes that have positively correlated log2 FC value, P-value; P<0.05, P<0.01*. FIGURE 3 \| Heat map for correlations of target genes expression FC using qPCR. The correlation heat map describes the combined Pearson correlation coefﬁcient based on the distance between the two gene expression value against all target groups. This shows that the heatmap will cluster together, genes that have positively correlated log2 FC value, P-value; P<0.05, P<0.01*. FIGURE 2 \| Box plots representing the differential expression of mRNA of thirteen genes. The expression of RIG-1, IL-10, IL-15 and TNFa were signiﬁcantly upregulated. Note that the expression RIG-1 was signiﬁcantly higher in WT as compared to LAV. Data represent the Log2 FC of ﬁve independent experiments. Signiﬁcance level was set at P value (p<0.05, p<0.001, p<0.0001). FIGURE 3 \| Heat map for correlations of target genes expression FC using qPCR. The correlation heat map describes the combined Pearson correlation coefﬁcient based on the distance between the two gene expression value against all target groups. This shows that the heatmap will cluster together, genes that have positively correlated log2 FC value, P-value; P<0.05, P<0.01*. FIGURE 2 \| Box plots representing the differential expression of mRNA of thirteen genes. Expression Analysis The expression of RIG-1, IL-10, IL-15 and TNFa were signiﬁcantly upregulated. Note that the expression RIG-1 was signiﬁcantly higher in WT as compared to LAV. Data represent the Log2 FC of ﬁve independent experiments. Signiﬁcance level was set at P value (p<0.05, p<0.001, p<0.0001). FIGURE 2 \| Box plots representing the differential expression of mRNA of thirteen genes. The expression of RIG-1, IL-10, IL-15 and TNFa were signiﬁcantly upregulated. Note that the expression RIG-1 was signiﬁcantly higher in WT as compared to LAV. Data represent the Log2 FC of ﬁve independent experiments. Signiﬁcance level was set at P value (p<0.05, p<0.001, *p<0.0001). June 2021 \| Volume 12 \| Article 666543 Frontiers in Immunology \| www.frontiersin.org 5 SPPV Increases Sheep RIG-1 Expression In-Vitro Chibssa et al. poxviruses’ orthologs showed all essential residues contributing to E3 dsRNA binding (Supplementary Figure 2). positive correlation between the expression level of IL-18 and IL- 15 (P< 0.01). The expression of NF-kb positively and signiﬁcantly correlated with the expression of IL-10 (P< 0.05). positive correlation between the expression level of IL-18 and IL- 15 (P< 0.01). The expression of NF-kb positively and signiﬁcantly correlated with the expression of IL-10 (P< 0.05). In contrast, the N1L of SPPV and other capripoxvirus displayed considerable amino acid sequence divergence to the sequence of vaccinia. Nevertheless, amino acid residues 13, 16, 17, 30, 37, 44, 61 – 78, 89, 92, 98 -102, and 109 – 112 were either identical across all poxviruses or replaced with similar side groups e.g., isoleucine for Leucine in position 13. This prompted the protein quaternary structure modeling compared to the VACV N1L structure (Supplementary Figure 1). Sequence alignment of N1L protein from different poxviruses suggests that N1L is highly conserved between sheeppox virus (Djelfa, Roumania), lumpy skin disease virus, and goatpox virus. These substitutions do not affect the quaternary structure of the folded protein (Supplementary Figure 1). Flow Cytometry Analysis of IFNg in Sheep PBMC Cultured With SPPV Next, to validate mRNA expressions of immune markers, protein production by individual cell populations within the PBMC were analyzed through ﬂow cytometry. The IFNg producing cells were identiﬁed as a percentage of total live PBMC, monocytes, lymphocytes or CD4+, CD8+, and CD4/CD8 double-negative cells within the lymphocyte population (Figure 1). None of the subpopulations had a difference in the percentage of IFNg producing cells in the identiﬁed cell populations between WT or LAV treatments (Figure 4). Collectively, the IFNg production by PBMC detected with ﬂow cytometry was consistent with that determined through quantitative RT-PCR and showed that there was no disparity even among the subpopulation of cells. Quantiﬁcation of Active Virus We have analyzed the relative expression of thirteen selected genes, encoding for proteins involved in host innate responses to viral infections, following the infection of sheep PBMC with WT SPPV and LAV SPPV to understand better the interaction between SPPV and its host. The quantiﬁcation of replicating virus in PBMC incubated with WT virus reveals a higher viral load, close to a log difference, compared to LAV (Figure 5). This trend is maintained after removing monocytes from the whole PBMC. More than one log reduction was observed for both WT and LAV in CD14- cells (Figure 5). These changes were not statistically signiﬁcant after analysis was performed. RIG-1 expression was also measured in CD14- cells where an increase was observed when compared to CD14- control cultures (77.5 FC in WT and 77.9 F in LAV; Supplementary Figure 3). Among the mRNA of the pattern recognition receptors (PRRs), we have observed that RIG-1 was highly expressed in PBMC infected with WT and LAV, suggesting that RIG-1 may play an essential role in innate recognition of SPPV. As RIG-1 is a sensor for dsRNA (27), the high expression of this receptor in both WT and LAV suggests that dsRNA intermediates are produced during SPPV infection. Indeed, previous studies showed that some dsDNA viruses, such as poxviruses, that replicate and transcribe their genomes and assemble infectious N1L Protein in VACV and SPPV Modeling N1L Protein in VACV and SPPV Modeling To investigate the lack of downstream signal following the activation of RIG-1, including the absence of NF-kb and type I and type II interferon response, we analyzed the capripoxvirus orthologs of the vaccinia virus N1L and E3 proteins, two known inhibitors of the signaling to NF- kb. The multiple sequence alignment of SPPV E3 amino acid sequence with other FIGURE 5 \| Active viral replication in PBMC and CD14- cells. DNAse treated RNA was used a template for absolute quatiﬁcation of the RPO30 gene. Viral copies were calculated and the mean (n=5) plotted as box plots with GraphPad Prism 9. Analysis was performed using ANOVA. FIGURE 4 \| Flow cytometry analysis of the expression of IFNg in PBMC subpopulations. The box plots represent the percentage of IFNg expression in healthy sheep PBMC sub-population infected with either WT SPPV or LAV SPPV. FIGURE 5 \| Active viral replication in PBMC and CD14- cells. DNAse treated RNA was used a template for absolute quatiﬁcation of the RPO30 gene. Viral copies were calculated and the mean (n=5) plotted as box plots with GraphPad Prism 9. Analysis was performed using ANOVA. FIGURE 4 \| Flow cytometry analysis of the expression of IFNg in PBMC subpopulations. The box plots represent the percentage of IFNg expression in healthy sheep PBMC sub-population infected with either WT SPPV or LAV SPPV. FIGURE 5 \| Active viral replication in PBMC and CD14- cells. DNAse treated RNA was used a template for absolute quatiﬁcation of the RPO30 gene. Viral copies were calculated and the mean (n=5) plotted as box plots with GraphPad Prism 9. Analysis was performed using ANOVA. FIGURE 4 \| Flow cytometry analysis of the expression of IFNg in PBMC subpopulations. The box plots represent the percentage of IFNg expression in healthy sheep PBMC sub-population infected with either WT SPPV or LAV SPPV. June 2021 \| Volume 12 \| Article 666543 Frontiers in Immunology \| www.frontiersin.org 6 SPPV Increases Sheep RIG-1 Expression In-Vitro Chibssa et al. particles exclusively in the cytoplasm of cells produce dsRNA intermediates (28). The production of IFNg did not differ between WT and LAV in any of the cell populations when analyzed through ﬂow cytometry, which is consistent with mRNA expression data. However, the highest IFNg expression was seen in the monocyte cell population. It has already been shown that SPPV can infect monocytes (29). N1L Protein in VACV and SPPV Modeling We conﬁrmed this by observing a reduced replication of both WT and LAV in CD14- and no signiﬁcant difference in the expression of RIG-1 when monocytes are removed from culture. RIG-1 expression was signiﬁcantly higher in PBMC infected with WT than in those infected with LAV, suggesting that WT infection in PBMC produces more dsRNA intermediates than LAV. Our ﬁnding is consistent with the observation that active virus in infected PBMC is one log higher in WT infection than LAV, with this effect likely produced during the infection of monocytes. This suggests that the lower replication of LAV directly results in a lower number of dsRNA intermediates and consequent lower RIG-1 expression when compared to WT virus. This further suggests RIG-1 expression is a direct result of active viral replication yielding dsRNA intermediates. It was interesting to notice that among the tested PRRs, only RIG-1 was signiﬁcantly positively expressed, suggesting that host sensing of SPPV ds RNA intermediates is mostly through RLRs than TLRs. expression after sorting infected and non-infected cells. Interestingly, our data analysis shows a strong positive correlation between the PRR RIG-1 and IL-10, both positively signiﬁcantly expressed in WT and LAV SPPV infected PBMC. The strong positive correlation between RIG-1 and IL-10 suggests that the anti-inﬂammatory properties of IL-10 could mediate immune evasion of SPPV. Anti-inﬂammatory cytokines such as IL-10 create an inhibitory environment (34). On the other hand, RIG-1 negatively correlated with IFNg, a necessary cytokine for driving Th1 cell-mediated immunity to control viral infections. Curiously, the activation of RIG-1 did not lead to the subsequent expression of other signaling molecules of the RIG-1 pathway. For instance, our data suggested that the production of NF-kb and type I and type II interferon response were comparable between treated and control PBMC. The ﬂow cytometry results further supported the absence of speciﬁc expression after sorting infected and non-infected cells. Interestingly, our data analysis shows a strong positive correlation between the PRR RIG-1 and IL-10, both positively signiﬁcantly expressed in WT and LAV SPPV infected PBMC. The strong positive correlation between RIG-1 and IL-10 suggests that the anti-inﬂammatory properties of IL-10 could mediate immune evasion of SPPV. Anti-inﬂammatory cytokines such as IL-10 create an inhibitory environment (34). On the other hand, RIG-1 negatively correlated with IFNg, a necessary cytokine for driving Th1 cell-mediated immunity to control viral infections. N1L Protein in VACV and SPPV Modeling Curiously, the activation of RIG-1 did not lead to the subsequent expression of other signaling molecules of the RIG-1 pathway. For instance, our data suggested that the production of NF-kb and type I and type II interferon response were comparable between treated and control PBMC. The ﬂow cytometry results further supported the absence of speciﬁc IFNg production following PBMC infection with SPPV. A possible explanation is that SPPV-encoded virokines inhibited RIG-1 downstream signaling leading to a type 1 interferon response. For instance, SPPV possesses VACV orthologous genes encoding for E3, A52, and N1L proteins, all known to interfere with the downstream regulation of RIG-1 (26). Of interest, previous reports have shown that N1L promotes the virulence of VACV (35) through various mechanisms such the inhibition to signaling to NF-kb (24–26), as well as the IL-1b and TLR4 signaling cascades (25). The sequence alignment of the E3 showed the conservation of essential residues between SPPV and vaccinia virus. The quaternary structure modeling of SPPV N1L, in comparison to VACV, showed that the two proteins display similarities in their structures, suggesting that the SPPV ortholog of N1L protein could play the same function of inhibiting NF-kb signaling. This signaling is responsible for triggering pro-inﬂammatory activity in the host, indicating the importance of SPPV N1L as a virulence factor (36). There was a high inter-individual variability for cytokines compared to PRRs, though the levels of both cytokines and PPRs appeared to be characteristics of different donors. Indeed, in each donor, the cytokine levels and PRRs levels followed the same trend independently of the SPPV strain used for PBMC infection. Similar inter-individual variabilities in cytokine productions in human PBMC were previously reported and attributed to polymorphisms in genes that control cytokines expression (25, 30). Among the tested cytokines, the expressions of TNFa, IL-15, and IL-10 were all signiﬁcantly upregulated, suggesting that they may play an essential role in SPPV infections. A similar increase in IL-10 expression following SPPV infection in vivo has been reported (31). IL-10 is a critical anti-inﬂammatory cytokine secreted by monocytes following their infection with pathogens, and increased production of IL-10 following infection by other poxviruses has been previously reported (25, 32). In addition, an IL-10 gene homolog deﬁcient LSDV conferred cross-protective immunity when used as a live attenuated vaccine in both sheep and goats (33). These ﬁndings suggest the ability of SPPV to attenuate host immune responses during infection. N1L Protein in VACV and SPPV Modeling In conclusion, we have demonstrated that innate immunity is essential as a host defense response against SPPV infections by identifying critical innate mechanisms that follow PBMC exposure to SPPV. The ﬁndings will enable us to compensate for deﬁciencies in our knowledge of cellular mechanisms that activate innate immunity in sheep PBMC infected with SPPV. The data generated will also help develop inactivated vaccines for SPPV and other pox viral diseases in small and large ruminants by incorporating adjuvants that will yield the desired innate immune activation pathway. Similarly, the increase in TNFa production following poxvirus infection has been reported (25). It was interesting to note the upregulation of inﬂammatory cytokines IL-15 and TNFa in response to PBMC infection with SPPV. Virus infections result in an inﬂammatory environment with an increased IL-15 and TNFa and the subsequent recruiting of antigen-presenting cells to the infection site, leading to the induction of an adaptive immune response. However, IL-10 can inhibit those effects. This dichotomy of inﬂammatory and anti-inﬂammatory cytokine expressions could also result from the expression of different cytokines from infected and non- infected cells. Therefore, it would be worth looking at cytokine DATA AVAILABILITY STATEMENT The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession b ( ) b f d i th ti l /S l t M t i l The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material. number(s) can be found in the article/Supplementary Material. Frontiers in Immunology \| www.frontiersin.org REFERENCES 13. Rubins KH, Hensley LE, Jahrling PB, Whitney AR, Geisbert TW, Huggins JW, et al. The Host Response to Smallpox: Analysis of the Gene Expression Program in Peripheral Blood Cells in a Nonhuman Primate Model. PNAS (2004) 101(42):15190–5. doi: 10.1073/pnas.0405759101 1. Carn VM. Control of Capripoxvirus Infections. Vaccine (1993) 11 (13):1275–9. doi: 10.1016/0264-410X(93)90094-E 1. Carn VM. Control of Capripoxvirus Infections. 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Hamdi J, Bamouh Z, Jazouli M, Boumart Z, Tadlaoui KO, Fihri OF, et al. Experimental Evaluation of the Cross-Protection Between Sheeppox and Bovine Lumpy Skin Vaccines. Sci Rep (2020) 10(1):8888. doi: 10.1038/ s41598-020-65856-7 19. Kangethe RT, Pichler R, Chuma F, Cattoli G, Wijewardana V. Bovine Monocyte Derived Dendritic Cell Based Assay for Measuring Vaccine Immunogenicity In Vitro. Vet Immunol Immunopath (2018) 197:39–48. doi: 10.1016/j.vetimm.2018.01.009 7. World Organisation for Animal Health (OIE). (2021). AUTHOR CONTRIBUTIONS The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/ﬁmmu.2021. 666543/full#supplementary-material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/ﬁmmu.2021. 666543/full#supplementary-material CEL, RTK and VW conceptualized the work, TRC, RTK, RP, ELS and VW carried out the lab experiments, TRC, RTK, YL, VW and CEL analyzed and drafted the manuscript. All authors contributed to the article and approved the submitted version. CEL, RTK and VW conceptualized the work, TRC, RTK, RP, ELS and VW carried out the lab experiments, TRC, RTK, YL, VW and CEL analyzed and drafted the manuscript. All authors contributed to the article and approved the submitted version. Supplementary Figure 1 \| Ortholog modeling of N1L from SPPV Djelfa. Structural comparison between (A) N1L protein of Vaccinia virus (PBD 2I39) and (B) SPPV Djelfa ortholog predicted model, (C) Overlay of both structures, (D) Sequence alignment of N1L protein from different poxviruses. ACKNOWLEDGMENTS Supplementary Figure 2 \| Sequence alignment of E3 protein from different poxviruses. We thank Dr. Eveline Wodak (AGES) for the support in determining the health status of the animals and Dr. Bernhard Reinelt of Viechdoktorei, Tierarztpraxis Leithaprodersdorf OG for providing bovine blood. We also thank Dr. Hafsa Madani (Institut Supplementary Figure 3 \| RIG-1 expression in CD14- cells. The expression of RIG-1 was signiﬁcantly upregulated in CD14- cells. Note that the expression RIG-1 Supplementary Figure 3 \| RIG-1 expression in CD14- cells. The expression of RIG-1 was signiﬁcantly upregulated in CD14- cells. Note that the expression RIG-1 was similar in WT when compared to LAV. Data represent the Log2 FC of ﬁve independent experiments. RIG-1 was signiﬁcantly upregulated in CD14- cells. Note that the expression RIG-1 was similar in WT when compared to LAV. Data represent the Log2 FC of ﬁve independent experiments. ETHICS STATEMENT Ethical review and approval was not required for the animal study because the blood was collected by a local veterinarian Frontiers in Immunology \| www.frontiersin.org June 2021 \| Volume 12 \| Article 666543 7 SPPV Increases Sheep RIG-1 Expression In-Vitro Chibssa et al. (Viechdoktorei, Tierarztpraxis Leithaprodersdorf OG) and determined clinically healthy. The blood was collected as a part of randomized screening for Maedi-visna and Bluetongue Virus in sheep by the Austrian Agency for Health and Food Safety (AGES). National de la Médecine Vétérinaire, Laboratoire Central Vétérinaire; Algeria), Dr. Mehdi El Harrak (Biopharma, Morocco) for providing the SPPV WT and SPPV LAV samples respectively. REFERENCES Available at: https:// www.oie.int/en/disease/sheep-pox-and-goat-pox/ (Accessed June 1, 2021). 8. Kitching RP. Vaccines for Lumpy Skin Disease, Sheep Pox and Goat Pox. In: Brown F, Roth JA, editors. Vaccines for OIE List A and Emerging Animal Diseases. Dev Biol (2003) 114:161–7. doi: 10.1159/issn.1424-6074 20. Sassu EL, Kangethe RT, Settypalli TBK, Chibssa TR, Giovanni C, Wijewardana V. Development and Evaluation of a Real-Time PCR Panel for the Detection of 20 Immune Markers in Cattle and Sheep. Vet Immunol Immunopath (2020) 227:110092. doi: 10.1016/j.vetimm.2020.110092 9. McFadden G. Poxvirus Tropism. Nat Rev Microbiol (2005) 3:201–13. doi: 10.1038/nrmicro1099 21. Settypalli TBK, Lamien CE, Spergser J, Lelenta M, Wade A, Gelaye E, et al. One-Step Multiplex RT-Qpcr Assay for the Detection of Peste des petits ruminants virus, Capripoxvirus, Pasteurella multocida and Mycoplasma capricolum subspecies (ssp.) capripneumoniae. PloS One (2016) 11(4): e0153688. doi: 10.1371/journal.pone.0153688 10. Tuppurainen ESM, Pearson CR, Bachanek-Bankowska K, Knowles NJ, Amareen S, Frost L, et al. Characterization of Sheep Pox Virus Vaccine for Cattle Against Lumpy Skin Disease Virus. Antiviral Res (2014) 109(1):1–6. doi: 10.1016/j.antiviral.2014.06.009 22. PfafﬂMW. A New Mathematical Model for Relative Quantiﬁcation in Real- Time RT-PCR. Nucleic Acids Res (2001) 29(9):e45. doi: 10.1093/nar/ 29.9.e45 11. Norian R, Ahangaran NA, Vashovi HR, Azadmehr A. Evaluation of Humoral and Cell-Mediated Immunity of Two Capripoxvirus Vaccine Strains Against Lumpy Skin Disease Virus. Iran J Virol (2016) 10(4):1– 11. doi: 10.21859/isv.10.4.1 23. Wickham H. Ggplot2: Elegant Graphics for Data Analysis. Cham, Switzerland: Springer (2016). Available at: https://doi.org/10.1007/978-3-319-24277-4. 12. Chibssa TR, Grabherr R, Loitsch A, Settypalli TBK, Tuppurainen E, Nwankpa N, et al. A Gel-Based Pcr Method to Differentiate Sheeppox Virus Field Isolates From Vaccine Strains. Virol J (2018) 15(1):1–7. doi: 10.1186/s12985-018-0969-8 24. DiPerna G, Stack J, Bowie AG, Boyd A, Kotwal G, Zhang Z, et al. Poxvirus Protein N1L Targets the I-Kb Kinase Complex, Inhibits Signaling to NF-kB by June 2021 \| Volume 12 \| Article 666543 Frontiers in Immunology \| www.frontiersin.org 8 SPPV Increases Sheep RIG-1 Expression In-Vitro Chibssa et al. the Tumor Necrosis Factor Superfamily of Receptors, and Inhibits NF-kB and IRF3 Signaling by Toll-Like Receptors. J Biol Chem (2004) 279(35):36570–78. doi: 10.1074/jbc.M400567200 33. Boshra H, Truong T, Nfon C, Bowden TR, Gerdts V, Tikoo S, et al. A Lumpy Skin Disease Virus Deﬁcient of an IL-10 Gene Homologue Provides Protective Immunity Against Virulent Capripoxvirus Challenge in Sheep and Goats. Antiviral Res (2018) 123:39–49. doi: 10.1016/j.antiviral.2015.08.016 25. REFERENCES Zhang Z, Abrahams MR, Hunt LA, Suttles J, Marshall W, Lahiri DK, et al. The Vaccinia Virus N1L Protein Inﬂuences Cytokine Secretion in Vitro After Infection. Ann N Y Acad Sci (2005) 1056:69–86. doi: 10.1196/annals.1352.005 34. Subramanian Iyer S, Cheng G. Role of Interleukin 10 Transcriptional Regulation in Inﬂammation and Autoimmune Disease. Crit Rev Immunol (2012) 32(1):23–63. doi: 10.1615/CritRevImmunol.v32.i1.30 26. Smith GL, Talbot-Cooper C, Lu Y. How Does Vaccinia Virus Interfere With Interferon? Advs Virus Res (2008) 100:355–78. doi: 10.1016/bs.aivir.2018. 01.003 35. Bartlett N, Symons JA, Tscharke DC, Smith GL. The Vaccinia Virus N1L Protein Is an Intracellular Homodimer That Promotes Virulence. J Gen Virol (2002) 83(8):1965–76. doi: 10.1099/0022-1317-83-8-1965 27. Dixit E, Kagan JC. Intracellular Pathogen Detection by RIG-I-Like Receptors. Advs Immunol (2013) 117:99–125. doi: 10.1016/B978-0-12-410524-9.00004-9 36. Maluquer de Motes C, Cooray S, Ren H, Almeida GM, McGourty K, Bahar MW, et al. Inhibition of Apoptosis and NF-kb Activation by Vaccinia Protein N1 Occur Via Distinct Binding Surfaces and Make Different Contributions to Virulence. 28. Moss B. Poxvirus DNA Replication. Cold Spring Harb Perspect Biol (2013) 5: a010199. doi: 10.1101/cshperspect.a010199 PloS Pathog (2011) 7(12):e1002430. doi: 10.1371/journal.ppat.1002430 29. Embury-Hyatt C, Babiuk S, Manning L, Ganske S, Bowden TR, Boyle DB, et al. Pathology and Viral Antigen Distribution Following Experimental Infection of Sheep and Goats With Capripoxvirus. J Comp Pathol (2012) 146(2-3):106–15. doi: 10.1016/j.jcpa.2011.12.001 Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. 30. Yaqoob P, Newsholme EA, Calder PC. Comparison of Cytokine Production in Cultures of Whole Human Blood and Puriﬁed Mononuclear Cells. Cytokine (1999) 11(8):600–5. doi: 10.1006/cyto.1998.0471 Copyright © 2021 Chibssa, Kangethe, Berguido, Settypalli, Liu, Grabherr, Loitsch, Sassu, Pichler, Cattoli, Diallo, Wijewardana and Lamien. 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. y 31. Abu-El-Saad AAS, Abdel-Moneim AS. Modulation of Macrophage Functions by Sheeppox Virus Provides Clues to Understand Interaction of the Virus With Host Immune System. Virol J (2005) 2:22. doi: 10.1186/1743-422X-2-22 32. June 2021 \| Volume 12 \| Article 666543 REFERENCES Wong PS, Sutejo R, Chen H, Ng SH, Sugrue RJ, Tan BH. A System Based- Approach to Examine Cytokine Response in Poxvirus-Infected Macrophages. Viruses (2018) 10(12):692. doi: 10.3390/v10120692 June 2021 \| Volume 12 \| Article 666543 Frontiers in Immunology \| www.frontiersin.org 9
https://openalex.org/W3090558774	https://hal.inrae.fr/hal-03019741/file/2020_Monisha_Scientific%20Rep.pdf	English	null	Clinical utility of brain-derived neurotrophic factor as a biomarker with left ventricular echocardiographic indices for potential diagnosis of coronary artery disease	Scientific reports	2,020	cc-by	6,788	Clinical utility of brain-derived neurotrophic factor as a biomarker with left ventricular echocardiographic indices for potential diagnosis of coronary artery disease. K Monisha, Paramasivam Prabu, M Chokkalingam, Ram Murugesan, Dragan Milenkovic, Shiek Ahmed Clinical utility of brain-derived neurotrophic factor as a biomarker with left ventricular echocardiographic indices for potential diagnosis of coronary artery disease. K Monisha, Paramasivam Prabu, M Chokkalingam, Ram Murugesan, Dragan Milenkovic, Shiek Ahmed To cite this version: K Monisha, Paramasivam Prabu, M Chokkalingam, Ram Murugesan, Dragan Milenkovic, et al.. Clin- ical utility of brain-derived neurotrophic factor as a biomarker with left ventricular echocardiographic indices for potential diagnosis of coronary artery disease.. Scientific Reports, 2020, 10 (1), pp.16359. 10.1038/s41598-020-73296-6. hal-03019741 Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-03019741 https://hal.inrae.fr/hal-03019741v1 Submitted on 31 Mar 2021 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License www.nature.com/scientificreports www.nature.com/scientificreports Clinical utility of brain‑derived neurotrophic factor as a biomarker with left ventricular echocardiographic indices for potential diagnosis of coronary artery disease OPEN K. G. Monisha1, Paramasivam Prabu2, M. Chokkalingam1, Ram Murugesan3, Dragan Milenkovic4 & Shiek S. S. J. Ahmed3* Brain-derived neurotrophic factor (BDNF) plays a central pivotal role in the development of the cardiovascular system. Recent evidence suggests that BDNF has adverse subclinical cardiac remodeling in participants with cardiovascular disease risk factors. Relating serum BDNF levels with two-dimensional echocardiographic indices will provide insights into the BDNF mediated pathophysiology in coronary artery disease (CAD) that may shed light upon potential diagnostic biomarkers. For the study, 221 participants were recruited and classified based on coronary angiogram examination as control (n = 105) and CAD (n = 116). All participants underwent routine blood investigation, two-dimensional echocardiography, and serum BDNF estimation. As a result, total cholesterol, triglyceride, low-density lipid, high-density lipid, HbA1c (glycosylated hemoglobin), serum creatinine, eosinophils, lymphocyte, monocytes, neutrophils, and platelets were significantly elevated in CAD individuals compared to controls. Notably, the serum BDNF was significantly lower in individuals with CAD (30.69 ± 5.45 ng/ml) than controls (46.58 ± 7.95 ng/ml). Multivariate regression analysis showed neutrophils, total cholesterol, left ventricular mass index, mitral inflow E/A ratio, and pulmonary vein AR duration were associated with low BDNF in CAD. Four independent support vector machine (SVM) models performed to ensure the BDNF level in the classification of CAD from healthy controls. Particularly, the model with serum BDNF concentration and blood parameters of CAD achieved significant improvement from 90.95 to 98.19% in detecting CAD from healthy controls. Overall, our analysis provides a significant molecular linkage between the serum BDNF level and cardiovascular function. Our results contribute to the emerging evidence of BDNF as a potential diagnostic value in CAD that might lead to clinical application. Coronary artery disease (CAD) is one of the significant causes of mortality worldwide. Endothelial dysfunc- tion, inflammation, and atherosclerotic plaque formation lead to CAD1. Such events accelerated by diabetes, hypertension, smoking, and obesity2. Although these risk factors predict cardiovascular events, the molecules and their mechanism involved in CAD development are still under investigation. In the modern era, several 1Department of Cardiology, Chettinad Hospital and Research Institute, Chettinad Health City, Kelambakkam, Tamil Nadu 603103, India. 2School of Medicine, Department of Neurology, University of New Mexico Health Sciences Center, University of New Mexico, Albuquerque, USA. 3Drug Discovery and Multi‑Omics Laboratory, Faculty of Allied Health Sciences, Chettinad Academy of Research and Education, Kelambakkam, Tamil Nadu 603103, India. 4Université Clermont Auvergne, INRAe, UNH, Clermont‑Ferrand, France. Clinical utility of brain‑derived neurotrophic factor as a biomarker with left ventricular echocardiographic indices for potential diagnosis of coronary artery disease OPEN email: shiekssjahmed@ gmail.com \| https://doi.org/10.1038/s41598-020-73296-6 Scientific Reports \| (2020) 10:16359 www.nature.com/scientificreports/ Table 1. Characteristics of the study population in anthropometric and blood parameters. Significance at p < 0.05. Clinical parameters Without CAD With CAD p value Age (years) 54.49 ± 11.40 57.25 ± 10.44 0.062 BMI (kg/m2) 22.64 ± 2.27 26.23 ± 3.05 < 0.001* Systolic BP (mmHg) 121.37 ± 13.60 123.70 ± 17.40 0.271 Diastolic BP (mmHg) 81.61 ± 12.52 82.99 ± 13.18 0.426 Platelet (LAC/C.mm) 2.18 ± 0.77 2.91 ± 1.05 < 0.001* Basophil (%) 0.82 ± 0.43 0.88 ± 0.66 0.4 Eosinophil (%) 5.06 ± 2.20 6.53 ± 3.13 < 0.001* Lymphocyte (%) 29.38 ± 7.73 36.27 ± 14.59 < 0.001* Monocyte (%) 5.66 ± 2.02 7.85 ± 2.59 < 0.001* Neutrophil (%) 52.24 ± 12.67 57.89 ± 17.71 < 0.001* HbA1c (%) 4.73 ± 0.38 6.42 ± 1.16 < 0.001* HDL cholesterol (mg/dL) 47.12 ± 9.00 35.81 ± 8.24 < 0.001* LDL cholesterol (mg/dL) 84.27 ± 15.07 132.85 ± 36.95 < 0.001* TGL cholesterol (mg/dL) 96.29 ± 18.43 142.16 ± 59.04 < 0.001* T.Cholesterol (mg/dL) 131.25 ± 24.77 194.69 ± 66.03 < 0.001* Serum creatinine (mg/dL) 0.84 ± 0.23 1.06 ± 0.40 < 0.001* BDNF (ng/mL) 46.58 ± 7.95 30.69 ± 5.45 < 0.001* Table 1. Characteristics of the study population in anthropometric and blood parameters. Significance at p < 0.05. molecular techniques such as genomics, transcriptomics, proteomics, and metabolomics plays a crucial role to understand the molecular mechanism of CAD, which pave the way to discover molecular diagnostic markers3 molecular techniques such as genomics, transcriptomics, proteomics, and metabolomics plays a crucial role to understand the molecular mechanism of CAD, which pave the way to discover molecular diagnostic markers3 p y g In recent times several markers such as high sensitive c-reactive protein (hs-CRP), interleukins, monocyte chemoattractant protein-1 (MCP-1), TNF-α, serum amyloid A (SAA), vascular adhesion molecule 1 (VCAM- 1), selectins, vascular endothelial growth factor (VEGF), matrix metalloproteinases (MMPS) and soluble CD40 ligand is identified4. Of which, few are clinically useful to detect CAD, whereas other markers are neglected due to lack of direct involvement pathophysiological events of CAD5. For example, hs-CRP is one of the known inflammatory markers proven associated with two-dimensional echocardiography of CAD individuals6. There- fore, any new marker needs to be cross-linked with the gold standard imaging techniques making it useful for clinical application. Clinical utility of brain‑derived neurotrophic factor as a biomarker with left ventricular echocardiographic indices for potential diagnosis of coronary artery disease OPEN Brain-derived neurotrophic factor (BDNF) is a neurotrophic protein expressed in the central and peripheral circulation7. Although BDNF is a neurotrophic protein, it plays a vital role in the cardiac system8. Importantly, BDNF involved in the development of heart9, endothelial cells10, vascular smooth muscle cells11, macrophages12, lymphocytes13 and atherosclerotic vessels11. Recent research emphasizes that BDNF induces oxidative stress by activating oxidase enzyme in the coronary artery vasculature14. Low serum BDNF was observed in patients with CAD that causing future coronary events15. In general, the ventricular function has been assessed by the two- dimensional echocardiographic imaging recommended by the American Society of Echocardiography (ASE)16. Relating serum BDNF levels with echocardiographic indices may provide the involvement of BDNF in cardiac systems, which may be useful in developing a potential alternative method for CAD diagnosis. Recently Bahls et al., 2019 showed the association between low BDNF and echocardiographic indices in participants with tra- ditional risk factors of future CAD17. However, the role of BDNF with cardiac left ventricular function and its ability to classify CAD from healthy controls is less well known.h y y y This study aimed to investigate the relationship between serum BDNF levels with biochemical and echocar- diographic indices in 221 participants for its utility as a CAD marker. We identified a significant molecular link- age between the BDNF with biochemical changes and echocardiographic indices associated with cardiovascular function. Further, we construct an automated CAD detection model using a support vector machine with BDNF showing improved accuracy making a rapid decision in CAD. BDNF and CAD associated clinical and echocardiography parameters. To determ BDNF and CAD associated clinical and echocardiography parameters. To determine the associa- tion between serum BDNF levels and clinical parameters, the CAD patients were quartile grouped as low and high (described in the method section). The BDNF concentration with ≤ 29.91 ng/ml considered as a low BDNF group, and those with levels > 29.92 ng/ml are considered a high BDNF group. The Supplementary Table A1 shows the characteristics of collected blood parameters and echocardiographic indices in low and high BDNF group. Elevated platelets, basophils, eosinophils, lymphocytes, monocytes, neutrophils, LDL, TGL, total choles- terol, HbA1c, and serum creatinine were observed in low BDNF group of CAD patients. In contrast, significant decreased BMI and HDL noticed in low BDNF groups compared to the high BDNF group. Similarly, the echo- cardiographic indices in low BDNF shows evaluated Biplane LVEF, PV AR (m/s), GLS LVEF, and decreased LVMI, MV E/A ratio, IVRT (ms), and PV S/D ratio compared to the high BDNF group. The statistical analysis of blood parameters and echocardiographic indices confirms the significance between the low and high BDNF groups, except LAD, MV S E/e’ ratio, and L E/e’ ratio (Supplementary Table A2). Multivariate regression analysis. We performed a multivariate regression analysis to determine the blood parameters influencing low BDNF concentration in CAD (Table 3). The neutrophils (β = − 0.494, p ≤ 0.001) and total cholesterol (β = − 0.407, p ≤ 0.001) were noticed to contribute low BDNF concentration in CAD with a model fit measure of r = 0.725 and r-square = 0.526. Similarly, the echocardiographic indices (Table 4), LVMI (β = 0.3380, p ≤ 0.001), MV E/A (β = − 0.3751, p ≤ 0.001), PV AR (β = − 0.3444, p ≤ 0.001) and Biplane LVEF (β = 0.3467, p = 0.010) were significantly linked with low BDNF concentration in CAD with a model fit measure r = 0.826 and r-square = 0.683. Overall, the analysis suggests low BDNF concentration is the measure of both blood (neutrophils and total cholesterol) and echocardiographic indices (LVMI, MV E/A, PV AR, and Biplane LVEF) in CAD. Support vector machine classification of CAD from control. Support vector machine (SVM) based classification model was developed to determine the influence of BDNF as a biomarker in predicting CAD. Prior to disease classification, we use SVM Attribute Evaluator with the ranker method to select most contributing predictor variables for CAD from clinical and echocardiographic indices. Results P i Patients characteristics and comparative analysis. The clinical and anthropometric characteristics of the participants involved in this study was illustrated in Table 1. All descriptive data expressed as mean ± stand- ard deviation for control and CAD. All participants in control group are free from risk factors, Whereas the CAD group contains 10.2% of hypertensive, 15.3% of diabetes mellitus and 5.5% of smokers. However, most CAD participants in CAD are free from the risk factors. Further, the statistical t-test analysis showed a significant change in BMI, platelet, eosinophils, lymphocyte, monocytes, neutrophils, HbA1c (glycosylated hemoglobin), serum creatinine, low density lipid (LDL), high density lipid (HDL), triglyceride (TGL), and total cholesterol in CAD compared to control (Table 1). Mainly, serum BDNF was significantly (p ≤ 0.001) decreased in CAD (30.70 ± 5.4 ng/ml) compared to the control (46.580 ± 7.9 ng/ml). Although basophils, systolic, and diastolic pressure showed variations in CAD, but not statistically significant. Scientific Reports \| (2020) 10:16359 \| https://doi.org/10.1038/s41598-020-73296-6 www.nature.com/scientificreports/ Table 2. Echocardiographic indices of the participants with CAD and without CAD. Biplane LVEF (biplane left ventricular ejection fraction); LAD (left atrial diameter), LVMI (left ventricular mass index), MV E/A (Mitral inflow E/A ratio), Le’ /Se’(mitral lateral/septal peak myocardial early diastolic velocity), IVRT (isovolumetric relaxation time), PV AR (pulmonary vein AR duration), PV S/D (pulmonary vein systole / diastole ratio) and GLS EF (global longitudinal strain). Significance at p < 0.05. Echo indices Without CAD With CAD p value Biplane LVEF (%) 61.08 ± 3.01 44.75 ± 7.01 < 0.001* LAD (cm) 3.25 ± 0.30 3.45 ± 0.51 < 0.001* LVMI (g/m2) 82.31 ± 24.67 100.69 ± 24.35 < 0.001* MV E/A 1.06 ± 0.30 0.64 ± 0.45 < 0.001* S E/e’ 8.42 ± 1.01 7.20 ± 1.28 < 0.001* L E/e’ 10.46 ± 1.04 7.62 ± 1.14 < 0.001* IVRT (ms) 80.81 ± 16.34 114.57 ± 23.61 < 0.001* PV AR (m/s) 32.19 ± 5.80 27.78 ± 5.24 < 0.001* PV S/D 0.69 ± 0.23 1.29 ± 0.57 < 0.001* GLS EF (%) − 16.18 ± 0.47 − 13.94 ± 1.27 < 0.001* Table 2. Echocardiographic indices of the participants with CAD and without CAD. Results P i Biplane LVEF (biplane left ventricular ejection fraction); LAD (left atrial diameter), LVMI (left ventricular mass index), MV E/A (Mitral inflow E/A ratio), Le’ /Se’(mitral lateral/septal peak myocardial early diastolic velocity), IVRT (isovolumetric relaxation time), PV AR (pulmonary vein AR duration), PV S/D (pulmonary vein systole / diastole ratio) and GLS EF (global longitudinal strain). Significance at p < 0.05. Table 2. Echocardiographic indices of the participants with CAD and without CAD. Biplane LVEF (biplane left ventricular ejection fraction); LAD (left atrial diameter), LVMI (left ventricular mass index), MV E/A (Mitral inflow E/A ratio), Le’ /Se’(mitral lateral/septal peak myocardial early diastolic velocity), IVRT (isovolumetric relaxation time), PV AR (pulmonary vein AR duration), PV S/D (pulmonary vein systole / diastole ratio) and GLS EF (global longitudinal strain). Significance at p < 0.05. Table 2. Echocardiographic indices of the participants with CAD and without CAD. Biplane LVEF (biplane left ventricular ejection fraction); LAD (left atrial diameter), LVMI (left ventricular mass index), MV E/A (Mitral inflow E/A ratio), Le’ /Se’(mitral lateral/septal peak myocardial early diastolic velocity), IVRT (isovolumetric relaxation time), PV AR (pulmonary vein AR duration), PV S/D (pulmonary vein systole / diastole ratio) and GLS EF (global longitudinal strain). Significance at p < 0.05. Assessment of echocardiography indices. Next, we assessed the echocardiographic indices in 221 par- ticipants. Increased left atrial diameter (LAD), left ventricular mass index (LVMI), isovolumetric relaxation time (IVRT), pulmonary vein systole / diastole ratio (PV S/D) and decreased biplane left ventricular ejection fraction (LVEF), Mitral inflow E/A ratio (MV E/A ratio), mitral septal peak myocardial early diastolic velocity (S E/e′ ratio), mitral lateral peak myocardial early diastolic velocity (L E/e′ ratio), pulmonary vein AR duration (PV AR), and global longitudinal strain (GLS LVEF) were observed in the CAD compared to the control. The statistical analysis confirms the variation in echocardiographic indices of CAD compared to control (Table 2). BDNF and CAD associated clinical and echocardiography parameters. To determ The BMI, HbA1c, HDL, LDL, and Total cholesterol were identified as the five most important blood predictor variables of CAD. Whereas in echo- cardiographic indices, Biplane LVEF, LAD, L E/e, IVRT, and GLS LVEF were determined as important indices for CAD prediction (Table 5). To check influence of BDNF in predicting CAD, four independent SVM models were developed, the model-A was trained with BMI, HbA1c, HDL, LDL, and Total cholesterol (Table 5). The model-B contains Biplane LVEF, LAD, L E/e, IVRT, and GLS LVEF echocardiographic indices (Table 5). The model-C was trained with model-A attributes along with BDNF concentration. Similarly, model-D was trained with model-B attributes along with BDNF concentration. The accuracy, True Positive (TP) rate, False Positive (FP) rate, precision, recall, F-measure, and Receiver operating characteristic (ROC) of each model shown in Table 5. All SVM models showed accuracy over 90%. For instance, model-A showed 90.95% accuracy, whereas adding the serum BDNF concentration in model-C showed 98.19% improved accuracy in detection CAD. As Scientific Reports \| (2020) 10:16359 \| https://doi.org/10.1038/s41598-020-73296-6 www.nature.com/scientificreports/ Table 3. Multivariate regression analysis showing low BDNF contributing blood parameters in CAD. Data are represented with β and p values with 95% confidence interval. Significance at p < 0.05. Predictor Univariate analysis Multivariate analysis Coefficient correlation p value 95% confidence interval β 95% confidence interval p value Age (years) 0.06 0.63 − 0.075 to 0.04 BMI (kg/m2) 0.11 0.38 − 0.12 to 0.30 Systolic BP (mmHg) 0.05 0.70 − 0.04 to 0.03 Diastolic BP (mmHg) 0.03 0.79 − 0.04 to 0.05 Platelet (LAC/C.mm) 0.11 0.40 − 0.38 to 0.92 Basophil (%) 0.04 0.76 − 0.754 to 1.03 Eosinophil (%) 0.04 0.73 − 0.28 to 0.20 Lymphocyte (%) 0.02 0.87 − 0.03 to 0.04 Monocyte (%) 0.12 0.35 − 0.13 to 0.37 Neutrophil (%) 0.61 < 0.001* − 0.11 to 0.05 − 0.494 − 0.09 to 0.04 < 0.001* HDL cholesterol (mg/dL) 0.01 0.93 − 0.08 to 0.09 LDL cholesterol (mg/dL) 0.12 0.34 − 0.01 to 0.02 TGL cholesterol (mg/dL) 0.01 0.91 − 0.0098 to 0.01 T.Cholesterol (mg/dL) 0.54 < 0.001* − 0.02 to 0.01 − 0.407 − 0.020 to 0.007 < 0.001* HbA1c (%) 0.013 0.92 − 0.52 to 0.58 Serum Creatinine (mg/ dL) 0.12 0.3 − 1.03 to 2.72 Table 3. Multivariate regression analysis showing low BDNF contributing blood parameters in CAD. Data are represented with β and p values with 95% confidence interval. Discussion h b Similarly, altered BDNF expression in human atheromatous intima, adventitia, macrophages, and smooth muscle cells in atherosclerotic coronary arteries14. These results suspect us to investigate the association of serum BDNF with echocardiography indices in CAD.i g p y Multivariate regression analysis of blood parameters showed a significant contribution of neutrophils and total cholesterol with low BDNF (≤ 29.91 ng/ml) in angiogram proven CAD patients. Aleksandra Sustar et al., 2019 confirm the significant association of low BDNF with total cholesterol in coronary heart disease. Similar result was noticed in the Chinese population that relates low BDNF level with cardiovascular disease risk factors22. In addition to total cholesterol concentration, the increased neutrophils was noticed associated with low BDNF in CAD individuals. Neutrophils play a vital role in CAD, which promotes atherothrombotic mechanisms leading to cause myocardial infarction. Halade et al., 2013 confirm the interdependency of BDNF with neutrophils in recruiting macrophages into the infarcted region of BDNF haploinsufficient mice25. Besides, neutrophils involved in the release of reactive oxygen species, which activates the endothelium to deposit additional neutrophils for plaque formation. Simultaneously, our multivariate regression analysis of low BDNF group showed a significant association with echocariographic indices, including LVMI, MV E/A, and PV AR in angiogram proven CAD patients. Recently, Martin Bahls et al., 2019 demonstrated the role of BDNF on left ventricular cardiac remodeling in individuals with the risk of cardiovascular disease17. However, Martin Bahls et al., 2019 did not justify the role of BDNF on left ventricular dysfunction in the CAD population by comparing it with healthy control. Whereas, our results showed significant evidence of low BDNF in CAD that correlates with the echocardiograohic indices compared to healthy control. Overall, our analysis suggests low BDNF concentration is the representative of both blood parameters (neutrophils and total cholesterol) and echocardiographic indices (LVMI, MV E/A, PV AR and Biplane LVEF) in CAD. p We construct SVM models which showed significant improvement in detecting CAD with BDNF as one of the attributes (Table 5). Recently, Akella et al., 2020 use a variety of machine learning algorithms and achieved maximum accuracy of 93% in detecting CAD26. Interestingly our study showed benefit of adding the BDNF as one of the attribute to SVM models that represents blood parameters (neutrophils and total cholesterol) and echocardiography indices (LVMI, MV E/A, PV AR, and Biplane LVEF) in CAD (Tables 3, 4). Discussion h b BDNF has been extensively studied to promote neurogenesis18 and also plays a dominant role in the cardiovascu- lar system19. BDNF expressed in endothelial cells, vascular smooth muscle cells, macrophages, lymphocytes, and atherosclerotic vessels14. Framingham Heart Study 2015 suggests that high BDNF concentration was associated with a decreased risk of CVD and mortality15. A recent study suggests the involvement of BDNF in oxidative stress in coronary artery vasculature and atherosclerotic plaque formation20. Despite various studies, the associa- tion of BDNF in CAD with non-invasive cardiovascular imaging techniques like two-dimension echocardiogra- phy imaging has not been reported. Therefore, we investigated the involvement of BDNF in CAD compared to healthy control. Elucidating the association of BDNF with CAD associated parameters may enable utilization of BDNF as a possible diagnostic biomarker for CAD. Particularly, establishing the relationship between alerted serum BDNF level and echocardiographic indices will help develop a potential alternative method for CAD diagnosis. This is the first study conducted in South Indian ethnicity to assess the association of serum BDNF levels with clinical parameters and echocardiography indices in CAD. We segmented our study design into three- fold. First, we confirmed the significant changes in blood parameters, imaging indices, and BDNF levels in CAD compared to controls. Second, we use multivariate regression analysis to determine the blood parameters and imaging indices influencing low BDNF concentration in CAD. Finally, we generate SVM models to determine the influence BDNF in improving the classification of CAD from control by including and excluding the BDNF attribute while training and testing the models. Our present study showed decreased serum BDNF in CAD which is in agreement with Eyileten et al., 2016, confirming the decreased serum BDNF that correlates with VCAM1 and soluble P-selectin in CAD21. Similarly, Aleksandra Sustar et al., 2019 reports lower BDNF in the CAD associated with an increased risk of cardiovas- cular events and mortality22. Additionally, BDNF showed a significant association with traditional risk factors, including diabetes, hypertension, smoking, physical activity, and obesity23. Similar results observed in our study relating low BDNF with a lipid profile and body mass index in CAD (Supplementary Table A1). Also, our results follows the similar outcome of Jiang H et al., 2011showing association with increased LDL, TGL, and decreased HDL levels with lower BDNF concentration in angina pectoris24. Interestingly, Ejiri et al., 2005 report altered BDNF in the coronary circulation between coronary sinus and aorta in patients with angina. BDNF and CAD associated clinical and echocardiography parameters. To determ Significance at p < 0.05. Table 4. Multivariate regression analysis showing low BDNF contributing Echocardiographic indices in CAD. Data are represented with β and p values with 95% confidence interval. Indicate significance at p < 0.05. Predictor Univariate analysis Multivariate analysis Coefficient correlation p value 95% confidence interval β 95% confidence interval p value Biplane LVEF (%) 0.45 < 0.001* 0.09 to 0.30 0.35 0.03 to 0.26 0.01* LAD (cm) 0.38 0.003* 0.752 to 3.5 0.13 − 0.19 to 1.69 0.117 LVMI (g/m2) 0.52 < 0.001* 0.034 to 0.09 0.33 0.01 to 0.05 < 0.001* MV E/A 0.32 0.012* 0.80 to 6.36 − 0.37 − 2.63 to− 0.97 < 0.001* S E/e’ 0.04 0.743 − 0.86 to 1.21 L E/e’ 0.01 0.897 − 1.40 to 1.59 IVRT (ms) 0.18 0.163 − 0.01 to 0.06 PV AR (m/s) 0.61 < 0 .001* 0.18 to 0.38 0.34 0.06 to 0.24 < 0.001* PV S/D 0.12 0.338 − 0.95 to 2.73 GLS EF (%) 0.45 < 0.001* − 1.62 to 0.497 0.05 − 0.47 to 0.71 0.686 Table 4. Multivariate regression analysis showing low BDNF contributing Echocardiographic indices in CAD. Data are represented with β and p values with 95% confidence interval. *Indicate significance at p < 0.05. Table 5. Model describing the parameters associated with the accuracy in classifying CAD from healthy control. TP true positive, FP false positive, ROC receiver operating characteristic. Model Predictor variables TP rate FP rate Precision Recall F-measure ROC area Accuracy (%) Model-A BMI, HbA1c, HDL, LDL, and Total cholesterol 0.911 0.086 0.913 0.910 0.91 0.912 90.95 Model-B Biplane LVEF, LAD, L E/e, IVRT, and GLS LVEF 0.986 0.012 0.987 0.986 0.986 0.986 98.64 Model-C BDNF, BMI, HbA1c, HDL, LDL, and Total cholesterol 0.986 0.015 0.987 0.986 0.986 0.986 98.19 Model-D BDNF, Biplane LVEF, LAD, L E/e, IVRT, and GLS LVEF 1 0 1 1 1 1 100 Table 5. Model describing the parameters associated with the accuracy in classifying CAD from healthy control. TP true positive, FP false positive, ROC receiver operating characteristic. expected, the model-B with echocardiographic indices revealed high (98.64%) accuracy as the echocardio- graphic imaging is the important tool that routinely used in detecting CAD. However, the model-D with serum BDNF concentration as an additional attribute to the other echocardiographic parameters of model-B resulted 100% accuracy in detecting CAD. https://doi.org/10.1038/s41598-020-73296-6 Scientific Reports \| (2020) 10:16359 \| www.nature.com/scientificreports/ Methods i l Quantification of serum BDNF. Overnight fasting blood (4 ml) was collected from the participants in BD vacutainer Plus Plastic Serum Tubes. The samples were immediately centrifuged at 3000 RPM for 15 min to separate serum from other cellular material and stored at − 80 °C for future analysis. Serum BDNF levels (ng/ ml) were measured using the ELISA kit (R&D SYSTEMS, USA) following the manufacturer’s instructions. The BDNF concentrations were measured based on the optical density (OD) curve using known standards concen- tration provided within the kit. In addition, the cellular and biochemical parameters such as complete blood count, lipid profile, HbA1c, and serum creatinine were determined by standard laboratory techniques as a part of routine blood tests. Statistical analysis. Student t-test was performed to confirm the statistical significance of variables in CAD compared to healthy controls. Serum BDNF concentration in CAD was stratified into quartiles as low and high levels to determine its relationship with clinical and echocardiography indices. In brief, we divide CAD patients into quartiles by merging first and second quartile to have cut-off ≤ 29.91 ng/ml, represented as low BDNF group (n = 58). The third and fourth quartiles are merged obtaining a cut-off above 29.92 ng/ml, desig- nated as high BDNF group (n = 58). Further, the multivariate regression analysis was performed to confirm the major contributing clinical and echocardiography indices associated with low serum BDNF levels in the CAD. All statistical analysis was performed using SPSS software (version 21), and the significance was considered based on p value < 0.05. Classification based on the machine learning algorithm. Support Vector Machine (SVM) is one of the efficient and widely used supervised machine learning algorithms for disease classification27. Prior to classi- fication, the SVM Attribute Evaluator with ranker method28 was used to select most (top five) contributing blood parameter and echocardiographic indices to achieve maximum accuracy in CAD prediction. Further, we use SVM in Weka software29 to generate four SVM models (model-A, B, C, and D) with selected attributes from the data set (116 CAD and 105 control) for training and testing of each model. The predictor variables for model-A contains BMI, HbA1c, HDL, LDL, and Total cholesterol (Table 5). Whereas the model-B contains LVEF, LAD, L E/e, IVRT, and GLS LVEF, a top five echocardiographic indices for CAD prediction (Table 5). Methods i l Patient selection. The protocol of this study was approved by the institutional human ethical committee of the Chettinad Academy of Research and Education (IHEC/10-17/Proposal No:372). All experiments were per- formed in accordance with relevant guidelines and regulations. The individuals presenting angina pectoris were recruited from the Chettinad Super Specialty Hospital between October 2018 to June 2019. Written informed consent, demographic information, and health status was recorded for each participant by questionnaire. Based on the collected data, the 221 participants were selected following the exclusion and inclusion criteria. Exclusion criteria: the participants with the previous history of arterial revascularization such as stenting and bypass graft- ing, myocardial infarction (MI), heart failure (following the New York Heart Association Classification), valvu- lar heart disease, pericardial diseases, idiopathic cardiomyopathy, acute or chronic infectious diseases, severe systemic disorders (malignancy, Immune diseases) and mental disorders. Inclusion criteria: (1) the participants belonging to south Indian origin between age 25 and 70 years, (2) the participants of both genders with angina, and (3) the physiological status was proven by coronary angiogram for the participant with CAD and without CAD (healthy controls, ≤ 30% lesion). Two‑dimensional echocardiography and coronary angiogram examination. Transthoracic two- dimensional echocardiography was performed for all participants by qualified echocardiography technologist (KGM: one of the author) using the Esaote [MyLab 25Gold] following the American Society of Echocardiogra- phy guidelines. The echocardiography indices such as left ventricular (LV) mass index, biplane left ventricular ejection fraction (LVEF), early transmitral flow velocity (E), late transmitral flow velocity (A), mitral inflow E/A ratio, isovolumetric relaxation time, mitral lateral/septal peak myocardial early diastolic velocity (Le’ and Se’), pulmonary vein AR duration (PV AR), pulmonary vein systole/diastole (PV S/D) ratio, and global longitudinal strain (GLS) were calculated to assess the cardiovascular function. Simultaneously, the coronary angiogram was performed by a qualified cardiologist (CM: one of the author) using contrast agents (omnipaque and visipaque) to determine coronary artery lesions using the GE INOVA 2100-IQ PLUS with ADW DICOM. Based on angio- gram report, the study participants were classified as 1) CAD patients (lesion > 30% in the primary coronary artery or its major branches) and 2) healthy controls (lesion ≤ 30%). Of those 221 participants, 116 were CAD presenting single vessel (n = 41), double vessel (n = 39), and triple vessel disease (n = 36), while the other 105 participants with proven angiogram negative were designated as healthy control. Discussion h b Although our find- ings provides significant improvement in CAD diagnostic research, there are few limitations that to be considered before its clinical utility. First, this study includes only the South Indian population. Second, we did not follow-up on the CAD patients. Third, the changes in BDNF levels was not studied on the improvement after the treatment of CAD. Alternatively, strength of the this study need to be acknowledged that (1) selection of participants both control and CAD has been proven with coronary angiogram. (2) this study integrates the blood parameters and echocardiography indices to develop a diagnostic method that has been strengthened with a machine learning algorithm showing better accuracy in detecting CAD. g g y g In conclusion, our study has presented a novel approach for determining the association of decreased serum BDNF with blood parameters and echocardiography indices of CAD. The machine learning (SVM) algorithm was developed to determine accuracy of BDNF with blood parameters and echocardiography indices for disease classification in CAD and healthy controls. Although all our SVM models showed better accuracy in disease classification, the model-C and model-D will be significantly improve detecting CAD using serum BDNF concen- tration. Therefore, our results, along with machine-based disease classification, has demonstrated the emerging evidence of BDNF in the prediction of CAD with the best accuracy value that may pave the way towards bench side clinical application. Scientific Reports \| (2020) 10:16359 \| https://doi.org/10.1038/s41598-020-73296-6 www.nature.com/scientificreports/ Methods i l The attributes for model-C contains BDNF concentration as an additional attribute along with the other five predictor variables of model-A (Table 5). Similarly, the model-D contains BDNF concentration as additional attribute of model-B for CAD (Table 5). prediction. Each data set was represented with a class attribute of “CAD” or “control” for 221 instances designated based on the coronary angiogram. A tenfold cross-validation method was adopted to meas- ure an unbiased prediction of the models. The performance of each model was assessed based on the accuracy, true positive (TP) rate, false positive (FP) rate, precision, recall, F-measure, and receiver operating characteristic (ROC). Comparing the models based on accuracy would enable us to explore the importance of BDNF in the classification of CAD from controls. https://doi.org/10.1038/s41598-020-73296-6 Scientific Reports \| (2020) 10:16359 \| www.nature.com/scientificreports/ Received: 4 July 2020; Accepted: 26 August 2020 References Jiang, H., Liu, Y., Zhang, Y. & Chen, Z. Y. Association of plasma brain-derived neurotrophic fact and prognosis in angina pectoris. Biochem. Biophys. Res. Commun. 415, 99–103 (2011).l ang, H., Liu, Y., Zhang, Y. & Chen, Z. Y. Association of plasma brain-derived neurotrophic factor and cardiovascular risk factors nd prognosis in angina pectoris. Biochem. Biophys. Res. Commun. 415, 99–103 (2011).l 24. Jiang, H., Liu, Y., Zhang, Y. & Chen, Z. Y. Association of plasma brain derived neurotrophic factor and cardiovascular risk fa and prognosis in angina pectoris. Biochem. Biophys. Res. Commun. 415, 99–103 (2011).l p g g p p y , ( ) 25. Halade, G. V. et al. 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PLoS ONE 12, e0178686 (2017). 3. Kurajoh, M. et al. Plasma brain-derived neurotrophic factor concentration is a predictor of chronic kidney disease in patients with cardiovascular risk factors—Hyogo Sleep Cardio-Autonomic Atherosclerosis study. PLoS ONE 12, e0178686 (2017). Zh & Ch Z A f l b d d h f d d l k f j p p y p cardiovascular risk factors—Hyogo Sleep Cardio-Autonomic Atherosclerosis study. PLoS ONE 12, e0178686 (2017). 24. Competing interests h p g The authors declare no competing interests. Scientific Reports \| (2020) 10:16359 \| https://doi.org/10.1038/s41598-020-73296-6 www.nature.com/scientificreports/ Additional information Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-73296-6. Correspondence and requests for materials should be addressed to S.S.S.J.A. Reprints and permissions information is available at www.nature.com/reprints. 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https://openalex.org/W2983597516	http://pdf.blucher.com.br/engineeringproceedings/siintec2019/32.pdf	Portuguese	null	METODOLOGIAS PARA QUALIFICAÇÃO TECNOLÓGICA APLICADA AO DESENVOLVIMENTO DE PRODUTO: UMA REVISÃO SISTEMÁTICA	Blucher Engineering Proceedings	2,019	cc-by	3,304	1 Centro universitário SENAI CIMATEC, Avenida Orlando gomes 1845, Salvador-BA; pedromartins175@gmail.com 2 Centro universitário SENAI CIMATEC, Avenida Orlando gomes 1845, Salvador-BA Resumo: Este trabalho tem como objetivo caracterizar a produção científica sobre o processo de qualificação tecnológica aplicada ao desenvolvimento de produto e, para isso, foi realizada uma revisão sistemática com o objetivo de identificar os principais trabalhos na área, identificando metodologias e ferramentas aplicadas. Foram identificados 12 estudos que evidenciam o uso da qualificação tecnológica na etapa de desenvolvimento de produto, cujos resultados mostram a diversidade de metodologias para a qualificação tecnológica, com apenas um estudo utilizando de métricas relacionadas a necessidades dos clientes, demonstrando a necessidade de mais pesquisa nessa linha. Palavras-Chave: Qualificação de tecnologias, Desenvolvimento de produto, Incertezas da Inovação. METODOLOGIAS PARA QUALIFICAÇÃO TECNOLÓGICA APLICADA AO DESENVOLVIMENTO DE PRODUTO: UMA REVISÃO SISTEMÁTICA Pedro Martins de Oliveira1, Valter Estevão Beal2 1 Centro universitário SENAI CIMATEC, Avenida Orlando gomes 1845, Salvador-BA; pedromartins175@gmail.com 2 Centro universitário SENAI CIMATEC, Avenida Orlando gomes 1845, Salvador-BA 1 Centro universitário SENAI CIMATEC, Avenida Orlando gomes 1845, Salvador-BA; pedromartins175@gmail.com 2 Centro universitário SENAI CIMATEC, Avenida Orlando gomes 1845, Salvador-BA Keywords: Technology Qualification, Product Development, Innovation uncertities. 1. INTRODUÇÃO O processo de desenvolvimento de produto é uma atividade criativa que requer sólidos conhecimentos em engenharia, por isso o processo de desenvolvimento integrado de produto consiste na utilização de métodos estruturados, não para limitar a criatividade da equipe de projeto, mas para entender as necessidades dos clientes e trazer um conceito que traga resultados inovadores [1] Algumas ferramentas podem ser utilizadas para auxiliar neste processo, criando de maneira sistemática soluções para problemas cotidianos em escritórios de projeto. Esses métodos sistemáticos são chamados de DFX (design for excelency), que consiste em uma família de métodos de projeto para solucionar algum problema relacionado a um fator que ele dever ter, sendo o mais famoso deles o DFMA (Design for manufacturing and assembly), que traz regras e métodos para o desenvolvimento de um produto levando em consideração os fatores de montagem e fabricação [2]. Além dessas ferramentas é também comum o uso nos produtos de tecnologias que não foram testadas em ambiente relevante, sendo necessário um processo de qualificação da tecnologia, para isso as guias para práticas de qualificação de novas tecnologias são expostas pela DNVRP-A203. Este procedimento explica como identificar, descrever e gerenciar riscos oriundos das incertezas tecnológicas [3]. O motivo de atenção para a qualificação tecnológica é que, muitas vezes, o desenvolvimento destas tecnologias são custosos, trazem riscos para o projeto e para o produto final [4]. O estado da prática expõe diretrizes para o avanço da tecnologia em um projeto, e alguns estudos mostram ferramentas para remediar essas incertezas que são desenvolvidas no processo de desenvolvimento de produto [5]. Entretanto fica evidente a falta de literatura que relaciona os requisitos e necessidades dos clientes como métricas para o avanço da maturidade tecnológica. O objetivo deste trabalho é analisar as contribuições das pesquisas produzidas acerca de metodologias aplicadas na qualificação tecnológica no processo de desenvolvimento de produto, com vistas em comprimento de requisitos dos clientes no processo de avanço da maturidade da tecnologia. METHODOLOGIES FOR TECHNOLOGICAL QUALIFICATION APPLIED TO PRODUCT DEVELOPMENT: A SYSTEMATIC REVIEW Abstract: This paper aims to characterize the scientific production about the technological qualification process applied to product development. For this, a systematic review was carried out to identify the main works in the area, identifying methodologies and applied tools. Twelve studies were identified that show the use of technological qualification in the product development stage. The results shows the diversity of methodologies for technological qualification, with only one study using metrics related to customer needs, demonstrating the need for further research in this area. words: Technology Qualification, Product Development, Innovation uncertities. Keywords: Technology Qualification, Product Development, Innovation uncertities. análise e avaliação de todos os estudos incluídos na revisão; produção de uma síntese das informações e resultados encontrados e apresentação da revisão. análise e avaliação de todos os estudos incluídos na revisão; produção de uma síntese das informações e resultados encontrados e apresentação da revisão. As bases de dados selecionadas foram as seguintes: Science direct, IEEE explore e One petro. Os descritores escolhidos foram: “Technology qualification”, “product development” e “requirements management”, reordenando-os utilizando dos operadores lógicos “and” e “or”. Os critérios de inclusão foram: artigos que abordavam o processo de qualificação da tecnologia como um processo integrado ao desenvolvimento de produto, utilizando métricas comuns de confiabilidade ou de requisitos de projetos no período entre 2009 e 2019, disponibilizados na íntegra na língua inglesa ou portuguesa e na base de dados da One Petro somente artigos submetidos a revisão por pares participaram do estudo. O processo de exclusão partiu da metodologia exposta na figura 1: exclusão dos duplicados, análise dos títulos e palavras chaves (Necessário, pois o banco de dados da One Petro não permite pesquisar somente por título e palavras chaves) e por final análise dos resumos e métricas utilizados. Figura 1 – Sistemática de exclusão dos artigos. Figura 1 – Sistemática de exclusão dos artigos. Além desses critérios de inclusão, foram inseridos critérios de qualidade para os artigos, que foram: O caso pode ser aplicado em um processo de desenvolvimento de produto integrado e requisitos de projetos ou necessidades dos clientes foram métricas nos resultados. 3. RESULTADOS E DISCUSSÃO A partir deste métodos ficaram 12 artigos selecionados mostrado na tabela 1. Além desses critérios de inclusão, foram inseridos critérios de qualidade para os artigos, que foram: O caso pode ser aplicado em um processo de desenvolvimento de produto integrado e requisitos de projetos ou necessidades dos clientes foram métricas nos resultados. Além desses critérios de inclusão, foram inseridos critérios de qualidade para os artigos, que foram: O caso pode ser aplicado em um processo de desenvolvimento de produto integrado e requisitos de projetos ou necessidades dos clientes foram métricas nos resultados. 2. METODOLOGIA Este estudo foi desenvolvido por meio de uma pesquisa bibliográfica e contemplou aspectos relacionados ao uso de métodos de qualificação tecnológica aplicada ao desenvolvimento de produto. Trata-se de um estudo de revisão sistemática da literatura, cuja construção passou pelas seguintes etapas: identificação do objeto de estudo; formulação da questão norteadora do estudo; identificação das bases de dados a serem consultadas; definição dos descritores; definição dos critérios de inclusão e exclusão dos trabalhos; 3. RESULTADOS E DISCUSSÃO A partir deste métodos ficaram 12 artigos selecionados mostrado na tabela 1. Tabela 1: Relação dos artigos coletados na pesquisa. Descritores Science direct Total / Incluídos na pesquisa IEEE Total / Incluídos na pesquisa One Petro Total / Incluídos na pesquisa “Product development” and “Technology qualification 7 / 2 0 1 / 0 “Technology qualification” and “requirements” 33 / 6 6 / 1 13 / 3 Total de artigos inclusos 8 1 3 É possível observar um aumento das publicações entre 2014 e 2016 na Figura 2. Este número vem crescendo pela necessidade da indústria de óleo e gás de novas tecnologias que possam ser usadas em ambientes em maior altura de lâmina d’agua e necessitando de um método sistemático para a qualificação dessas tecnologias. Figura 2: Número de artigos no período investigado Após a análise dos artigos selecionados foi percebido a falta de consenso em processos de qualificação tecnológica aplicada a desenvolvimento de produto. Alguns trabalhos partem de uma abordagem voltada à confiabilidade utilizando de ferramentas de uso conhecido como FMEA (failure mode anlysis), enquanto outra parcela utiliza de questionários e entrevistas com especialistas para identificação da maturidade tecnológica. Poucos estudos levantam as necessidades dos clientes como métrica para o avanço da maturidade da tecnologia. A seguir a síntese da análise dos artigos selecionados para o estudo. E. Hessen et al, (2013) [6] utilizou das recomendações da norma Det Norske V it (DNV) lifi ã d b d d dió id d b l Tabela 1: Relação dos artigos coletados na pesquisa. Descritores Science direct Total / Incluídos na pesquisa IEEE Total / Incluídos na pesquisa One Petro Total / Incluídos na pesquisa “Product development” and “Technology qualification 7 / 2 0 1 / 0 “Technology qualification” and “requirements” 33 / 6 6 / 1 13 / 3 Total de artigos inclusos 8 1 3 É possível observar um aumento das publicações entre 2014 e 2016 na Figura 2. Este número vem crescendo pela necessidade da indústria de óleo e gás de novas tecnologias que possam ser usadas em ambientes em maior altura de lâmina d’agua e necessitando de um método sistemático para a qualificação dessas tecnologias. Figura 2: Número de artigos no período investigado Tabela 1: Relação dos artigos coletados na pesquisa. É possível observar um aumento das publicações entre 2014 e 2016 na Figura 2. absorção como modo de prever a criticidade dos modelos preliminares desenvolvidos. O artigo traz ênfase na validação destes sub-modelos que confrontam os valores disponíveis em dados experimentais da literatura e dados obtidos através dos sub- modelos. R. Proskovics et al, (2016) [7] propôs uma metodologia para avaliar os riscos de subestruturas de turbinas eólicas flutuantes, O autor separa em etapas, como avaliação dos riscos tecnológicos, análise da composição tecnológica, categorização da tecnologia, riscos à saúde, segurança e meio ambiente, riscos na manufatura e comercialização. O presente estudo não possuí aplicações da metodologia e nem métricas. M. Sabet (2013) [8] propôs uma metodologia baseada em objetivo para a qualificação tecnológica utilizando de opiniões de especialistas e satisfação do comprimento desses objetivos. O artigo participou em dois estudos de casos na área industrial de óleo e gás; foi efetuado a coleta de dados construindo o modelo de objetivo através de coleta das informações técnicas do portfólio da indústria refinando as informações com entrevistas com especialistas. T. Myhrvold et al. (2009) [9] propõe uma metodologia para a qualificação tecnológica de tecnologias de captura de dióxido de carbono em três fases: desenvolvimento das diretrizes, o teste das diretrizes em estudos de casos e mudança das diretrizes para uma versão final. Como resultado é a proposta da metodologia. H. Barkhtiary-Dvijany, T.Myhrvold (2013) [10] fizeram uma comparação dos métodos disponíveis de avaliação da maturidade tecnológicas. Os autores criticam a necessidade de analisar a maturidade tecnológica trazendo poucos benefícios em melhorar a tecnologia e propõem um método combinado com uma abordagem baseado em riscos. H. Barkhtiary-Dvijany, T.Myhrvold (2013) [10] fizeram uma comparação dos métodos disponíveis de avaliação da maturidade tecnológicas. Os autores criticam a necessidade de analisar a maturidade tecnológica trazendo poucos benefícios em melhorar a tecnologia e propõem um método combinado com uma abordagem baseado em riscos. A. Aal (2016) [11] Expõe que tecnologias avançadas de semicondutores não são capazes de satisfazer as necessidades da indústria automotiva, propõe um método e testa experimentalmente avaliando o efeito de cargas mecânicas como maneira de simular o envelhecimento desses eletrônicos como um método de projeto voltado a resiliência. B. Goo et al, (2019) [12] utilizam da ferramenta FMECA (failure mode and criticallity analysis) em conjunto com projeto axiomático para o melhoramento de um sistema de suprimento de gás natural liquefeito. 3. RESULTADOS E DISCUSSÃO Este número vem crescendo pela necessidade da indústria de óleo e gás de novas tecnologias que possam ser usadas em ambientes em maior altura de lâmina d’agua e necessitando de um método sistemático para a qualificação dessas tecnologias. É possível observar um aumento das publicações entre 2014 e 2016 na Figura 2. Este número vem crescendo pela necessidade da indústria de óleo e gás de novas tecnologias que possam ser usadas em ambientes em maior altura de lâmina d’agua e necessitando de um método sistemático para a qualificação dessas tecnologias. p q ç g Figura 2: Número de artigos no período investigado Após a análise dos artigos selecionados foi percebido a falta de consenso em processos de qualificação tecnológica aplicada a desenvolvimento de produto. Alguns trabalhos partem de uma abordagem voltada à confiabilidade utilizando de ferramentas de uso conhecido como FMEA (failure mode anlysis), enquanto outra parcela utiliza de questionários e entrevistas com especialistas para identificação da maturidade tecnológica. Poucos estudos levantam as necessidades dos clientes como métrica para o avanço da maturidade da tecnologia. Figura 2: Número de artigos no período investigado Figura 2: Número de artigos no período investigado Após a análise dos artigos selecionados foi percebido a falta de consenso em processos de qualificação tecnológica aplicada a desenvolvimento de produto. Alguns trabalhos partem de uma abordagem voltada à confiabilidade utilizando de ferramentas de uso conhecido como FMEA (failure mode anlysis), enquanto outra parcela utiliza de questionários e entrevistas com especialistas para identificação da maturidade tecnológica. Poucos estudos levantam as necessidades dos clientes como métrica para o avanço da maturidade da tecnologia. Após a análise dos artigos selecionados foi percebido a falta de consenso em processos de qualificação tecnológica aplicada a desenvolvimento de produto. Alguns trabalhos partem de uma abordagem voltada à confiabilidade utilizando de ferramentas de uso conhecido como FMEA (failure mode anlysis), enquanto outra parcela utiliza de questionários e entrevistas com especialistas para identificação da maturidade tecnológica. Poucos estudos levantam as necessidades dos clientes como métrica para o avanço da maturidade da tecnologia. A seguir a síntese da análise dos artigos selecionados para o estudo. E. Hessen et al, (2013) [6] utilizou das recomendações da norma Det Norske Veritas (DNV) para a qualificação de um absorvedor de dióxido de carbono em larga escala. Foi um processo baseado em risco utilizando de modelagem do processo de qualificação tecnológica, teste de aceitação pela fábrica, teste funcional e teste de integração. M. Downes , L. Nguyen (2013) [15] utilizam de ferramentas de confiabilidade para o avanço da maturidade do projeto e separam quais atividades devem ser feitas nas respectivas maturidades tecnológicas. Em uma etapa inicial o processo é análise das funções críticas e testes analíticos e experimentais para a diminuição de incertezas. Em uma etapa em que o Sistema é mais maduro utiliza-se de DOE (Design for experiment) e física da falha para a otimização do projeto. Este estudo utilizou como experimento um fuso aplicado à indústria armamentícia. Algumas ferramentas comuns ao desenvolvimento de produto são utilizadas como o QFD (quality function deployment), que é usado para traduzir as necessidades dos combatentes em requisitos de projetos. P. Vella et al,(2018) [16] propõem um método para avaliar a maturidade tecnológica de processos micros e nanos de manufatura e parte da seguinte metodologia: definir a escala de maturidade, identificação de indicadores de maturidade, desenvolvimento de questionários para identificação de projetos de pesquisa e desenvolvimento, coleta dos questionários em pesquisadores trabalhando em diferentes projetos no portfólio das empresas, análise dos resultados para obter os esforços necessários da área de pesquisa e desenvolvimento ao longo da escala de maturidade. T. Albert (2015) [17] propõe um método para avaliar a maturidade tecnológica através do método de blocos de textos de modo rápido, como coleta de dados. A primeira etapa foi a coleta de informações de tecnologias similares para delinear em relações a tecnologias semelhantes em que será analisada a maturidade para formulação de um questionário preliminar, só então os resultados desses questionários foram confrontados com relação as tecnologias fora da área de pesquisa, para excluir certos termos na pesquisa. O processo proposto pelo autor é separado em duas fases: a construção do projeto axiomático, como uma etapa conceitual em conjunto com o cliente e aplicar o FMECA para identificação dos riscos e se são riscos que não podem ser ignorados. S. Yasseri et al, (2018) [13] comparam os estados da prática como as normas API 17 Q e N e as normas DNV-RP-A203 quanto a qualificação tecnológica e propõem um método integrado em utilizar os métodos propostos pelas normas em um processo de desenvolvimento de produto integrado. Storstenvik (2016) [14] propõe um método modular para qualificação tecnológica e utiliza esse método para um Sistema de compressão subsea, separando o produto a ser qualificado em módulos e utilizando a seguinte sistemática: teste da ão pela fábrica, teste ] utilizam de ferramen separam quais ativida . Em uma etapa inicial s e experimentais pa ma é mais maduro utiliza otimização do projeto. dústria armamentícia. A são utilizadas como o Q r as necessidades do m um método para a nos de manufatura e uridade identificação 5. REFERÊNCIAS 5. REFERÊNCIAS 1 G. Pahl, W. Beitz, J. Feldhusen, and K.-H. G. Grote, Engineering Design, Third. Darmstadt: Springer Science and Business Media, 2015. 1 G. Pahl, W. Beitz, J. Feldhusen, and K.-H. G. Grote, Engineering Design, Third. Darmstadt: Springer Science and Business Media, 2015. 2 J. Harkonen and J. Hyysalo, “Design for eXcellence in high tech companies Design for eXcellence in high tech companies Keywords : Design for eXcellence , Product development , Requirements,” no. December 2008, 2008. 10 H. Bakhtiary-Davijany and T. Myhrvold, “On methods for maturity assessment of CO2 capture technologies,” Energy Procedia, vol. 37, pp. 2579– 2584, 2013. 4. CONCLUSÃO Este artigo teve como objetivo identificar a produção científica que aborda a relação de desenvolvimento de produtos e qualificação tecnológica. A pesquisa mostra que a maioria dos estudos utiliza de ferramentas comuns de identificação de riscos, como o FMEA (failure mode analysis) e FTA (fault tree analysis) e poucos desses trabalhos possuem estudos de casos para embasar os resultados e são somente propostas de metodologias. Por fim, nesta revisão sistemática foi identificado apenas um artigo que leva em consideração necessidades de clientes, sendo somente de clientes internos, mostrando lacunas dos processos de qualificação tecnológica, utilizando de ferramentas de gerenciamento de requisitos para acompanhar o avanço da maturidade tecnológica com o comprimento dos requisitos impostos pelos clientes. 5. REFERÊNCIAS 17 T. Albert, M. G. Moehrle, and S. Meyer, “Technology maturity assessment based on blog analysis,” Technol. Forecast. Soc. Change, vol. 92, pp. 196–209, 2015. 11 A. Aal, “Reliability management — The central enabler for advanced Technologies in Automotive,” Microelectron. Reliab., vol. 64, pp. 13–18, 2016. 12 B. Goo, J. Lee, S. Seo, D. Chang, and H. Chung, “Design of reliability critical system using axiomatic design with FMECA,” Int. J. Nav. Archit. Ocean Eng., vol. 11, no. 1, pp. 11–21, 2019. 13 S. Yasseri, H. Bahai, and R. Yasseri, “Reliability Assurance of Subsea Production Systems: A Systems Engineering Framework,” Int. J. Coast. Offshore Eng., no. January, 2018. 14 A. Storstenvik, “Subsea Compression - Designing and Building a Subsea Compressor Station,” 2016. 3 G. L. DNV, “Recommended Practice DNV-RP-203 ‘Technology Qualification,’” 2013. 4 P. H. Kim, R. Kotha, S. P. L. Fourné, and K. Coussement, “Taking leaps of faith: Evaluation criteria and resource commitments for early-stage inventions,” Res. Policy, vol. 48, no. 6, pp. 1429–1444, 2019. 5R. Coelho, R. Daltry, V. Dobbin, E. Lachaud, and I. Miller, “Design Process and Validation of an Autonomous Surface Vehicle for the Offshore Industry,” 2015. 6 E. T. Hessen, H. Bakhtiary-Davijany, and T. Myhrvold, “Process modelling in risk-based qualification of large-scale CO2absorption processes,” Energy Procedia, vol. 37, pp. 2802–2810, 2013. 7 R. Proskovics, G. Hutton, R. Torr, and M. N. Scheu, “Methodology for Risk Assessment of Substructures for Floating Wind Turbines,” Energy Procedia, vol. 94, no. January, pp. 45–52, 2016. 8 M. Sabetzadeh, D. Falessi, L. Briand, and S. Di Alesio, “A goal-based approach for qualification of new technologies: Foundations, tool support, and industrial validation,” Reliab. Eng. Syst. Saf., vol. 119, pp. 52–66, 2013. 9 T. Myhrvold, K. Helle, K. Johnsen, and A. Hussain, “Development of a guideline for the qualification of CO2 capture technology,” Energy Procedia, vol. 1, no. 1, pp. 1527–1534, 2009. 10 H. Bakhtiary-Davijany and T. Myhrvold, “On methods for maturity assessment of CO2 capture technologies,” Energy Procedia, vol. 37, pp. 2579– 2584, 2013. 14 A. Storstenvik, “Subsea Compression - Designing and Building a Subsea Compressor Station,” 2016. 15 M. R. Downes and L. P. Nguyen, “Reliability engineering efforts at U.S. Army Armaments Research Development and Engineering Center,” Proc. - Annu. Reliab. Maintainab. Symp., 2013. 16 P. C. Vella, S. S. Dimov, R. Minev, and E. B. Brousseau, “Technology maturity assessment of micro and nano manufacturing processes and process chains,” Proc. Inst. Mech. Eng. Part B J. Eng. Manuf., vol. 232, no. 8, pp. 1362–1383, 2018.
https://openalex.org/W2162091268	https://academicjournals.org/journal/AJAR/article-full-text-pdf/A11612B34083.pdf	English	null	The effects of agricultural subsidies applied in Turkey on sunflower cultivation area and the producers income	African journal of agricultural research	2,013	cc-by	4,765	Vol. 8(12), pp. 1059-1065, 4 April, 2013 DOI: 10.5897/AJAR11.1344 ISSN 1991-637X ©2013 Academic Journals http://www.academicjournals.org/AJAR African Journal of Agricultural Research Vol. 8(12), pp. 1059-1065, 4 April, 2013 DOI: 10.5897/AJAR11.1344 ISSN 1991-637X ©2013 Academic Journals http://www.academicjournals.org/AJAR African Journal of Agricultural Research African Journal of Agricultural Research African Journal of Agricultural Research Full Length Research Paper Arif Semerci Department of Agricultural Economics, Faculty of Agriculture, Mustafa Kemal University, Tayfur Sökmen Kampüsü, Antakya, Hatay, Turkey Department of Agricultural Economics, Faculty of Agriculture, Mustafa Kemal University, Tayfur Sökmen Kampüsü, Antakya, Hatay, Turkey Accepted 14 January, 2013 Sunflower takes an important place in terms of raw materials provided for the oil industry and of added value, as well as bring-in income for producers in Turkey’s economy. Sunflower is an important source of income by standing the main product in alternation of the wheat in the sense of manufacturers in Thrace Region which constitutes the European part of Turkey. In this study, the effect of premium support application on sunflower cultivation area and production amount in Thrace where approximately 65% of sunflower production in Turkey is met and also the effect of support payments on producer income are investigated. In this study, it is concluded that premium supports are inefficient in increasing sunflower cultivation area and production whereas it is concluded that it has an important role on producer income and on the determination of the market price. In fact, from a survey that was carried out, it emerges that the area-based support (diesel oil, fertilizer etc. support) together with premium support increase the revenue of sunflower up to 27.74% per area unit and the gross profit in proportion to 98.87%. This study indicates that in order to increase the sunflower production in Turkey the amounts of sunflower premium support, diesel oil and fertilizer should be rearranged in compliance with current market conditions. Moreover, as well as for other agricultural products, the use of certified seed should also be included into agricultural support programmes, production on well irrigated areas should be widened and the use of oil crops varieties with high oil ratio should be extended. Key words: Oil crops, sunflower, agricultural subsidy, agricultural income, gross profit. Full Length Research Paper The effects of agricultural subsidies applied in Turkey on sunflower cultivation area and the producers’ income Full Length Research Paper INTRODUCTION Alike food sector, agriculture sector has a significant role in national economics because of its contribution to employment and income, its role in meeting the needs of industrial raw materials and its direct or indirect benefits on exports. According to 2009 data, in Turkey the agricultural sector account for 51 billion US$ of gross domestic product and sunflower is one of the leading Alike food sector, agriculture sector has a significant role in national economics because of its contribution to employment and income, its role in meeting the needs of industrial raw materials and its direct or indirect benefits on exports. According to 2009 data, in Turkey the agricultural sector account for 51 billion US$ of gross domestic product and sunflower is one of the leading agricultural products for manufacturers and oil industry especially in Thrace, at the European side of the Turkey (MARA, 2011a, b).Turkey which accounts for 2.46% of sunflower world cultivated area and 3.26% of production is among the top ten countries for production of sunflower according to production data from the year 2009 (Anonymous, 2011a). Corresponding author. E-mail: arifsemerci69@gmail.com. Tel: Tel: +90(326)2455845. Fax: +9 1060 Afr. J. Agric. Res. production might affect the production diversity (Střeleček et al., 2009). A Premium support system is an alternative support model that can be applied by assuring stability in the sense of prices and the producer in agriculture of Turkey, depending on the research work of Sahinoz et al. (2007). In a study published by the Vegetable Oils and Fats Industrialists Association, sunflower production is supposed to be decreasing at an average annual rate of 6% in the period 2010 to 2013 whether premium supports which is being paid to oil seed producers in Turkey will not be paid (Koc, 2005). In another study which investigates the effects of premium support systems in terms of agricultural subsidies in Turkey, it is educed that the subsidy got by farmers has significant effects on increasing the sunflower cultivated area and premium supports paid for sunflower are efficient in protecting the producers against to market prices (Erdal and Erdal, 2008). Sunflower which accounts for 3.60% of field crops cultivated areas in Turkey, also meets approximately 55% of vegetable oil production (TURKSTAT, 2011; Ozcelik and Fidan, 2003). Besides, 4.99% of 2,328,731 farmers in the country are working on sunflower production (MARA, 2011a). INTRODUCTION Despite the high agricultural production potential, the self-sufficiency rate of sunflower in our country is only 46.60% (Anonymous, 2011b). In 2008, 3 billion US$ of vegetable oil and oil seeds was imported in order to meet the current deficit (Anonymous, 2009). ) Because of the low elasticity of demand, exposure to climate conditions, intense economic instability (price and income) of agricultural sector, government interventions are carried on cultivation areas and the sector is diversely supported across the globe (Gunaydin, 2006). Turkey is one of the countries supporting the agricultural sector in line with the various agricultural policies implemented in accordance with the developments in the world (Zemheri, 2010). Use of certified seeds, premium support system, diesel oil and fertilizer compose are the main factors of agricultural subsidy studies in Turkey especially since the early 2000’s in order to ensure an increase in the production of products which are in short supply. However, it is not exactly known the extent these practises reflect to manufacturer agricultural income and how the agricultural production is affected. Recently, agricultural production support policies applied in Turkey have actually changed a lot are deeply changing. A new model of agricultural production and support model (Turkey Agricultural Basin Production and Support Model) has just begun to be implemented in Turkey. The aims of the new model are; determination of the agricultural basins, preparation of a clear agricultural inventory, provision of opportunities for production planning, and making projections of demand for the future (Sahin et al., 2010). By the study carried out in Thrace where approximately 65% of sunflower for oil industry is produced in Turkey, the effects of premium supports on sunflower cultivation areas and amount of production with the aim of increasing oleaginous seeds production was investigated. In addition to this, in this study, it is also put forth the contribution of support payments to manufacturer prosperity for increasing sunflower production. In some studies conducted in various countries, support of agricultural production and the effects of these supports on agricultural production, farmer’s welfare and the trade of agricultural products were analyzed (Hennessy, 1998; Mayrand et al., 2003; Anderson et al., 2006; Keeny, 2009). Some studies reside in Turkey related to agricultural support effects on sunflower production, reflection to producer income and foreign trade regime (Gaytancioglu, 1999; Koc, 2005; İlkdogan, 2008). INTRODUCTION The amount of resources allocated to support agricultural production varies depending on the development levels of countries. Direct payments are the largest item with 74% of agriculture subsidies (approximately €55 billion) in the European Union’s budget in 2008. Direct payments are allocated with a share of 99% to agricultural markets, 0.5% to fisheries sector and 0.5% to plant and animal health. The second largest item which has a 23.5% share with €12.9 billion is rural development. The amount of direct subsidies that USA farmers have received in the years from 1996 and 2001 is 70 billion US$. In the period from 2002 to 2008, the farmers are estimated to have benefited from direct state assistance of 100 billion US$ and 80 billion US$ is planned to be distributed before 2012 (Acar and Bulut, 2009). The amount of resources allocated to agricultural subsidies in 2010 in Turkey is given in the Table 1.Premium support payments in Turkey in 2010 account for 36.95% of agricultural support payments and 24.57% of total agricultural subsidies. The share of total agricultural support payments stood at 0.82% of Gross Domestic Product. The support payments for development of oil seed production in 2008 in Turkey are given based on the products in Table 2. In terms of premium supports the price sunflower seed is ranked second in oil seed support payments in Turkey. According to a study based on a ‘Theoretical Microeconomic Model’ whose data were obtained from 1,000 agricultural enterprices in Norway, it is concluded that agricultural subsidies have significantly positive effects on input use and the level of output (Henningsen et al., 2009). As a result of research conducted in Czech Republic, despite the country’s agriculture having a competitive market, in comparison to some of European countries it emerges that agricultural subsidies have low share in producer income. In this study, it is determined that the lower percentages of support level weaken the competitiveness of agricultural area, and the regeneration and modernization of agriculture. As a result, it is concluded that the subsidies which are applied according to type of RESULTS AND DISCUSSION Σ ( Nh Sh)2 n = N2 D2 + Σ Nh (Sh)2 Σ ( Nh Sh)2 n = N2 D2 + Σ Nh (Sh)2 n MATERIALS AND METHODS The primary data used in this study were obtained by question- 1061 Semerci Table 1. Funds Allocated to Agricultural Support in Turkey (2010). Criteria US$ Premium supports 1.340.745.000 Agricultural support payments 3.627.382.000 Implementation of low interest rate credit 487.536.000 Total agricultural support payments 5.455.663.000 Total gross domestic product 665.891.300.000 Kaynak: MARA, General Directorate of Agricultural Production and Improvement, 2009. Table 1. Funds Allocated to Agricultural Support in Turkey (2010). Table 2. Premium support payments on oil seed plants and olive. Products Number of enterprices Production area (ha) Amount due to supports (ton) Sum of supports (US$) Product rate in sum of support (%) Safflower 292 1.399 1.733 248.552 0.04 Corn for grain 65.929 383.650 3.557.650 46.460.339 8.18 Canola 1.610 6.868 16.704 2.403.263 0.42 Cotton Unseed 69.208 441.581 1.905.437 422.260.035 74.35 Soybean 950 5.199 16.180 2.647.695 0.47 Sunflower 89.984 420.505 705.216 92.108.143 16.22 Olive 5.126 27.610 13.845 1.810.883 0.32 Total 227.973 1.286.812 6.216.765 567.938.910 100.00 Resource: MARA, General Directorate of Agricultural Production and Improvement, 2009 naires from specified 571 agricultural enterprises in 53 locations on the European side of provinces of Edirne, Kırklareli, Tekirdağ, İstanbul and Çanakkale with Stratified Random Sampling Method in production period of 2009. In determination of stratified random sampling method, two different techniques are commonly applied by agricultural economic studies. These are: Neyman Method and Proportional Distribution Method (Erkan and Cicek, 1996). Because of the incompliance of sunflower cultivation areas in locations and enterprises to normal distribution, Neyman Method is used in this research. The equality of Neyman Method is given below (Yamane, 1967). In the determination of effects of supports on producer welfare applied for sunflower production; Trakya Birlik’s purchase price is taken as product price and the support unit price applied in 2009 by the Ministry of Agriculture and Rural Affairs is taken as the support unit prices. However, in the study, besides the purchase price of sunflower and premium support price, also the unit price variations of diesel fuel, fertilizer (20-20-0 compound fertilizer), seed and agricultural pesticides were researched between the years 1999 to 2010. Relationship between premium support, cultivated area and production amount of sunflower Price variation occured in the factors that determine product cost, in premium support, and price of Years Fuel (US$/lt) Fertilizer (US$/kg) Pesticide (US$/lt) Seed (US$/kg) Sunflower (US$/kg) Premium support (US$/kg) 1999 0.369 0.170 2.948 5.528 0.268 0.029 2000 0.622 0.333 4.000 6.370 0.293 0.048 2001 0.415 0.311 3.111 3.457 0.278 0.022 2002 0.664 0.155 4.019 5.480 0.326 0.046 2003 0.998 0.203 4.706 10.338 0.426 0.080 2004 1.149 0.265 5.191 11.864 0.478 0.096 2005 1.445 0.277 4.817 12.597 0.504 0.130 2006 1.563 0.281 3.874 13.029 0.511 0.141 2007 1.948 0.421 5.127 18.799 0.876 0.171 2008 1.863 0.727 4.624 16.515 0.673 0.125 2009 1.639 0.348 4.684 18.066 0.642 0.141 2010 1.922 0.399 5.178 19.417 0.777 0.149 Resource: MARA, Costs of agricultural products in Trakya Region 1999-2010. Kirklareli Ataturk Soil and Water Resources Research Institute. Kirklareli. Resource: MARA, Costs of agricultural products in Trakya Region 1999-2010. Kirklareli Ataturk Soil and Water Resources Research Institute. Kirklareli. decline at the ratio of 10.88% on average of sunflower cultivation areas to 559.456 ha before supported period (1988 to 1998) in comparison with the supported period (1999 to 2010). In other words, the premium support applied for oil sunflower since 1999 is inadequate in preventing the reduction of the cultivation areas which began before the support period. This circumstance also shows that the amount of support given to agricultural production is not in a level sufficient to promote the production. Therefore, the enhancement of sunflower plantations can be possible by providing continuous support and satisfactory reorganization to current conditions. As an example, in early 2000s the support policy applied to corn has successfully resulted in making Turkey self-sufficient over a period of 3 to 5 years whereas before 2000 it was foreign-dependent for corn production. (536 Businesses) expressed their opinion that Premium Support applied by Agricultural and Rural Affairs should be continued. The expectation of 69.52% of companies (390 enterprises) for Premium Support unit prices of oil sunflower as a product of the year 2010 was 20.17 cent/kg. However, the expounded Premium Support unit price was 30% lower than the expected value of producers, that is to say 14.12 cent/kg. This shows that the value of stated unit price is far from the manufacturer expectations. Relationship between premium support, cultivated area and production amount of sunflower In the formula; n = sample size, Nh = number of units in layer h (frequency of farm size);S h = standard deviation of layer h; N = total number of units; D = d/z; d = a certain percentage of deviation from average(1%-5%-10%), and z = degrees of freedom in t- distribution scale (N-1) and expresses “t value” belongs to a certain confidence limit (90%-95%-99%). In this study, in the period from 1988 to 2010, it is investigated whether there is a relationship between non- supported period of oil sunflower (1988 to 1998) and supported period (1999 to 2010) on sunflower cultivation area and the amount of production. For this purpose, the dummy variable for the unsupported period (1988 to 1998) is taken to be “0” and for the supported period (1999 to 2010) is taken to be “1”. Within the scope of the research, the effects of premium supports applied in Turkey on the cultivation areas and production amount of sunflower for the next production period were investigated. To that end, taking account of two periods as premium supported and unsupported, the relationship between cultivation areas and production amount was investigated with “correlation analysis” by using SPSS programme (Green et al., 2000). With this purpose, sunflower production periods in Turkey were divided into two parts; unsupported (1988 to 1998) and premium supported (1999 to 2008). The dummy variable is taken to be “0” in 1988 to 1998 period and taken to be “1” in 1999 to 2008 period. The correlation between dummy variable related to support and sunflower production areas is found to be significant, and the correlation between the dummy variable related to support and the amount of sunflower production is found to be statistically insignificant in this study (Table 3). This situation can be explained with the Afr. J. Agric. Res. 1062 Table 3. The relationship between premium support periods and nonsupported periods with cultivation areas and production amount of sunflower in Turkey. Table 3. The relationship between premium support periods and nonsupported periods with cultivation areas and production amount of sunflower in Turkey. Variable Criteria Dummy variable Sunflower production area (ha) Correlation -0.535( ) Significance level 0.008() Observation numbers 23 Sunflower production quantity (ton) Correlation 0.092 Significance level 0.677() Observation numbers 23 P < 0.01; **Non-significant. Table 4. Price variation occured in the factors that determine product cost, in premium support, and price of sunflower. Relationship between premium support, cultivated area and production amount of sunflower In a study carried out on the subject, excepting premium support, diesel fuel and fertilizer support paid in Turkey within the scope of agricultural support is concluded to be insufficient (Ozcelik and Ozer, 2007). The variation occured between 1999 to 2010 on the prices of sunflower production input and the premium support which has been granted since 1999 in Turkey is given in Table 4. When the data is analysed, it is conspicuous that especially the price of diesel fuel input has increased 5.2 times in the period 1999 to 2010, while p As a part of the research, attitude and the level of expectations of manufacturers to “Premium Support” are also analysed. 97.25% of manufacturers in the survey 1063 Semerci Table 5. Effects of sunflower support payments to producer welfare on enterprices in field of survey (2009). Table 5. Effects of sunflower support payments to producer welfare on enterprices in field of survey (2009). Table 5. Effects of sunflower support payments to producer welfare on enterprices in field of survey (2009). Sunflower’s income (excluding subsidizing payments) Number of enterprises (survey) 571 Sunflower production area (ha) 5.349.75 Production (ton) 9.487.08 Yield (ton/ha) 1.773 Price (US$/ton) 504.27 Gross production value (US$/ha) 894.24 Variable expenses (US$/ha) 575.54 Gross profit (US$/ha) 318.70 Sunflower’s income (including subsidizing payments) Fertilizer support (US$/ha) 36.98 Fuel support (US$/ha) 36.98 Premium support(US$/ton) 140.00 Price (sales price+Premium support price) (US$/ton) 644.27 Gross production value (US$/ha) 1.142.29 Total supports (US$/ha) 322.18 Variable expenses( US$/ha) 575.54 Gross profit (US$/ha) 640.71 Share of subsidies in Gross Profit (%) 50.28 Sunflower’s income (excluding subsidizing payments) Sunflower’s income (excluding subsidizing payments) Number of enterprises (survey) 571 Sunflower production area (ha) 5.349.75 Production (ton) 9.487.08 Yield (ton/ha) 1.773 Price (US$/ton) 504.27 Gross production value (US$/ha) 894.24 Variable expenses (US$/ha) 575.54 Gross profit (US$/ha) 318.70 Sunflower’s income (including subsidizing payments) Fertilizer support (US$/ha) 36.98 Fuel support (US$/ha) 36.98 Premium support(US$/ton) 140.00 Price (sales price+Premium support price) (US$/ton) 644.27 Gross production value (US$/ha) 1.142.29 Total supports (US$/ha) 322.18 Variable expenses( US$/ha) 575.54 Gross profit (US$/ha) 640.71 Share of subsidies in Gross Profit (%) 50.28 The variations generated by support applications on Gross Production Values and Gross Profit of enterprises are examined by calculating the sunflower income both in per supported and unsupported unit area (ha) with the help of data acquired from 571 agricultural enterprises in scope of the research. Conclusion Among the farms of the survey, Gross Production Value of sunflower reaches on average the level of 1,142.29 US$ per hectare with an increase of 27.74% on the unsupported level in consideration of the average yield (1.773 ton/ha) and of the payments of oil seed supports. In conjunction with the support payments, sunflower Gross Profit reaches up to the level of 640.71 US$/ha with a growth of 98.87%. The share of subsidies in “Gross Production Value of New Sunflower” of which was calculated in consideration of supports is 50.28%. This rate clearly reveals the contribution and importance of support payments on Gross Profit and even in Gross Production Value of sunflower. Other studies in this issue reveal that although support payments (premium supports Relationship between premium support, cultivated area and production amount of sunflower In the study, the average sunflower yield of enterprises was calculated as 1.773 tons/ha. In case that the support is disregarded, the average GPV of sunflower is calculated to be 894.24 US$/ha, variable costs calculated to be 575.54 US$/ha and Gross Production Value is calculated to be 318.70 US$/ha (Table 5). the price increase for the same period was recognized to be 3.5 times for seed, 2.3 times for fertilizer and 1.8 times for pesticides. Despite the 5.1 times increase on unit price of premium support in 2010 in comparison with 1999, the increase in purchasing price of the sunflower seed remained at the level of 2.9 times. Because of the diesel fuel and the product prices lower than the seeds which take important part of the costs, fluctuations are observed on sunflower cultivations areas. In the other studies carried in the region, in accordance with the evidences identified in the study, it is concluded that the purchase price of sunflower has significantly decreased in real terms in response to real increase especially in the price of diesel fuel (Aksoy and Gaytancioglu, 1996; Semerci and Kaya, 2010). the price increase for the same period was recognized to be 3.5 times for seed, 2.3 times for fertilizer and 1.8 times for pesticides. Despite the 5.1 times increase on unit price of premium support in 2010 in comparison with 1999, the increase in purchasing price of the sunflower seed remained at the level of 2.9 times. Because of the diesel fuel and the product prices lower than the seeds which take important part of the costs, fluctuations are observed on sunflower cultivations areas. In the other studies carried in the region, in accordance with the evidences identified in the study, it is concluded that the purchase price of sunflower has significantly decreased in real terms in response to real increase especially in the price of diesel fuel (Aksoy and Gaytancioglu, 1996; Semerci and Kaya, 2010). (2011b). Anonymous (2011b). Available at: Anonymous (2011b). http:///www.tuik.gov.tr/agriculture/database http:///www.tuik.gov.tr/agriculture/database ) Today, agriculture is a sector that should be supported because of its nature especially in developing countries. Turkey is one of the countries that have special emphasis on support of agricultural production. Agricultural support systems which are carried out in Turkey are in accordance with international rules. However, over time, due to the insufficiency of agricultural support policies, innovations of systems and fundamental changes have become inevitable. In the light of developments in recent years, a new agricultural production support system “Turkey Agricultural Basin Production and Support Model” was adopted since 2011 in Turkey. The basic expectations from the new system implemented are to provide an increase in producer income and availability to meet shortage of products supply (oil seeds etc.) with domestic production by supporting them in higher levels as well as an increase in agricultural production and productivity. Erdal G, Erdal H (2008). The Effects of premium payment in Turkey agricultural supports system. Gaziosmanpasa Univ. J. Agric. Fac. 25(1):41-51. Erkan O, Cicek A (1996). Research and sampling methods in agricultural economics. GOP University. Agricultural Faculty Publishing. 12(6). Tokat. Turkey. p.72. Gaytancioglu O (1999). Economic anaysis of the agricultural politics applied on sunflower and the yield loss owing to warehousing. Trakya University. Tekirdag Agriclyural. Faculty, Tekirdag. University. Tekirdag Agriclyural. Faculty, Tekirdag. G S S S Green SB, Salkind NJ, Akey TM (2000). Using SPSS for windows, analyzing and understanding data. Second Edition. Prentice-Hall Inc. Upper Saddle River. New Jersey.USA. pp. 243-252. pp y pp Gunaydin G (2006). Agriculture sector in Turkey. J. Agric. Eng. 76- 77:12-27 lkdogan U (2008). Development of oilseed trade in the world and EU an evaluation in terms of Turkey. EU Expertise Thesis. Ministry of Agriculture and Rural Affairs of Turkey. Foreign Affairs and EU Coordination Department.Ankara. p. 128 Hennessy AD (1998).The Production effects of agricultural income support policies under uncertainity. Am.J.Agric. Econ.80:46-57. Henningsen A, Kumbhakar S, Lien G (2009). Econometric analysis of the effects of subsidies on farm production in case of endogenous input quantities. AAEA & ACCI Joint Annual Meeting. Milwaukee. Wisconsin. USA. p y Agricultural subsidies for producers in Turkey comprise approximately 6% of total agricultural production value and 0.82% of Gross Domestic Product. (2011b). In this case, as of now, the usage of support tools and field based supports and also the compliance with the rule of “de minimis” constitute a hindrance for the country. However, both the study findings and the evidences of previous researches demonstrate that premium supports and supports unit prices of diesel fuel and fertilizer are insufficient to increase sunflower production and they thereby reveal that unit support prices should definitely be increased. Keeny R (2009). Transfer efficiency and distributional impacts of U.S. farm support: evidence from a macro–micro simulation. Am. J. Agric. Econ. 91(5):1289–1295. Koc A (2005). Oilseed sector (seed, pulp and oil) status: Abatement of customs tariff rates and effects of premium supports. Vegetable Oils and Fats Industrialists Association, Hazar Advertising Printing, Publication Ankara 7:108. MARA (2011a). 2010 Current data of Ministry of Agriculture and Rural Affairs of Turkey. General Directorate for Agricultural Production an Development. Ankara. p.29. MARA (2011b). 2010 Annual Report of Tarsim Agricultural Insurance Pool. Ministry of Agriculture and Rural Affairs and Prime Ministry Undersecretariat of Treasury and Foreign Trade. Ankara. p. 7. This study carried out proves that the premium support applied in Turkey has no effect on increasing the cultivation areas of sunflower. It only shows that the supports of diesel fuel and fertilizers have significant increasing effects on producer income and decreasing costs for sunflower with premium supports. In conse- quence of the study, dissemination of production in watery conditions, encouragement usage of varieties with high oil content, usage of appropriate fertilizer according to results of soil tests, and the seed use should be proposed to be incorporated to agricultural subsidy system for sunflower production like in other products. Mayrand K, Dionne S, Paquin M, Le Bell IP (2003). The Economic and environmental impacts of agricultural subsidies: An assessment of the 2002 US Farm Bill & Doha Round. Unisféra International Centre. Canada. Ozcelik A, Fidan H (2003). Importance of sunflower regarding to economy of Turkey. Turkey First Oil Seeds, Vegetable Oils and Technologies Symposium. pp.92-102. Ozcelik AO, Ozer O (2007). The Evaluation of supporting payments to farmers for fuel and chemical fertilizer in Turkey. J. Agric. Sci. 13 (1)1-8 Sahin I, Semerci A, Kaya Y, Citak N (2010). Determining efficiency of subsidizing policies and productivity on sunflower production. MARA. Tagem (08-R&D-6). p. 354. Sahinoz A, Cagatay S, Teoman O (2007). (2011b). Discussion of the Applicability of Premium Support System as Agricultural Support in Turkey and Economic Analysis of the System. MARA. Agricultural Economics Research Institute. Publ. Ankara. 155:75. Effects of applied agricultural supports to producer welfare in sunflower production Sunflower in Thrace is ranked as the second in the production pattern of agricultural enterprises with a ratio of 42.74% after the wheat. The contribution of sunflower to enterprises total agricultural income is calculated to be approximately 30%. These data clearly reveal the importance of sunflower for the producers in the field of research (Sahin et al., 2010). In this context, as well as the contribution of premium support and other support applications to welfare of sunflower producers, product cost reduction effect is also important. Afr. J. Agric. Res. Afr. J. Agric. Res. 1064 global agricultural subsidies and market access. World Trade Rev. 5(3):357-376. global agricultural subsidies and market access. World Trade Rev. 5(3):357-376. and field-based supports) are directly not efficient on increasing sunflower production, they play an important role on the formation of healthier sunflower purchase prices and on farmer’s income. It also reveals that abolition of support payments will result in an absolute decrease on sunflower production (Erdal and Erdal, 2008; Koc, 2005). ( ) Anonymous (2009). Agriculture in Turkey with ecomonomical indicators. MARA. Agricultural Economics Research Institute. Project Ankara. 176:41-102. Anonymous (2011a). (http:///www.fao.org/FAOSTAT-Agriculture /production/crops) p p ) Anonymous (2011b). Available at: http:///www tuik gov tr/agriculture/database at: p p ) Anonymous (2011b). g y p Anderson K, Martin W, Valenzuela E (2006). The relative importance of Englowood Cliffs. N. Jersey.USA. (Please provide page number). Zemheri O. (2010). Recent Developments in Agricultural Support Policies. The J. Minis. Agric. Rural Affairs. (Turk Tarım) 2010:195. Ankara. p. 18. REFERENCES Semerci A, Kaya Y (2010). The Component of production cost in sunflower and its relationships with input prices. Int. Rev. Appl. Econ. 5(1-2):139-146. Acar M, Bulut E (2009). Recent developments in agricultural support policies in Turkey and the world. S.U. IIBF. J. Soccal Econ. Res. 9 (17)1-19 Střeleček F, Zdeněk R, Lososová J (2009). Comparison of agricultural subsidies in the Czech Republic and in the selected states of the European Union. Agric. Econ–Czech 55(11):519-533 ( ) Aksoy S, Gaytancioglu O (1996). Research on the effects of increases in agricultural input prices to product prices. I. Agricultural Economics Congress of Turkey, İzmir. p. 2. p g ( ) TURKSTAT (2010). Statistical indicators 1923-2009. Turk. Stat Inst. Publ. Ankara 3493:170. p g ( ) TURKSTAT (2010). Statistical indicators 1923-2009. Turk. Stat Inst Publ. Ankara 3493:170. Yamane T (1967). Elementary sampling theory. Prentice-Hall Inc. Publ. Ankara 3493:170. Yamane T (1967). Elementary sampling theory. Prentice-Hall Inc. g y, p Anderson K, Martin W, Valenzuela E (2006). The relative importance of Anderson K, Martin W, Valenzuela E (2006). The relative importance of Yamane T (1967). Elementary sampling theory. Prentice-Hall Inc. 1065 Semerci
https://openalex.org/W4362561053	https://figshare.com/articles/journal_contribution/Supplementary_Figure_S3_from_Constitutive_IDO1_Expression_in_Human_Tumors_Is_Driven_by_Cyclooxygenase-2_and_Mediates_Intrinsic_Immune_Resistance/22539958/1/files/40003297.pdf	English	null	Supplementary Figure S3 from Constitutive IDO1 Expression in Human Tumors Is Driven by Cyclooxygenase-2 and Mediates Intrinsic Immune Resistance	null	2,023	cc-by	282	Suppl. Fig. 3. Number of COX-2 transcripts per 4000 GAPDH transcripts + + + - - - A66 (p110αi) TGX221 (p110βi) MK2206 (AKTi) + + - - MF63 (mPGES1i) GW627368X (EP4i) 0.4 0.8 1.2 1.6 0.0 Fig. S3 (related to Fig 4) : Effect of PI3K inhibitors on COX-2 expression in KUL98-MELA cells. KUL98-MELA cells were treated with 10μM MF63, 5 μg/ml GW627368X, 20μM A66, 30μM TGX221 or 10μM MK2206 for 72h. RNA was extracted and expression of COX-2 was analysed by RT-qPCR. (Data represented are Mean+SD; the experiment was repeated 3 times) Suppl. Fig. 3. Number of COX-2 transcripts per 4000 GAPDH transcripts + + + - - - A66 (p110αi) TGX221 (p110βi) MK2206 (AKTi) + + - - MF63 (mPGES1i) GW627368X (EP4i) 0.4 0.8 1.2 1.6 0.0 Fig. S3 (related to Fig 4) : Effect of PI3K inhibitors on COX-2 expression in KUL98-MELA cells. KUL98-MELA cells were treated with 10μM MF63, 5 μg/ml GW627368X, 20μM A66, 30μM TGX221 or 10μM MK2206 for 72h. RNA was extracted and expression of COX-2 was analysed by RT-qPCR. (Data represented are Mean+SD; the experiment was repeated 3 times) Suppl. Fig. 3. Suppl. Fig. 3. Number of COX-2 transcripts per 4000 GAPDH transcripts + + + - - - A66 (p110αi) TGX221 (p110βi) MK2206 (AKTi) + + - - MF63 (mPGES1i) GW627368X (EP4i) 0.4 0.8 1.2 1.6 0.0 Fig. S3 (related to Fig 4) : Effect of PI3K inhibitors on COX-2 expression in KUL98-MELA cells. KUL98-MELA cells were treated with 10μM MF63, 5 μg/ml GW627368X, 20μM A66, 30μM TGX221 or 10μM MK2206 for 72h. RNA was extracted and expression of COX-2 was analysed by RT-qPCR. (Data represented are Mean+SD; the experiment was repeated 3 times)
https://openalex.org/W4389241841	https://isg-journal.com/isjea/article/download/562/314	Ukrainian	null	Евристичне управління дискретними системами енергетики	International Science Journal of Engineering & Agriculture	2,023	cc-by	4,466	Евристичне управління дискретними системами енергетики Олександр Іванович Брунеткін Кафедра програмних і комп’ютерно-інтегрованих технологій, Національний університет «Одеська політехніка», Одеса, Україна ORCID 0000-0002-6701-8737 Олександр Іванович Брунеткін Кафедра програмних і комп’ютерно-інтегрованих технологій, Національний університет «Одеська політехніка», Одеса, Україна ORCID 0000-0002-6701-8737 Олександр Валерійович Бабенко Кафедра програмних і комп’ютерно-інтегрованих технологій, Національний університет «Одеська політехніка», Одеса, Україна ORCID 0009-0009-9197-1446 Олександр Валерійович Бабенко Кафедра програмних і комп’ютерно-інтегрованих технологій, Національний університет «Одеська політехніка», Одеса, Україна ORCID 0009-0009-9197-1446 3. Мета та задачі дослідження Завдання управління можна сформулювати в такий спосіб: забезпечити оптимальний баланс між як технологічного процесу, надійністю, ефективністю та вартістю. При цьому слід, що довкілля може змінюватися непередбачуваним чином. Розглянемо поточний стан системи через рівні інтервали часу. В межах інтервалу структура системи змінюється, тобто. обладнання не включається та не вимикається, та працює в номінальному режимі. Таким чином, завдання керуючого алгоритму – отримати план перемикання обладнання, що відповідає оптимальному вирішенню з точки зору обраного критерію. 1. Вступ Накладемо на процес управління обмеження та умови. − Забезпечення максимально можливої якості, тобто мінімізувати відхилення поточного параметра від заданого. − Забезпечення максимально надійної надійності обладнання. − Забезпечення максимально надійної надійності обладнанн − Використання процесу управління найбільш ефективного устаткування. − Забезпечення надійності обладнання не нижче задано. − Система має в міру можливості збереження працездатності в умовах обмеженості енергоресурсів. 2. Об'єкт і предмет дослідження Об'єкт дослідження: система управління теплопостачанням із змінною структурою технічних засобів об'єкта управління. Предмет дослідження: математичне забезпечення розв’язання задачі оптимізації автоматизованої систем управління процесами теплопостачання із змінною структурою технічних засобів. Для цитування цієї статті: Брунеткін Олександр, Бабенко Олександр. Евристичне управління дискретними системами енергетики. International Science Journal of Engineering & Agriculture. Vol. 2, No. 6, 2023, pp. 65-72. doi: 10.46299/j.isjea.20230206.08 j j Надійшла до редакції: 01 листопада 2023 р.; Схвалено: 30 листопада 2023 р.; Опубліковано: 01 грудня 2023 р. Анотація: Розглянуто евристичне управління дискретними системами енергетики, в основу якого покладено цільову функцію оптимізаційного розв’язання задачі для системи теплопостачання із змінною структурою технічних засобів. Цільова функція поєднує в собі такі властивості як якість процесу, ефективність обладнання, його надійність і вартість ресурсів, що дозволяє комплексно оцінювати процес функціонування системи на основі техніко-економічних показників при обмеженнях на ресурси і визначати найкраще рішення у разі їх зміни. В основу математичної моделі для забезпечення управління системою теплопостачання по вибору структури об'єкта управління покладено використання генетичного алгоритму, що дозволяє досягти раціонального використання енергоресурсів і мінімізувати час розрахунків. Запропоновано три керуючі алгоритми пошуку оптимального рішення. Запропоновано комплексний критерій оптимального управління структурою системи на основі техніко-економічних показників. Розроблено кілька варіантів керуючих алгоритмів, що визначають оптимальну структуру технічних засобів, використовуючи генетичний алгоритм. Кожен із варіантів має свої особливості. Якщо у першому варіанті реалізовано релейне управління обладнанням та введення обмежень на ресурси та значення вагових коефіцієнтів критерію управління здійснює оператор, то у другому — їх значення автоматично оцінюється на основі наявності енергоресурсів з урахуванням їх майбутніх надходжень, а також непередбачених подій, що впливають на роботу системи теплопостачання. У третьому варіанті під час виконання управління за попередньо розрахованим планом паралельно ведеться розрахунок наступного плану з урахуванням зміни потужності устаткування. Для зменшення кількості перемикань обладнання при оптимізації структури системи розроблено четвертий алгоритм, в якому управління здійснюється не лише зміною структури системи, а й зміною завдань регуляторів теплопередаючих технічних засобів. Ключові слова: Евристичне управління, керування зміною структури, критерій оптимального керування, генетичний алгоритм, система теплопостачання, цільова функція Брунеткін Олександр та ін.: Евристичне управління дискретними системами енергетики 66 5. Метод формування плану перемикання обладнання Складність вирішення завдань формування плану переключення обладнання передбачається тим, що немає чіткої залежності оптимальної структури системи від її поточного і необхідного стану. Так як цільова функція може досягати різних альтернативних структур системи, в кожен момент ми вимагаємо необхідності вибору, наслідки якого можна оцінити тільки в майбутньому. Беремо до уваги, що система теплопостачання складається з трьох об'єктів (два газових котли і ТНУ), кожен з яких може бути або включений, або виключений, отримуємо 2 3 = 8 можливих наявних систем в кожен момент часу. Відповідно безліч з N інтервалів дає нам можливі плани переключення обладнання. Очевидно, що простий перебір усіх варіантів може зайняти занадто багато часу. Таким чином, виникає питання, як для прийнятного часу знайти оптимальне або близьке до оптимального рішення. Можна провести визначені аналогії між вирішуваною задачею і задачами планування розподілу ресурсів. Найбільш близькою за змістом є задача про створення раціону [1]. р у р ур р р р у [ ] Отже, формуємо основні властивості вирішуваної задачі: Отже, формуємо основні властивості вирішуваної задачі: Отже, формуємо основні властивості вирішуваної задачі: р у р у езна кількість альтернативних рішень; — величезна кількість альтернативних рішень; — величезна кількість альтернативних рішень; — немає інформації про залежність плану переключення обладнання від поточного стану системи; — цільова функція може мати безліч локальних екстремумів. — цільова функція може мати безліч локальних екстремумів. — цільова функція може мати безліч локальних екстремумів Аналіз різних літературних джерел [2,3,4,5,6] показав, що для вирішення подібних завдань добре використовуються генетичні алгоритми. Тому було вирішено використовувати їх для отримання плану переключення обладнання, достатньо близького до оптимального з точки зору обраного критерію дискретності функція , аргументами якої є плани перемикання технічних засіб х протягом t . В остаточній формі цільова функція відбувається вигляду : 𝐽(𝑥; 𝑡) = √𝑤𝑟𝑅н 2(𝑥; 𝑡) + 𝑤𝑞𝑄н 2(𝑥; 𝑡) + 𝑤𝑠𝑆н 2(𝑥; 𝑡) + 𝑤𝑒𝐸н 2(𝑥; 𝑡) (1) (1) 𝐽(𝑥; 𝑡) = √𝑤𝑟𝑅н 2(𝑥; 𝑡) + 𝑤𝑞𝑄н 2(𝑥; 𝑡) + 𝑤𝑠𝑆н 2(𝑥; 𝑡) + 𝑤𝑒𝐸н 2(𝑥; 𝑡) (1) де: 𝑤𝑟, 𝑤𝑞, 𝑤𝑠, 𝑤𝑒 – вагові коефіцієнти, які приймаються експертним методом; R(t) – ймовірністю відмови системи; Q(t) – якістю підтримувати заданої температура; S(t) – вартістю енергоресурсів; E(t) – ефективністю перетворення енергії. При описі генетичних алгоритмів використовуються визначення, використані з генетики. Наприклад, мова іде про популяцію осіб, а в якості базових понять застосовуються ген, хромосома, генотип, фенотип, аллель. Також підходять цим термінам визначення з технічної лексики, зокрема, ланцюг, двоїчна послідовність, структура [7,8]. 4. Аналіз літератури Аналіз різних літературних джерел показав, що для вирішення подібних завдань добре використовувати евристичне управління дискретними системами. Теоретичні дослідження задач оптимізації проводили В.М. Глушков, А.Г. Івахненко, Р. Белман, Л.С. Понтрягін, Л.А. Барський, В.З. Козін, А.С. Давідкович та ін. Розв’язанню оптимізаційних задач в галузі теплоенергетики присвятили свої роботи Е.В. Сарнацький, Р.Р. Авезов, Р.В. Жесан, С.П. Плєшков [2,3,4,5,6]. Сучасні наукові досягнення довели, що розв’язання завдань теплопостачання може бути отримано шляхом використання взаємозамінного обладнання, яке працює незалежно одне від одного при паралельному підключенні. Це робить доцільним об'єднання його в рамках єдиної системи для отримання оптимального співвідношення якості продукції при мінімізації вартості витрачених ресурсів за рахунок постійної зміни структури як теплогенеруючих, так і теплопередаючих технічних засобів [2,3,4]. Проведений аналіз показав, що під оптимальним керуванням розуміється керування структурою регулятора і оптимізацією його поточних налаштувань. Аналіз літературно- патентних джерел показав, що дослідники в галузі автоматизації звернули увагу на завдання керування зміною структури технічних засобів порівняно давно, на етапі класифікації автоматизованих систем, однак у відкритій літературі не виявлено ніяких алгоритмів керування структурою складної системи для підвищення ефективності [5,6]. International Science Journal of Engineering & Agriculture 2023; 2(6): 65-72 67 5. Метод формування плану перемикання обладнання Дуже важливим поняттям в генетичних алгоритмах вважається функція пристосовності, інакше звана функцією оцінки. Вона представляє міру здатності даної особи в популяції. Ця функція відіграє головну роль, оскільки дозволяє оцінити ступінь пристосовності окремих осіб у популяції та вибрати з них найбільш пристосовані (тобто мають найбільше значення функції пристосовності) відповідно до еволюційного принципу виживання «сильніших» (краще всього пристосованих). Функція пристосованості також отримала свою назву безпосередньо з генетики. Вона надає сильний вплив на функціонування генетичних алгоритмів і повинна мати точне і правильне визначення. У завданнях оптимізації функція пристосовності, як правило, оптимізується і називається цільовою функцією. В задачах мінімізації цільова функція перетворюється, і проблема зводиться до максимізації. В теорії управління функція пристосування може приймати вид функції погрішності, а в теорії ігор – функції вартості. На кожній ітерації генетичного алгоритму пристосованість особи кожної даної популяції оцінюється за допомогою функцій працездатності, і на цій основі створюється наступна Брунеткін Олександр та ін.: Евристичне управління дискретними системами енергетики 68 популяція осіб, складових безліч потенційних рішень проблем, наприклад, завдань оптимізації [9,10]. Чергова популяція в генетичному алгоритмі називається поколінням, а до знову створеної популяції особи застосовується термін «нове покоління» або «покоління потомків». Метод пошуку найкращого рішення у вигляді класичного генетичного алгоритму наступний. Шаг 1. Початок, введення даних. , ізація, вибір вихідної популяції хромосом. Шаг 2. Ініціалізація, вибір вихідної популяції хромосом. Шаг 3. Оцінка здатності хромосом в популяції. Шаг 4. Перевірка умов пристосовності хромосоми, якщо умова виконана, здійснюємо відбір кращої за критерієм хромосоми і завдання пошуку вважається виконане, кінець. Шаг 5. Умову не виконано, відсутні пристосовані хромосоми. Шаг 4. Перевірка умов пристосовності хромосоми, якщо умова виконана, здійснюємо відбір кращої за критерієм хромосоми і завдання пошуку вважається виконане, кінець. Шаг 5. Умову не виконано, відсутні пристосовані хромосоми. Шаг 4. Перевірка умов пристосовності хромосоми, якщо умова виконана, здійснюємо відбір кращої за критерієм хромосоми і завдання пошуку вважається виконане, кінець. Шаг 5 Умову не виконано відсутні пристосовані хромосоми Шаг 5. Умову не виконано, відсутні пристосовані хромосоми. Шаг 6. Продовжуємо селекцію. Шаг 6. Продовжуємо селекцію. Шаг 7. Застосовуємо генетичні оператори. Шаг 8. Створюємо нову популяцію хромосом, з новими властивостями. . Створюємо нову популяцію хромосом, з новими властивостями. Шаг 9. Переходим на Шаг 3. Розглянемо застосування генетичного алгоритму для вирішення завдань складання плану переключення обладнання. Перш за все, необхідно закодувати своє рішення у вигляді послідовності генів. У кожен момент часу система може знаходитися в одному з 8-ми різних станів, що визначають комбінації включеного обладнання. 5. Метод формування плану перемикання обладнання Приймемо, що 1 відповідає ввімкненому складу одиниць обладнання, а 0 — вимкнутому, тому можна скласти наступну таблицю кодування наявних систем. Очевидно, що в двійковій системі стану системи теплопостачання відповідні числа від 0 до 7. Якщо прийняти, що кожен ген буде відповідати інтервалу часу. Наприклад, ген, що має значення 3, буде відповідати подвійному представленню 011, що означає вимкнуто котел №1, і ввімкнене обладнання, котел №2 і ТНУ. Таблиця 1. Таблиця кодування станів системи. Код стану Котел №1 Котел №2 ТНУ 0 0 0 0 1 0 0 1 2 0 1 0 3 0 1 1 4 1 0 0 5 1 0 1 6 1 1 0 7 1 1 1 Послідовність генів утворює хромосому. Довжина хромосоми визначається кількістю N інтервалів. Таким чином, послідовність генів в будь-якій хромосомі являє собою план переключення обладнання. Пошук структури методом генетичного алгоритму починається з формування початкової популяції хромосом. У цьому випадку ця популяція складалася з 10 хромосом і формується випадковим чином [11,12]. Для оцінки працездатності кожної хромосоми використовувався вираз (1). Для цього моделювалася робота системи відповідно до плану перемикання обладнання, даної конкретної хромосоми, і розраховувалися всі необхідні компоненти. Критерій зупинки алгоритму це умова незмінності найбільш пристосованої хромосоми протягом 50 циклів. Проведений аналіз за викладеним методом підтверджує, що генетичні алгоритми дозволяють знаходити у величезній кількості альтернативних прийнятних рішень за розумний час. Так навіть при International Science Journal of Engineering & Agriculture 2023; 2(6): 65-72 69 69 N=1000 середній час пошуку рішення склало всього 500 с за цей час, як повний перебір усіх варіантів зайняв би 3,210996 с. N=1000 середній час пошуку рішення склало всього 500 с за цей час, як повний перебір усіх варіантів зайняв би 3,210996 с. Головний же висновок полягає в тому, що, вибрав конкретну конфігурацію ЕОМ, призначену для моделювання, і задаючись прийнятним часом пошуку рішення, залежно від рис. 1 можна визначити максимальне значення параметра N . З іншої сторони представляється доцільним проводити моделювання на період, що перевищує тривалість самого тривалого перехідного процесу системи. В наслідок цього, розділив на величину N знайдену на рис. 1 можна визначити значення інтервалу 𝛥𝜏: З іншої сторони представляється доцільним проводити моделювання на період, що перевищує тривалість самого тривалого перехідного процесу системи. В наслідок цього, розділив на величину N знайдену на рис. 1 можна визначити значення інтервалу 𝛥𝜏: 𝛥𝜏≤ 𝜏пп 𝑁. (2) (2) 𝛥𝜏≤ 𝜏пп 𝑁. Так, наприклад, вибрав максимальний час пошуку рішення рівний 120 секундам на рис. 1 знаходимо N ≈ 500. 5. Метод формування плану перемикання обладнання З попередніх досліджень встановлено, що максимальна тривалість переходного процесу в системі теплопостачання становить приблизно 9500 с. В наслідок цього: 𝛥𝜏≤ 9500 500 = 19 с. Ґрунтуючись на отриманому результаті, подальше моделювання системи теплопостачання проводилося при 𝛥𝜏= 15 с. 6. Результати розробки керуючих алгоритмів системи У ході роботи по створенню керуючого алгоритму були запропоновані різні варіанти, що відрізняються складністю і потенційними можливостями. Управляючий варіант алгоритму 1. Це простий варіант алгоритму, який у подальшому був використаний у процесі імітаційного моделювання. Послідовність дій наступна. Шаг. 1. Введення вихідних даних. Значення ваги, що визначають і кількість пріоритетних ресурсів. Шаг. 1. Введення вихідних даних. Значення ваги, що визначають і кількість пріоритетних ресурсів. ур Шаг. 2. Формування плану переключення обладнання генетичним алгоритмом. Шаг. 3. Реалізація плану. Шаг. 3. Реалізація плану. Шаг. 4. Перевірка умов реалізації плану, якщо план не реалізований перехід на Шаг 3. і і б Шаг. 5. План реалізований, переходимо на перевірку умов завершення роботи, якщо немає, то переходимо на Шаг 1. , р Шаг. 6. Робота завершена. Вихід із алгоритму. р Шаг. 6. Робота завершена. Вихід із алгоритму. 0 100 200 300 400 500 600 700 800 900 0 500 1000 1500 N t, с Рис. 1. Залежність середнього часу пошуку рішення від кількості інтервалів N. Рис. 1. Залежність середнього часу пошуку рішення від кількості інтервалів N. Для простого алгоритму можна представити у вигляді нескінченого циклу. У цьому випадку введення обмежених ресурсів і значення вагових коефіцієнтів критеріїв оптимального управління здійснює оператор. Цей алгоритм може бути використаний для реалізації 70 Брунеткін Олександр та ін.: Евристичне управління дискретними системами енергетики автоматизованого управління або ж як система підтримки прийняття рішень. Хоча перший варіант керуючого алгоритму досить точний для проведення імітаційного моделювання системи теплопостачання, з практичної точки зору він малопридатний [13,14]. Управляючий алгоритм варіант 2. Необхідно виключити людський фактор на кожному кроку алгоритму. Тому був розроблений більш інтелектуальний варіант керуючого алгоритму, який дозволяє автоматично оцінювати значення вагових коефіцієнтів складових критеріїв (1) на підставі поточної наявності первинних енергоресурсів, їх майбутніх поступлень, а також усіх непередбачених флуктуацій, що впливають на роботу параметрів системи. Послідовність дій наступна. 1. Введення вихідних даних. Визначення доступних ресурсів. Шаг. 1. Введення вихідних даних. Визначення доступних ресурсів. 2. Рішення задачі розподілу ресурсів. Визначення кількості кожного ресурсу н рацію. у у р Шаг. 3. Формування плану переключення обладнання генетичним алгоритмом без урахування кількості ресурсів. Визначення дефіциту кожного ресурсу. Шаг. 4. Визначення дефіциту кожного ресурсу. д ф ц у р ур у Шаг. 5. Зміна вагових коефіцієнтів критеріїв оптимального управління пропорційно величині відповідного дефіциту ресурсу. ф у р ур у Шаг. 6. Формування уточненого плану переключення обладнання генетичним алгоритмом з урахуванням кількості ресурсів. Шаг. 7. План реалізації. р 8. Перевірка умов реалізації плану, якщо план не реалізований перехід на Шаг 6. 6. Результати розробки керуючих алгоритмів системи Шаг. 8. Перевірка умов реалізації плану, якщо план не реалізований перехід на Шаг 6. Шаг. 9. Реалізований план переходу на введення змін у кількість доступних ресурсів у результаті виникнення зовнішніх і внутрішніх збурень перевірку переходимо на Шаг 2. Шаг. 10. План реалізовано, збурення відсутні. Вихід із алгоритму. Шаг. 10. План реалізовано, збурення відсутні. Вихід із алгоритму. Перевага даного алгоритму полягає в тому, що тільки в системі спостерігається дефіцит будь-якого ресурсу, відбувається збільшення відповідних вагових компонентів в критеріях управління, що, в свою чергу, приводить до зниження поточних витрат ресурсу. З запропонованого підходу в цьому алгоритмі реалізована спроба об'єднання АСУП і АСУ ТП. На верхньому рівні вирішується класична задача планування розподілу ресурсів. На нижньому — приймається рішення про те, як слід переключити конкретне обладнання для того, щоб досягти оптимального значення цільової функції по використанню ресурсів. Управляючий алгоритм варіант 3. Далі відзначимо, що в запропонованих алгоритмах основні розрахункові витрати проходять в момент формування плану. Після того, як сформований план, розрахункова потужність ЕОМ не використовується. У підсумку ми тратимо кілька хвилин на формування плану, який реалізується протягом кількох годин. З метою задіяння вимірювальних і розрахункових можливостей ЕОМ в процесі виконання плану була запропонована наступна концепція [15]. Припустимо, що обладнання системи теплопостачання працює не в релейному режимі, а на різних рівнях потужності. Для визначеності приймемо, що такі рівні 5: 0, 25, 50, 75 і 100%. З розрахункової точки зору завдання пошуку оптимального плану істотно ускладнюється, так як кількість варіантів тепер (53)N =125N. З іншої сторони, маневрування потужності дозволить більш гнучко використовувати ресурси всередині системи теплопостачання. Крім того, як уже було сказано, є великий потенціал не використаного часу роботи ЕОМ. Тому перспективним представляється гібридний підхід. На початку формується попередній план переключення обладнання виходячи з того, що воно працює в релейному режимі. Далі, поки цей план реалізується, паралельно формується поточний план, який передбачає роботу обладнання на різних рівнях потужності. Таким чином, з'являється можливість більш гнучкого управління, що покращує ефективність. International Science Journal of Engineering & Agriculture 2023; 2(6): 65-72 71 7. Висновки Вернадського. Серія: Технічні науки, 2021, Том 32 (71) Ч. 1 № 2 с. 170 – 179. р у , ( ) 4) Максимов М.В., Максимова О.Б., Мінчев Д.С. Методи та моделі управління системою зі зміненою структурою об'єктів теплопостачання. Вчені записки ТНУ імені В.І. Вернадського. Серія: Технічні науки, 2021, Том 32 (71) Ч. 1 № 2 с. 170 – 179. р у , , ( ) 5) Давидов В.О. аналіз надійності ПО АСУТП / В. О. Давидов, О.Б. Максимова, А.А. Шевчук // Холодильна техніка і технологія. — Одесса, 2011. — Вип . 6(134). — С. 86 — 91. р у , , ( ) 5) Давидов В.О. аналіз надійності ПО АСУТП / В. О. Давидов, О.Б. Максимова, А.А. Шевчук // Холодильна техніка і технологія. — Одесса, 2011. — Вип . 6(134). — С. 86 — 91. 6) Проектування спеціалізованих інформаційно-вичислювальних систем: Учеб. пособие по спец. ЕОМ і АСУ/ Смирнов Ю.М., Воробьов Г.Н., Потапов Е.С., Сюзев В.В.; Під ред. Смирнова Ю.М. — М.: Вищ . шк ., 1984. — 359 с. 6) Проектування спеціалізованих інформаційно-вичислювальних систем: Учеб. пособие по спец. ЕОМ і АСУ/ Смирнов Ю.М., Воробьов Г.Н., Потапов Е.С., Сюзев В.В.; Під ред. Смирнова Ю.М. — М.: Вищ . шк ., 1984. — 359 с. 7) Рутковская Д., Пилиньский М., Рутковский Л. Нейронные сети, генетические алгоритмы и нечеткие системы: пер. с польск. И.Д. Рудинского . — М.: Горяча лінія — Телеком, 2006. — 452 с. 7) Рутковская Д., Пилиньский М., Рутковский Л. Нейронные сети, генетические алгоритмы и нечеткие системы: пер. с польск. И.Д. Рудинского . — М.: Горяча лінія — Телеком, 2006. — 452 с. 8) Ammar, S. Characteristics and features of a performance evaluation model using a multilevel fuzzy rule-based system [Текст] / S. Ammar, R. Wright. // International Journal of Technology, Policy and Management, Vol. 3 No. 3/4, 2003. – P. 301-321. 8) Ammar, S. Characteristics and features of a performance evaluation model using a multilevel fuzzy rule-based system [Текст] / S. Ammar, R. Wright. // International Journal of Technology, Policy and Management, Vol. 3 No. 3/4, 2003. – P. 301-321. y g 9) Cricelli, L. Modelling the competition of an HNO versus an MVNO in the mobile telecommunications industry [Текст] / L. Cricelli, M. Grimaldi and N. L. Ghiron // International Journal of Technology, Policy and Management, Vol. 9 No. 3, 2009.– P. 277-295. y g 9) Cricelli, L. 7. Висновки 1. Вперше запропонований комплекс алгоритмів управління системою теплопостачання зі зміненою структурою технічних засобів, який дозволяє реалізувати оптимальне управління за рахунок зміни структури об'єкта управління. 1. Вперше запропонований комплекс алгоритмів управління системою теплопостачання зі зміненою структурою технічних засобів, який дозволяє реалізувати оптимальне управління за рахунок зміни структури об'єкта управління. 2. Пошук оптимального рішення в розробленому комплексі керуючих алгоритмів здійснюється з використанням методу пошуку оптимуму «генетичний алгоритм», який дозволить у фактично без кінцевій множині варіантів знайти рішення, близьке до оптимального за припустимий часовим інтервалом. р у р 3. Пошук оптимального рішення залежить від обмеженості ресурсів, які можуть задаватися оператором, або розраховуватися системою управління на базі інформації про плановану кількість і час надходження ресурсів. 3. Пошук оптимального рішення залежить від обмеженості ресурсів, які можуть задаватися оператором, або розраховуватися системою управління на базі інформації про плановану кількість і час надходження ресурсів. 4. Запропонований комбінований алгоритм пошуку оптимального рішення, який полягає в поєднанні швидко розрахованого попереднього рішення, заснованого на релейному режимі регулювання обладнання з наступним точним рішенням, розрахованого у процесі реалізації попереднього рішення, і заснованого на припущеннях щодо можливості регулювання потужності обладнання. Список літератури: 1) Максимова О.Б. Методика розрахунку критеріїв управління структурою складних технічних систем / О.Б. Максимова, В.О. Давидов, В.М. Тонконогий // Вісник Сумського державного університету. Серія Технічні науки. — 2011. — №3. — С. 19-23. Список літератури: С со л ера ур : 1) Максимова О.Б. Методика розрахунку критеріїв управління структурою складних технічних систем / О.Б. Максимова, В.О. Давидов, В.М. Тонконогий // Вісник Сумського державного університету. Серія Технічні науки. — 2011. — №3. — С. 19-23. 2) Максименко І.М. Розробка принципів цілорічного управління установкою теплопостачання на основі методів об'єктно-орієнтованого аналізу / И. Н. Максименко, Ю. К. Тодорцев // Вісник Одеської державної академії будівництво та архітектури . — Вип . 17. — 2005. — С. 251 — 253. 2) Максименко І.М. Розробка принципів цілорічного управління установкою теплопостачання на основі методів об'єктно-орієнтованого аналізу / И. Н. Максименко, Ю. К. Тодорцев // Вісник Одеської державної академії будівництво та архітектури . — Вип . 17. — 2005. — С. 251 — 253. 3) Максимова О.Б. Исследование адаптивних самоорганізующихся автоматизованих систем забезпечення комфортной температури / О.Б. Максимова, В.О. Давыдов , В.М. Тонконогий // Тр . Одес . політехн . ун -та. — Одесса, 2011. — Вип . 2(36). — С. 155-160. р у , ( ) 4) Максимов М.В., Максимова О.Б., Мінчев Д.С. Методи та моделі управління системою зі зміненою структурою об'єктів теплопостачання. Вчені записки ТНУ імені В.І. 7. Висновки Control Eng., 2000, 214. – P. 469-480 14) Dixon, R. The ALSTOM benchmark challenge on gasifier control [Текст] / R. Dixon, A. Pike, and M. Donne // Proc. Inst. Mech. Eng. I, J. Syst. Control Eng., 2000, 214. – P. 389-394. 14) Dixon, R. The ALSTOM benchmark challenge on gasifier control [Текст] / R. Dixon, A. Pike, and M. Donne // Proc. Inst. Mech. Eng. I, J. Syst. Control Eng., 2000, 214. – P. 389-394. 15) Taylor, C. J. Proportional-integral-plus (PIP) control of the ALSTOM gasifier problem [Текст] / C. J. Taylor, A. P. McCabe, P. C. Young, and A. Chotai // IMECHE Proc., J. Syst. Control Eng., 2000, 214. – P. 469-480 15) Taylor, C. J. Proportional-integral-plus (PIP) control of the ALSTOM gasifier problem [Текст] / C. J. Taylor, A. P. McCabe, P. C. Young, and A. Chotai // IMECHE Proc., J. Syst. Control Eng., 2000, 214. – P. 469-480 Heuristic control of discrete power systems Oleksandr Brunetkin Department of Software and Computer-Integrated Technologies, Odesa Polytechnic National University, Odesa, Ukraine ORCID 0000-0002-6701-8737 7. Висновки Modelling the competition of an HNO versus an MVNO in the mobile telecommunications industry [Текст] / L. Cricelli, M. Grimaldi and N. L. Ghiron // International Journal of Technology, Policy and Management, Vol. 9 No. 3, 2009.– P. 277-295. gy y g 10) Kondratenko, Y. P. Modelling and optimisation of tanker’s cargo process in real fuzzy conditions [Текст] / Y. P. Kondratenko, L. P. Klymenko, V. Y. Kondratenko and D. M. Pidopryhora // Lectures on Modeling and Simulation. AMSE Periodicals, Series A, Vol. 9, Palma de Majorca, Balearic Islands, Spain, 2008.– P. 122-133. 10) Kondratenko, Y. P. Modelling and optimisation of tanker’s cargo process in real fuzzy conditions [Текст] / Y. P. Kondratenko, L. P. Klymenko, V. Y. Kondratenko and D. M. Pidopryhora // Lectures on Modeling and Simulation. AMSE Periodicals, Series A, Vol. 9, Palma de Majorca, Balearic Islands, Spain, 2008.– P. 122-133. Брунеткін Олександр та ін.: Евристичне управління дискретними системами енергетики 72 11) Zadeh, L. A. Fuzzy sets [Текст] / L. A. Zadeh // Information and Control. – Vol. 8, 1965. – P. 338-353. 11) Zadeh, L. A. Fuzzy sets [Текст] / L. A. Zadeh // Information and Control. – Vol. 8, 1965. – P. 338-353. 11) Zadeh, L. A. Fuzzy sets [Текст] / L. A. Zadeh // Information and Control. – Vol. 8, 1965. – P. 338-353. 12) Zimmermann, H. J. Fuzzy Set Theory and Its Applications [Текст] / H. J. Zimmermann. – Kluwer Academic Publishers, Boston/Dordrecht/London, 1992. 12) Zimmermann, H. J. Fuzzy Set Theory and Its Applications [Текст] / H. J. Zimmermann. – Kluwer Academic Publishers, Boston/Dordrecht/London, 1992. 13) Ротштейн, А. П. Интеллектуальные технологии идентификации: нечеткие множества, генетические алгоритмы, нейронные сети. [Текст] / А. П. Ротштейн. – Винница: "УНІВЕРСУМ-Вінниця", 1999. – 300 с 13) Ротштейн, А. П. Интеллектуальные технологии идентификации: нечеткие множества, генетические алгоритмы, нейронные сети. [Текст] / А. П. Ротштейн. – Винница: "УНІВЕРСУМ-Вінниця", 1999. – 300 с ) , у ф , генетические алгоритмы, нейронные сети. [Текст] / А. П. Ротштейн. – Винница: "УНІВЕРСУМ-Вінниця", 1999. – 300 с 14) Dixon, R. The ALSTOM benchmark challenge on gasifier control [Текст] / R. Dixon, A. Pike, and M. Donne // Proc. Inst. Mech. Eng. I, J. Syst. Control Eng., 2000, 214. – P. 389-394. 15) Taylor, C. J. Proportional-integral-plus (PIP) control of the ALSTOM gasifier problem [Текст] / C. J. Taylor, A. P. McCabe, P. C. Young, and A. Chotai // IMECHE Proc., J. Syst. Oleksandr Babenko Oleksandr Babenko Department of Software and Computer-Integrated Technologies, Odesa Polytechnic National University, Odesa, Ukraine ORCID 0009-0009-9197-1446 Oleksandr Babenko Department of Software and Computer-Integrated Technologies, Odesa Polytechnic National University, Odesa, Ukraine ORCID 0009-0009-9197-1446 Abstract: The heuristic management of discrete energy systems is considered, which is based on the objective function of the optimization solution of the problem for the heat supply system with a variable structure of technical means. The objective function combines such properties as the quality of the process, the efficiency of the equipment, its reliability and the cost of resources, which allows you to comprehensively evaluate the process of functioning of the system on the basis of technical and economic indicators with restrictions on resources and to determine the best solution in case of their change. The basis of the mathematical model for ensuring the control of the heat supply system by choosing the structure of the control object is the use of a genetic algorithm, which allows achieving rational use of energy resources and minimizing calculation time. Three control algorithms for finding the optimal solution are proposed. A comprehensive criterion for optimal management of the system structure based on technical and economic indicators is proposed. Several variants of control algorithms have been developed that determine the optimal structure of technical means using a genetic algorithm. Each of the options has its own characteristics. If in the first option relay control of the equipment is implemented and the introduction of restrictions on resources and the value of the weighting factors of the control criterion is carried out by the operator, then in the second option their value is automatically estimated based on the availability of energy resources, taking into account their future receipts, as well as unforeseen events affecting the operation of the system heat supply In the third option, during the execution of control according to a previously calculated plan, the calculation of the next plan is carried out in parallel, taking into account the change in the capacity of the equipment. In order to reduce the number of equipment switches while optimizing the system structure, a fourth algorithm was developed, in which control is carried out not only by changing the system structure, but also by changing the tasks of heat transfer technical means regulators. y y g g g Keywords: Heuristic control, structure change control, optimal control criterion, genetic algorithm, heat supply system, objective function
https://openalex.org/W2890418226	https://socialwork.journals.ac.za/pub/article/download/649/599	English	null	VIOLATION OF HUMAN RIGHTS OF OLDER PERSONS IN SOUTH AFRICA. THE CASE OF LAVELA OLD AGE CENTRE, NTSELAMANZI, EASTERN CAPE PROVINCE, SOUTH AFFRICA	Social work	2,018	cc-by	8,013	Social Work/Maatskaplike Werk Vol 54 No 3; Issue 2 http://socialwork.journals.ac.za/pub doi:http://dx.doi.org/10.15270/54-3-649 Social Work/Maatskaplike Werk Vol 54 No 3; Issue 2 http://socialwork.journals.ac.za/pub doi:http://dx.doi.org/10.15270/54-3-649 Social Work/Maatskaplike Werk Vol 54 No 3; Issue 2 http://socialwork.journals.ac.za/pub doi:http://dx.doi.org/10.15270/54-3-649 VIOLATION OF HUMAN RIGHTS OF OLDER PERSONS IN SOUTH AFRICA. THE CASE OF LAVELA OLD AGE CENTRE, NTSELAMANZI, EASTERN CAPE PROVINCE, SOUTH AFFRICA Social Work/Maatskaplike Werk Vol 54 No 3; Issue 2 http://socialwork.journals.ac.za/pub doi:http://dx.doi.org/10.15270/54-3-649 Social Work/Maatskaplike Werk Vol 54 No 3; Issue 2 http://socialwork.journals.ac.za/pub doi:http://dx.doi.org/10.15270/54-3-649 Simon Kang’ethe There is no doubt that older persons face an environment bereft of resources, rights and dignity. This study was explorative and descriptive in design, with data collected from in-depth discussions with the elderly. Findings revealed diametrically opposed factors, with some older persons happily accessing an array of psychosocial benefits, while some had their grants mishandled or complained of the inadequacy of the grant. Others felt their rights to housing were largely being compromised. This paper recommends that the government prioritise the older persons’ state of housing and provide funding for old age centres to ensure that they become sources of psychosocial benefit for elderly members of society. Social Work/Maatskaplike Werk 2018:54(3) Social Work/Maatskaplike Werk 2018:54(3) BACKGROUND AND INTRODUCTION Aging is an entropic disorder that everyone has to prepare for, and contend with, as it affects all the body’s functionalities, whether social, physical, psychological or emotional (Magill, 1996 cited in Kang’ethe, 2009). The phenomenon of aging appears to have a larger effect in the developing world than the developed world. Tran (2012) contends that population aging, although a global phenomenon, is progressing faster in impoverished countries. This means that developing countries need to rethink and redirect resources to tackle aging if they are to uphold the rights of their elderly citizens. Despite the United Nations not adopting a standard criterion for inclusion in the older person category, generally, from age 60 onwards is used to refer to the older population (Bohman, Van Wyk & Ekman, 2009). While most countries in the developed world have accepted the chronological age of 65 years as an acceptable definition of an older person, developing countries peg the criterion with the age at which one can begin to receive pension benefits (Tangwe & Gutura, 2013). Despite the phenomenon of aging presenting both perfidious and pernicious circumstances, many researchers underscore the older person’s contribution to society (Madonsela, 2008; Lombard and Kruger, 2009), with caregiving, counselling and advisory services, inter alia, being key roles. In many African societies older people constitute the council of elders and society expects them to steer their communities onto the right path (Kang’ethe & Rhakudu, 2010). Importantly, in many developing countries, grandmothers are the primary nurturers and caregivers (Bohman, Van Wyk & Ekman, 2009). The elderly are revered as the vanguards of morality, a phenomenon most African countries need to rethink as societies appear to be slowly drifting into a bottomless pit of moral decadence (Kang’ethe & Khayundi, 2014). Historically, the elderly were treated with dignity (Afolayan, 2004). This was important for these societies as it was believed that they ushered in blessings to the younger members of their communities. Individuals who abandoned or failed to take good care of their older family or community members were frowned upon by society. According to African traditional religion, it was believed that such people were cursed by the societal deities and that they would find it difficult to succeed in life (Afolayan, 2004). Social Work/Maatskaplike Werk 2018:54(3) 283 VIOLATION OF HUMAN RIGHTS OF OLDER PERSONS IN SOUTH AFRICA. THE CASE OF LAVELA OLD AGE CENTRE, NTSELAMANZI, EASTERN CAPE PROVINCE, SOUTH AFFRICA Simon Kang’ethe BACKGROUND AND INTRODUCTION With the advent of modernisation, civilisation, migration to the cities, individualism and globalisation, as well as the change from socialistic life to a more capitalistic one, a money economy and a change from traditional governance to democratically elected governments, the role and place of the elderly has been shifting, with older people not being accorded the dignity and respect that they used to be afforded (Dhemba, 2013; Arowolo, 2010). According to the WHO (2002), neglect of the elderly constitutes deliberate withholding of food, personal and medical care, as well as failure to provide adequate shelter. Perhaps this is because some younger people consider older individuals a liability and a drain on taxpayers’ money (Batala, Gulwa, Gwebani & Rubushe, 2014). Today in South Africa there is evidence of an array of abuses meted to older persons. They also face various kinds of mistreatments and discrimination (Turok, 2006; Sargeant, 2007). Many youngsters use elderly women as maids to facilitate the upbringing of their children. Perhaps this phenomenon has been exacerbated by higher incidences of HIV/AIDS, demanding that they become caregivers for sick children and orphaned grandchildren (Lombard & Kruger, 2009). It needs to be said that the state and position of the elderly deserving respect is increasingly being eroded as modernisation, Westernisation, eurocentrism, migration and globalisation, cultural erosion and lifestyle changes, among an array of eclectic other factors, take root more deeply (Arowolo, 2010; Afolayan, 2004). Perhaps more needs to Social Work/Maatskaplike Werk 2018:54(3) 284 be done to advocate for the plight of the elderly and lobby societies to treat the aged with dignity and respect. The elderly are often the targets of abuse by both their children and their grandchildren, making them turn to the old age centres to get some warmth and dignity (Batala, et al., 2014). In South Africa older people are seen as soft targets by thieves aiming to either steal, or gain control of, their grant money. Many elderly people face dire financial hardship, largely because they have to share their meagre income with their intergenerational households (Lombard & Kruger, 2009). This has prompted many of them to move out to look for old age homes where they can live in peace and at least be afforded some psychosocial support. Benefits for these people are company, food and clothing, and having the opportunity to socialise with their peers. BACKGROUND AND INTRODUCTION There is sometimes the added bonus of access to recreational activities (Batala et al., 2014). It is good to note that the elderly are protected by the South African Constitution (Republic of South Africa, 1996 cited in Batala et al., 2014). The Constitution and the Bill of Rights enshrine the rights of all, irrespective of age, gender, sex, socio-economic position and culture (Department of Social Development (DSD), 2009). The elderly are protected in particular by the Older Person’s Act No. 13 of 2006 of South Africa. This act promotes the protection and wellbeing of older persons. South Africa is a signatory to the Madrid Plan of Action on aging (DSD, 2009), which is an indicator of the country’s stringent commitment to the welfare of the aged. To bolster the rights of the elderly, the country introduced the Social Assistance Amendment Act No. 6 of 2008, which allows men aged 63 to 64 to receive social grants. The provision of the Older Persons’ Home and Day Care Centres is proof that the government is committed to the rights of the aged (DSD, 2009). In this article the term ‘older persons’ and ‘the elderly’ have been used interchangeably. Problem Statement Old age is both entropic and pernicious, and presents various challenges for older individuals (Magill, 1996 cited in Kang’ethe, 2009). This is perhaps a result of their dependence and the attitudes of young people towards them. Among the older people linked to the Lavela Old Age Centre in Nzelamanzi village of Alice Township in the Eastern Cape is the perception that some older persons do not experience adequate care and/or love from their children and grandchildren. Usually poverty and its concomitant ramifications are a factor undermining the capability of their children to take care of them. Although the elderly have access to old age grants, they are often besieged by an array of challenges preventing them from utilising these grants for their own welfare. These grant recipients often have family members who take advantage of them and misuse their grants. The grant is also inadequate to meet an impoverished person’s basic needs. This researcher considers it pertinent to assess the perceptions of the elderly themselves regarding the environment in which they find themselves, and the respect and dignity, or lack thereof, accorded them by their children and others in their community. The outcome of such an analysis could be important to inform policy adjustments and improvements in the treatment and human rights of the elderly. STUDY AIM AND OBJECTIVES This study aimed to gain empirical insight into the human rights violations experienced by to older persons as seen through their eyes. Data were collected from the elderly linked to the Lavela Old Age Centre. This has provided insight into the different dynamics and perceptions of how the elderly persons’ rights to access an array of psychosocial support are violated and how they are generally treated by their close kin and the society they live in. The outcome could be important for policy makers who think that the elderly’s access to the old age grant is sufficient. Social Work/Maatskaplike Werk 2018:54(3) METHODS AND STUDY SETTINGS Conceptually, the research design is an architectural blueprint that the study follows (Creswell, 2014). The study was conducted according to a qualitative paradigm, approach and research design. The actual design used was that of a case study, where a few samples are richly interrogated in order to tap the subjects’ opinions, thinking, perceptions and insights pertaining to the phenomenon under study. In this case, assessing the perceptions of a few elderly people on how they perceive that the elderly are treated by their children and community members generally (Creswell, 2014). The design used was explanatory, descriptive and exploratory in nature. The aim of using this design is not to generalise the results, but to gain as much insight as possible into the phenomenon under study. This can then be a strong platform for additional research, either using a quantitative approach or mixed approaches. The researcher, complying with the ethos and principles of a qualitative approach, was the primary instrument for data collection and analysis (Creswell, 2014). This study utilised a semi-structured interview guide/schedule as a research instrument with open- ended questions that facilitated six in-depth interviews with elderly people. Conceptually, a research instrument is a platform of questions that the researcher utilises to elicit information from the research participants. The researcher believed he exhausted the samples to ensure he obtained adequate data to warrant the analysis. To collect data, this research applied a non-probability sampling methodology, but adopted a specifically purposive technique (Creswell, 2014). The study disregarded gender, but considered it pertinent to interview those who are 60 years and older and recipients of an old age grant. The researcher targeted the samples that were available and healthy enough to understand the questions posed by the researcher pertaining to the different abuses the elderly persons experienced at the hands of their children and other closer kin. Data collection was performed at the Lavela Old Age Centre in Nzelamanzi village of Alice Township in the Eastern Cape Province and was cross-sectional, implying that data were collected within a short period of time (De Vos, Strydom, Fouche and Delport, 2011). The number of elderly persons who frequent the centre varies from time to time and is largely determined by weather, with summer attracting as many as twenty, while during winter the number drops to as low as five. STUDY CONTEXTUALISATION The study has its roots a series of community engagement sessions that the researcher conducted for a period of three years (2015-2017) undertaken with the aim of psychosocially empowering the older persons of the Lavela Old Age centre. The older persons do an array of activities such as bead making, vegetable gardening, exercising and keeping one another company. The psychosocial empowerment Social Work/Maatskaplike Werk 2018:54(3) Social Work/Maatskaplike Werk 2018:54(3) 285 entailed bringing the older persons to share their life experiences, to share jokes and the unfortunate incidents they face such as various kinds of abuses they encounter at the hands of their children and other family members. Other psychosocial packages include encouraging the older persons to do some light vegetable gardening, cooking and eating together, and arranging and facilitating different professionals to visit and give them some educational talks. METHODS AND STUDY SETTINGS The researcher had prepared the Lavela pensioners with whom he had developed a rapport beforehand, as he frequently visits them as one of his bases for community engagement. This is to fulfil one of the core scholarly tasks that university lecturers have to carry out. The research therefore emanated from the outcomes of community engagement sessions. The data collection was conducted in IsiXhosa and the principal researcher, who cannot adequately comprehend and speak the language, sought the expertise of one of his research assistants, as well as a research mentee to carry out the interviews. The interviews were audiotaped and the proceedings interpreted later for the purpose of data analysis. The researcher, however, used his social work and research skills to assess the participants’ gestural temperaments as they engaged in in- depth interviews. On the unit of analysis, only six participants were involved in the in-depth interviews. The researcher considers the number to be sufficient because of the qualitative research approach that respects the magnitude of information generation more that the number of the samples involved (Creswell, 2014). The number is also considered adequate because the centre does not have a large membership. The number of people who frequent the centre rarely exceeds 20. Social Work/Maatskaplike Werk 2018:54(3) 286 DATA ANALYSIS Data analysis is a process of arranging and organising crude data into organised and consumable data that can be used to achieve the objectives of a study (Creswell, 2014). The research used a content thematic analysis method. This means the data are collected and refined and arranged into themes that inform the answers to the research questions. The content thematic analysis, informed by the work of Creswell (2014), entailed putting together piles and piles of crude data from the field, and then reorganising and sorting the data through the use of codes that gave rise to themes. To enrich the analysis, verbatim quotes and analogies in tandem with the researcher’s and his assistant’s interpretation of the participants’ temperaments were all used to inform the selection of themes. RESEARCH DOMAIN AND JUSTIFICATION OF CHOICE The data were collected from the participants in June 2015 at Lavela Old Age centre in Ntselamanzi village of Alice Township of Eastern Cape Province. The centre is around 3 to 5 km from the University of Fort Hare and Alice Township administrative centre. The researcher, through his community engagement sessions, sought insight into the perceptions of the members of the Lavela team regarding how they perceived the elderly were treated by their children and grandchildren and the wider community. He wanted to assess whether they felt their dignity was respected, or to what extent their rights as older persons were respected by others. As all the members who frequently attend the centre are older persons receiving an old age grant, the centre can be accepted as an appropriate research domain. ETHICAL AND LEGAL REQUIREMENTS This entails following all the requisite research protocols to ensure that the study is scientifically and administratively informed (Creswell, 2014; De Vos, et al., 2011). The researcher conceived the idea of this study as an outcome of community engagement sessions conducted in an attempt to psychosocially empower the elderly people linked to the Lavela Old Age Centre. The researcher and his assistant had letters from their head of department that allowed them to undertake community engagements. Therefore, no ethical clearance was required. The researcher had a good rapport with the elderly persons and, when they were asked to give written consent, they did so freely and offered to be research subjects. Pseudonyms were used to refer to the participants in order to protect their anonymity and maintain confidentiality. Social Work/Maatskaplike Werk 2018:54(3) Lavela Old Age Centre as a source of older persons’ psychosocial empowerment g p p y p Regarding the issue of how they perceived they are accorded dignity and their human rights are upheld, the participants gave immense credit to the Lavela Old Age Centre for enabling various aspects of their psychosocial empowerment, making them feel some sense of dignity. They indicated that the centre provided a forum for sharing life’s joys and sorrows with their peers and to encourage those besieged by challenges. It is a place where they can get some hot coffee and a free meal, and make contact with peers and friends. This makes them happier and satisfies some aspects on the lowest level of Maslow’s hierarchy of needs (Maslow, 1999 in Kang’ethe and Chivanga, 2015; Nyden, Petersson and Nystrom, 2003). The elderly also view the centre as a forum for education and hope amidst so many of life’s distractions. The visitors who came to the centre (such as the researcher and his assistant) endowed them with a sense of importance. The teaching/lessons/talks from various visitors make them happy and relieved boredom. This gives them motivation and make them widen their social networks and hence add happiness in their lives. This also widens the horizon of social capital (Singh & Srivastava, 2014; Kang’ethe, 2014). The following verbatim answers were given to interview questions: This centre, although it does not have water and electricity, is the only source of happiness some of use experience. God is good for giving us the centre. This centre, although it does not have water and electricity, is the only source of happiness some of use experience. God is good for giving us the centre. Each visitor comes with his/her lesson and teaching. We feel important that important people like you can come and spend time with the old people like ourselves. Each visitor comes with his/her lesson and teaching. We feel important that important people like you can come and spend time with the old people like ourselves. The centre makes us busy with our art and beadwork. We also spend time tending to our garden. We also spend time tending to our garden. Some us feed here and feel live is good. It is a forum for old persons’ friendly individuals to connect with us. We get various items such as clothing and food parcels. FINDINGS This section presents the results related to the attitudes, perceptions and insights of the six older persons who were interviewed. The piles of crude data were arranged into categories, using the code method, with the same thread of ideas. This led to the development of themes that formed the basis of the findings of this study. The first section of the findings will present the demographic characteristics of the six research participants. TABLE 1 DEMOGRAPHIC PROFILE OF THE PARTICIPANTS No Pseudonyms Age Gender Grant Recipient 1 Thanduxolo Bareng 69 Male Yes 2 Bala Baliso 70 Male Yes 3 Muthee 65 Female Yes 4 Pat 66 Female Yes 5 Jack 62 Male Yes 6 Esther 70 Female Yes DEMOGRAPHIC PROFILE OF THE PARTICIPANTS Social Work/Maatskaplike Werk 2018:54(3) Social Work/Maatskaplike Werk 2018:54(3) 287 The fact that three men and three women were interviewed for their insights pertaining to the abuses that the older persons were facing in the hands of their families presents a good coincidence that indicates the gender validity of the study. However, the researcher did not highlight the gender aspects of the participants and focused on their ability to provide relevant and data-rich information. Furthermore, their ages also added to the research validity in that they all qualified as older persons and were all grant recipients. The following table indicates the themes that emanated from data analysis. TABLE 2 TABLE OF EMERGENT THEMES Lavela Old Age Centre as a source of older persons’ psychosocial empowerment This indicates that the centre facilitates a lot of psychosocial activities for the elderly, such as sharing life’s challenges and joys, keeping them busy with artwork and gardening, and being educated by various visitors. The centre therefore helps the older people to sustain a sense of dignity and love. This finding is in agreement with the work of Batala et al., (2014) in Alice township in the Eastern Cape, who described an array of benefits that old age centres give to the elderly such as affording them company, food and clothing, and having the opportunity to socialise with their peers. If you report those taking the drugs, the police will break the oath of office by giving information to the drug takers, who will likely be onto the reporter’s neck. One then had better keep quiet. The culture of undisciplined youths contributes to denying the older persons their right to peace and dignity. The youths are responsible for the elderly citizens’ state of stress, apathy and despondency. Fighting drug and alcohol abuse is made more difficult by a lack of proficiency and professionalism among law enforcement agents. This mirrors the work of Makuyana (2018) in South Africa that points to the inefficiency of law enforcement officers in the war against both drug peddlers and abusers Social Work/Maatskaplike Werk 2018:54(3) Social Work/Maatskaplike Werk 2018:54(3) 288 Older persons treated with respect and dignity by their children/grandchildren Study findings indicated that some older persons are treated fairly by their children, especially those who are working. The fact that the older people enjoy visiting the old age centre does not mean they are maltreated by their children, but rather that they enjoy peer interaction and company, which has both psychosocial and recreational value for them. Some of the participants admitted they watch TV and enjoy other facilities that their children also enjoy. The following verbatim sentiments were expressed: Some older persons are treated with dignity and are given everything by their children. Some children know how to give their parents the rights and respect they deserve. children know how to give their parents the rights and respect they deserve. Some children do not have any problem with their parents, only that poverty makes the provision of their needs a difficult phenomenon and some older persons thinks they are disrespected. This is an indicator that most parents are accorded the requisite parental rights by their children and are treated with dignity, but that poverty disrupts the relationship between the older people and their children or grandchildren. This is evidence that societies still uphold the spirit of love and concern for the elderly. This is a demonstration of the younger generation embracing the spirit and philosophy of ubuntu (Afolayan, 2004). Older persons mistreated and mishandled by their children/grandchildren/significant others The study’s findings indicated that there are children and grandchildren who undermine and disrespect their older parents and grandparents. The situation is exacerbated by the culture of drug abuse among the youth, a phenomenon that makes the elderly very worried, stressed and bewildered. A participant associated the phenomenon of undisciplined youths with the fast dying culture of respect for older people. This is because of the unfortunate inclination of African societies increasingly to embrace euro- centrism or Westernisation at the expense of upholding and maintaining their cultures (Nomngcoyiya, 2018). The participants indicated they were afraid of reporting their drug-abusing children because the police have a tendency to protect the culprits who can then seek revenge. The following statements were made: Our youths are undisciplined and abuse drugs most of the time. We fear them as they verbally abuse us. The culture of abusing alcohol among the youths is too deeply entrenched in our societies today making them immensely irresponsible. Some are unashamedly abusing the aged individuals. It would be difficult to fight drug taking and abuse because the police are not serious. They protect the law breakers. If you report those taking the drugs, the police will break the oath of office by giving information to the drug takers, who will likely be onto the reporter’s neck. One then had better keep quiet. Inadequacy of the old age grant and mishandling by family members Findings indicated that the old age grant was not sufficient to meet the needs of the older persons and conflict ensued when their children claimed part of the grant money. This causes an environment of conflict between the children and their parents or grandparents. The respondents expressed the following sentiments: Social Work/Maatskaplike Werk 2018:54(3) Social Work/Maatskaplike Werk 2018:54(3) Social Work/Maatskaplike Werk 2018:54(3) 289 Why cannot the government give us enough money for the grant? We fight with our children and grandchildren because of the inadequacy of the grant. Most of our children and grandchildren are unashamedly asking us to give them our grant and we end up fighting. Some of us are even abused or have it taken away forcibly leaving us to succumb to hunger and starvation. The government should consider to award grants to our children who cannot find jobs. That can lessen the spate of conflicts and fights between them and ourselves. The scenario above indicates that older persons’ state of conflict is driven by the desperation and despondency emanating from the poverty of their children and grandchildren. This calls for the government to come up with poverty-alleviation programmes for the youth. Unequivocally, this state of conflict between the elderly and their children is a symptom and a reflection of a serious state of unemployment among the youth in South Africa, making them desperate and despondent to the extent of targeting the grants of elderly individuals (Gutura & Tanga, 2014). Older people’s right to housing largely compromised Some participants bemoaned the fact that elderly citizens generally live a substandard life. One participant led the researcher and his assistant to her one-roomed prefabricated house, where planks were falling off, and she indicated that life, especially during winter, was harsh. She also revealed that she has been living there since 1985 and the promise to give her another house has long since been forgotten. This participant also revealed that she uses a pit latrine. The following sentiments were expressed: The government hoodwinked us and it got its way. Imagine since 1985, this is the house I have been living with children who have grown and moved out. Look at this prefab room we call a house. Even a thief cannot struggle to find their way inside. We are living in shambles for decades. This is the life we know and internalised. Perhaps we will die in the same situation. We are living in shambles for decades. This is the life we know and internalised. Perhaps we will die in the same situation. This kind of living cannot have any dignity. Imagine the parents and adolescent children living in this one-roomed house. Where is dignity? Where are the rights of the older persons to housing? Because of the nature of this house (prefab one-roomed house), we are competing for space with very big rats from the bush. The scenario above presents the horrifying state of affairs in which some older persons are living. The situation of living in a squalid house that is both insecure and small points to a life bereft of adequate housing rights and dignity. According to Manomano (2013), this contravenes both national and international rights to a modest and decent housing. Furthermore, the fact that some aged people are still using pit latrines when most South Africans use modern flush toilets makes them feel desperate and despondent. They feel their rights to decent housing with even modest social amenities are largely being compromised (Manomano, 2013). DISCUSSION OF THE FINDINGS The availability of food also has a positive psychosocial impact, especially for those who are not adequately taken care of in their homes. It provides physical satisfaction that in turn impacts upon one’s social, psychological and emotional wellbeing. This improves ones psychosocial wellbeing and further enhances one’s social and physical functioning (Segal, Gerdes & Steiner, 2007). The provision of food helps to maintain body mass and is a key factor in the bottom level of Maslow’s hierarchy of needs (Nyden, Petersson & Nystrom, 2003). Study findings indicated diametrically opposed views, with some older people perceiving that they are treated with respect and dignity, while others felt the opposite to be true. Those participants who perceived that older persons are treated with dignity and that their rights are upheld are surely members of a community where younger people still respect the South African culture of past decades that embodied ample respect for older people (Lombard & Kruger, 2009; Dhemba, 2013). This is clear evidence that many young people in South Africa have survived the strong winds of cultural erosion and are still able to uphold the desirable cultural ethos of bestowing ample respect on the elderly (Afolayan, 2004). Families must fulfil the responsibilities provided for in the constitution of the country (Johns Hopkins University, 2012). In a study conducted in Alice townships to explore the circumstances that drive the elderly from their homes, some study participants indicated that they were treated with love by their children. This means that portions of society still uphold and implement the spirit of ubuntu (isiXhosa) or botho (Setswana) in maintaining the culture that has always demanded that the older individuals in the community be respected and treated with dignity (Batala et al., 2014). While some older people are treated with respect and dignity, some are maltreated and experience discrimination (Johns Hopkins University, 2012). Perhaps, as observed by Phillips, Ray & Marshall (2006), it is the fact that older persons have multifaceted needs that puts them at risk of abuse, neglect, poverty and institutionalisation. Old age is often accompanied by a reduced capacity for income generation as well as of poverty, loneliness, senility and a growing risk of debilitating illnesses (Dhemba, 2012, 2013). Research by Sargeant (2007) indicates that with increased modernisation and levels of development, discrimination based on age is increasing. DISCUSSION OF THE FINDINGS Findings indicate that the Lavela Old Age Centre was instrumental in affording the elderly various kinds of psychosocial support. They are fed there, share joys and sorrows, are involved in art and beadwork, run a gardening project and are exposed to visits from people who are older-person friendly. Importantly for the older persons’ social functioning, these psychosocial benefits allow them to adequately satisfy some of the first rank of Maslow’s hierarchy of needs (Nyden, Petersson and Nystrom, 2003). This also implies that the centre creates networks and widens the horizon of association and interaction between the elderly people themselves (Singh & Srivastava, 2014). The centre offers an opportunity to reduce or mitigate the effects of loneliness. To a large extent loneliness Social Work/Maatskaplike Werk 2018:54(3) 290 is a serious psychosocial deficit, because it is usually associated with some debilitating health conditions such as an increased risk of cardiovascular disease, fear and anxiety, elevated blood pressure, depression and even death (Singh & Srivastava, 2014). The opportunity for older persons to gather in a place specifically set aside for them also provides positive aspects of social interaction that creates a platform for enjoying, talking about and sharing issues and challenges pertaining to their lives. This increases the opportunities for developing social capital (Kang’ethe, 2014). It is therapeutic and has the positive effect of increasing their confidence, self-esteem and social functioning (Sheafor & Horejsi, 2008). The group dynamic has an emancipatory effect, making individuals allay the fear and anxiety associated with loneliness. The group effect allows older people to empower, console, advise or even counsel one another (Trevithick, 2009). The centre also provides an opportunity for the older persons to do some basic exercises to improve their health. This also has some psychosocial benefit. Exercise provides some psychosocial interventions and is instrumental in improving the older person’s social, psychological and emotional wellbeing. It also helps with balance, mobility, strength etc. Exercise for the elderly is also associated with decreased symptoms of depression and anxiety, and increased self-esteem (Blair, Sallis & Hutber 2012, 2012). With increasing age comes an increased risk of developing non-communicable chronic health conditions. In a recently published review Blair et al. (2012) emphasise the direct link between physical inactivity, low cardiovascular fitness and the occurrence of chronic health conditions. Social Work/Maatskaplike Werk 2018:54(3) DISCUSSION OF THE FINDINGS The elderly are also likely to face other forms of stigma and discrimination because of their disability and the fact that they frequently fall ill. The level of discrimination seems to be higher against women. Tran (2012) observed that women face age discrimination more than men, with older women having less access to education, health, earning capacity and limited access to rights of land ownership. Social Work/Maatskaplike Werk 2018:54(3) 291 Research findings indicate that the old age grant is rarely adequate to meet the needs of the recipients. This is because of the absence of any supplementary income to meet other life needs such as clothing (Gutura & Tanga, 2014). This is strange because South Africa is a country with one of the highest budgets for social welfare, with the state’s old age pension making up the lion’s share of the social welfare budget (Potts, 2011). It is unfortunate, therefore, that despite the old age grants constituting a significant chunk of the welfare assistance budget apparently, the recipients claim that it rarely meets their daily life needs (Potts, 2011). The situation is exacerbated by the spiralling inflation that the country has been facing. This researcher thinks that, with the country’s economy sliding towards international junk status, and the South African rand being unstable against the world’s major currencies, prices and inflation are bound to rise, making the old age grant increasingly unable to meet its objective of covering the basic needs of the elderly. It needs to be borne in mind that provision of the old age grant that currently stands at around R1,500 is a policy obligation for the government of South Africa. This is effected through the Social Assistance Act of 2004 and associated regulations that provide the legal framework for the administration of seven social grants, the old age grant being one of them (South Africa Social Security Agency, 2010). The government had bestowed the responsibility of administering these grants to the South African Social Security Agency (SASSA, 2010) (Tangwe & Gutura, 2013; South Africa Social Security Agency, 2010). Almost a third of all South Africans receive grants as their only source of income. It then follows that, as most elderly individuals also fall into the category of the poor, they most probably do not have any other source of income (Tangwe & Gutura, 2013). DISCUSSION OF THE FINDINGS Many elderly people have to rely on their children, grandchildren or a caregiver to buy them food and other necessities. This presents the opportunity for the money to be misappropriated and fail to supply the food that should last for a month until the next grant is due (Batala et al., 2014; South Africa Social Security Agency, 2010). Study findings indicate that elderly people are sometimes abused and their old age pension mishandled by their caregivers or children entrusted to take care of them. According to reports by the Department of Social Development (DSD, 2009), episodes of abuse and neglect of elderly people in their homes are on the rise. Perhaps the increase in poverty levels among South Africans generally, as well as cultural attrition, could be some of the significant causes of the abuse and abandonment the of the elderly. This researcher believes that the effect of poverty reflected in the statistics of rising unemployment explains the phenomenon of younger people mishandling the grants of their parents/grandparents (DSD 2009; Batala et al., 2014; Kang’ethe, 2015; Gutura & Tanga, 2014). A study by Batala et al. (2014) in Alice Township in the Eastern Cape found immense abuse and neglect of older persons, prompting them to leave their homes and be cared for by the old age centres. This researcher considers such abuse and neglect both pernicious and a human rights violation. It is unequivocally unethical and immoral for anybody with the responsibility of overseeing the implementation of the older individuals’ grants to be the one mishandling the grant. This leaves the older individuals vulnerable and despondent, a situation that can easily make them succumb to the fear and stresses associated with mistreatment, with potentially fatal consequences. According to research carried out by the Johns Hopkins University on older persons (Johns Hopkins University, 2012), the older persons have the inherent right to life and dignity, and the fact that they are too old to be productive should not be used to deny them the dignity and respect they deserve. Nevertheless, the government needs to be applauded for this cash transfer to the elderly as it strives to achieve the objective of poverty alleviation. In South Africa these old age grants, also known as state old age pensions, alongside the children’s grants, have a played pivotal role in mitigating the social and economic marginalisation of the poor. DISCUSSION OF THE FINDINGS They also contribute to the achievement of the country’s goal of redistribution of resources, especially to the black majority, who found themselves in dire economic circumstances in the apartheid era (Kang’ethe, Mulwayini & Manomano, 2015; Potts, 2011). Importantly, the grants offer the recipients an opportunity to sustain themselves as they make their way Social Work/Maatskaplike Werk 2018:54(3) 292 through the muddy and murky waters of life (Tangwe & Gutura, 2013). Currently, approximately 6 million elderly South Africans have access to the grant (SASSA, 2010). Ironically, access to grants appears to have perfidious effects on the productivity of household members. This is evidenced by empirical observation that the state old age pension grant has been instrumental in providing perverse incentives leading to dependency on state aid (Tangwe & Gutura, 2013). It is therefore critically important that screening of grant recipients be revised, as some grant money appears to be falling into the hands of undeserving individuals and motivating recipients and recipient households to depend on the grant rather than to seek employment (Tangwe & Gutura, 2013). Perhaps the worst challenge pertaining to the administration of the old age grant is the recipients’ inability to use the money themselves because of an array of challenges such as illiteracy and various kinds of health challenges usually associated with aging. This is because aging is an entropic disorder (Magill, 1996 cited in Kang’ethe, 2009). But disturbingly, it seems that some of the grant money is misused or abused by those who are supposed to be helping the old people (SASSA, 2010). This could explain why elderly people run away from their homes to the old age homes or day-care centres for the aged. Research by Batala et al. (2004) indicates that although the government gives grant money to the elderly, it is rarely adequate to meet their nutritional needs. Because of immense poverty and lack of any other income, there are so many other demands that compete for the same money. This is because the grant is their only source of income (SASSA, 2010). Study findings indicate that older persons have their rights compromised if they can only afford to stay in squalor and dilapidated housing bereft of security and where they are likely to become victims of the vagaries of weather (Johns Hopkins University, 2012). DISCUSSION OF THE FINDINGS Perhaps it needs to be emphasised that the rights to housing for all, including the elderly, are inalienably provided for through international housing instruments and protocols, in tandem with a country’s constitution (Manomano, 2013). These globalised instruments are of the utmost importance and hold that societies must recognise the contribution of the elderly. This is because they have value (Madonsela, 2008; Lombard and Kruger, 2009). In many traditional African societies where cultures are still respected, older community members are counsellors, arbitrators and caregivers (Kang’ethe, 2010). In South Africa, abusing old people is contrary to the provisions of the South African Constitution, which provides for the enjoyment of all the rights and freedoms of a human being (Manomano, 2013; UN Habitat, 2009). Studies by Johns Hopkins University (2012) indicate that in many parts of the globe inheritance laws deny widows the right to own or inherit property when their husbands die. This research indicates that a possible reason for some older people not easily acquiring houses is the long queues to apply. The phenomenon of malpractice surrounding the access to housing also poses a challenge. Perhaps this is why some study participants have lived in a one-roomed house since 1985, while waiting to be allocated a relatively modern and permanent RDP house (Kang’ethe & Manomano, 2014). They fear they may be called to meet their maker before being granted access to a decent house. CONCLUSION Older people unequivocally need to be respected and treated with love so that they can age with dignity and bless the youth as they face the challenges of life. If societies could follow the cultural dictates and ethos of the past that provided a special niche for the elderly, affording dignity to our aged citizens would not have to be enforced. This researcher believes that modernisation, eurocentrism and globalisation have dealt a huge blow to the traditional lifestyle that dignified the elderly, with the result that some suffer abandonment and neglect. 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https://openalex.org/W2063641341	https://zenodo.org/record/1498756/files/article.pdf	English	null	THE USE OF A HIGH FAT DIET IN THE TREATMENT OF DIABETES MELLITUS	Archives of internal medicine	1,921	public-domain	2,558	From the Department of Internal Medicine, Medical School, University of Michigan. 1. Newburgh, L. H., and Marsh, P. L.: The Use of a High Fat Diet in the Treatment of Diabetes Mellitus : First Paper, Arch. Int. Med. 26:657 (July) 1920. Downloaded From: http://archinte.jamanetwork.com/ by a Michigan State University User on 06/09/2015 THE USE OF A HIGH FAT DIET IN THE TREATMENT OF DIABETES MELLITUS SECOND PAPER: BLOOD SUGAR L. H. NEWBURGH, M.D., AND PHIL L. MARSH, M.D. ANN ARBOR, MICH. SECOND PAPER: BLOOD SUGAR L. H. NEWBURGH, M.D., AND PHIL L. MARSH, M.D. ANN ARBOR, MICH. In a previous communication 1 we discussed in outline the advan- tages of the use of a high fat diet in the treatment of diabetes mellitus. We reported briefly the results of an investigation of the effect of a diet whose energy came largely from fat, to which was added sufficient protein to maintain nitrogen balance and the minimal carbohydrate necessitated in making up a diet that a human being can eat over a long period of time. It was shown that with such a diet, glycosuria was avoided in severe diabetics, and that acidosis was not produced. , p The first paper stated the method employed and, in a general way, the results obtained. Freedom from glycosuria, however, does not necessarily mean normal glycemia. In this communication we shall deal with the effect of this type of diet on the blood sugar. yp Blood sugar determinations, sufficiently numerous to permit drawing conclusions concerning the effect of the diet on glycemia, are available in forty-five cases. We include in this grou[ every case in which such a series of determinations has been made, and have omitted only those whose blood sugar determinations have been too few to be of signifi¬ cance. A few patients left the hospital on higher diets than those shown in the tables, but as corresponding blood sugar determinations are not available, the tables for such individuals stop with the last blood sugar reading. g These cases are presented in four groups. The first three groups (Tables 1, 2 and 3), consisting of forty cases, show a satisfactory response of the blood sugar to the treatment. The fourth group (Table 4) comprises the five cases in which blood sugars did not reach a desirably low percentage. Of the forty satisfactory cases, those com¬ plicated by chronic nephritis have been brought together in Table 2, and those in which diets varied at times from our standard are presented in Table 3. Downloaded From: http://archinte.jamanetwork.com/ by a Michigan State University User on 06/09/2015 TABLE 1.—Cases Showing Satisfactory Response of Blood Sugar to Treatment TABLE 1.—Cases Showing Satisfactory Response of Blood Sugar to Treatment Case Day 19-391 Male 47 Osteomyelitis of foot 144 lbs. THE USE OF A HIGH FAT DIET IN THE TREATMENT OF DIABETES MELLITUS 19-537 Male 21 127 lbs. 19-567 Male 49 153 lbs. 19-264 remale 144 lbs. 19-108 Male, 54 Chronic myocarditis 162 lbs. 19-295 Male 53 160 lbs. Blood Sugar per Cent. 11 14 Iti 19 21 23 I 5 7 9 12 20 27 31 36 40 43 45 55 1 12 16 17 20 23 27 32 43 1 5 (i 18 29 38 37 45 5 6 S 10 46 IS 38 1 4 6 12 14 15 20 48 45 .50 52 53 55 57 62 70 0.082 0.090 0.070 0.130 0.109 O.IOO 0.100 0.120 0.300 0.211 0.400 0.176 0.175 0.140 ¿.'riè 0.187 0.125 0140 0.111 0.310 0.120 0.080 O.lfO 0.095 0.070 O.IOO 0.090 0.070 0.325 0.130 0.120 0.150 0.210 0.140 0.110 O.IOO 0.200 0.107 0.100 0.107 0.126 0.075 0.100 0.55 0.14 0.19 0.235 0.14 0.17 0.083 0.25 0.09 0.107 0.09 0.075 0.095 Pro¬ tein, Gm. 40 54 29 38 45 29 3(1 65 19 40 16 23 29 60 0.100 18 84 34 Fat, Gm. 225 240 135 135 42 1 5 200 140 WO 1411 152 95 155 125 160 17« Car¬ bohy¬ drate, Gm. Calories 10 13 18 10 8 10 10 111 10 10 10 40 10 31 13 1,370 2,350 2,400 1,200 1,475 1,500 500 1,475 1,700 2,100 1,400 1.500 960 1,425 1,550 1,450 960 1,600 1,025 1,150 1,600 1,700 Remarks 50 gm. bread added to diet one day; glycosurie Starvation 24 hours Left hospital and did not adhere strictly to diet ( ) Case Day Blood Sugar per Cent. Pro¬ tein, Gm. Fat, Gm. Car- bony- Calories (Irate, Gm. Remarks 19-306 Male 66 Osteomyelitii oí toot 144 lbs. 19-467 Female 52 181 lbs. 20-1 Male 60 174 lbs. 19-130 Male, 31 124 lbs. 20-458 Male, 65 121 lbs. 20-660 Female 60 114 lbs. 19-355 Female 20-376 Female, 5£ 135 lbs. 19-261 Female 61 151 lbs. 21-51 Male 73 19-163 Male 75 151 lbs. 20-753 Female 53 180 lbs. THE USE OF A HIGH FAT DIET IN THE TREATMENT OF DIABETES MELLITUS 1 7 10 13 17 23 24 34 35 38 12 44 49 56 58 12 16 28 10 1 8 9 13 17 18 19 30 11 15 17 1 7 9 13 1 5 8 1 4 5 6 7 9 11 17 41 1 4 5 6 7 8 0.550 0.200 0.110 0.09 0.190 0.187 0.140 0.100 0.140 0.120 0.110 0.100 0.15 0.15 6.13 6.14 0.19 0.15 0.16 0.225 0.190 0.145 0.18 0.13 0.35 0.11 0.10 0.14 0.33 0.30 0.163 0.136 0.125 0.270 0.136 0.24 6.17 0.11 0.450 0.125 0.160 0.145 0.17 0.136 0.130 0.275 0.145 0.130 0.180 0.16O 0.14U 0.120 0.3O 6.18 10 25 16 16 36 42 47 16 23 16 27 18 28 34 55 1!) 32 0.13 0.16 16 16 16 50 15 55 16 25 30 55 95 II« 221· 245 25? 100 146 2113 90 ISO 170 210 90 145 2O0 95 14«' 95 235 l«i 135 ISO 185 180 236 16 16 l.i 11 15 12 1» 16 14 20 35 16 14 11 10 16 lo '8 10 lo 10 20 25 80 »80 1,300 086 1,000 2,400 2,660 1,000 1,400 1,000 1,000 950 1,400 1,800 2,200 925 1,500 2,000 960 1,400 960 2,400 1,000 1,450 1,650 2,200 SA» 1,400 1,800 2,400 Diet had been re¬ stricted previous to entrance for opera¬ tion for cataracts Left against advfce Unexplained héma¬ turie; refused cyst- oscopy and left 30 min. after meal Dietetic erroT 7:00 p. m. Ate candy Downloaded From: http://archinte.jamanetwork.com/ by a Michigan State University User on 06/09/2015 TABLE 1.—Cases Showing Satisfactory Response of Blood Sugar to Treatment—(Continued) TABLE 1.—Cases Showing Satisfactory Response of Blood Sugar to Treatment—(Continued) TABLE 1.—Cases Showing Satisfactory Response of Blood Sugar to Treatment—(Continued) No. 20 Case 27 20-759 Male 48 162 lbs. 20-558 Male 35 152 lbs. 20-653 Male 22 Restricted before entrance 118 lbs. 20-882 Male 141 lbs. 20-738 Female 56 131 lbs. 20-703 Male 46 168 lbs. 20-688 Male 153 lbs. 21-8 Male 138 lbs. 21-9 . Female 18 155 lbs. 21-31 Female 57 158 lbs. 2. Meyers, V. C., and Bailey, C. V.: The Lewis and Benedict Method for the Estimation of Blood Sugar, with Some Observations Obtained in Disease, J. Biol. Chem. 24:147, 1916. Bing, H. J., and Jakolson, B.: Blutuntersuchungen unter normalen u. einigen pathologische Verh\l=a"\ltnissen,Deutsch. Arch. f. klin. Med. 113:571, 1914. Hopkins, A. R.: Studies in the Concentration of Blood Sugar in Health and Disease as Determined by Bang's Micromethod, Am. J. Med. Sc. 149:254, 1915. THE USE OF A HIGH FAT DIET IN THE TREATMENT OF DIABETES MELLITUS Day 1 5 15 17 28 1 3 7 10 18 15 17 22 31 41 43 141 13 16 18 •>5 82 1 3 5 7 9 12 15 1 2 4 5 4 5 9 11 13 15 18 22 27 1 I 5 8 10 14 17 19 :·:, 8 12 Blood Sugar per Cent. 0.85 0.21 0.14 0.11 0.37 0.12 0.07 o.io 0.10 0.11 0.08 0.11 0.08 0.14 0.07 0.13 0.12 0.30 0.19 0.18 0.16 0.11 o.os 0.19 0.20 0.09 0.10 0.18 0.17 0.09 ¿.¿ß' 0.18 0.12 ' 0.267 0.220 0.120 0.180 0.120 0.130 0.180 0.150 0.120 0.220 0.120 0.420 0.17 0.18 0.14 0.18 0.27 0.125 0.16 0.12 Pro¬ tein, Gm. 16 25 SO 16 .35 16 25 30 10 25 30 16 25 30 55 16 25 16 16 25 45 16 Fat, Gm. 90 135 180 180 230 18;. 180 135 ISO 185 180 230 90 135 135 90 135 ISO 180 90 135 180 Car¬ bohy¬ drate, Gm. 14 14 20 25 25 30 Calories 20 25 20 25 20 25 30 14 14 20 25 10 14 20 !»*! 900 1,400 1,800 1,800 2,400 1,400 1,800 1,400 1,800 1,400 1,800 2,400 900 1,400 1,400 14 900 20 1,400 1,900 1,900 900 1,400 1,800 Dietetic error Drank 2glasses milk without permission Menstruation Downloaded From: http://archinte.jamanetwork.com/ by a Michigan State University User on 06/09/2015 The twenty-eight cases contained in Table 1 show that a high fat diet such as we have used is capable of bringing the blood sugar down to normal and keeping it at that level during the period of observation. TABLE 2.—Response to Treatment of Blood Sugar in Diabetics with Marked Nephritis No. 30 Case Day 19-371 Male 47 158 lbs. 19-438 Female 19-218 Female 19-50 Female 56 168 lbs. 19-131 Female 60 131 lbs. 19-84 Female, 51 175 lbs. 21-19 Male 76 133 lbs. 1 2 8 12 13 IS 19 22 23 26 37 1 3 8 9 10 17 21 27 37 1 3 7 10 13 1 5 6 9 10 14 18 28 Blood Sugar per Cent. 0.205 0.115 0.092 0.136 0.130 0.190 0.180 0.125 0.200 0.185 0.200 0.125 0.130 0.380 0.232 0.150 0.150 0.140 0.140 0.135 0.100 0.18 0.11 0.12 0.145 0.145 0.125 0.425 0.115 0.120 0.30 11.15 0.22 0.20 0.18 0.125 Pro¬ tein, Gm. Downloaded From: http://archinte.jamanetwork.com/ by a Michigan State University User on 06/09/2015 THE USE OF A HIGH FAT DIET IN THE TREATMENT OF DIABETES MELLITUS 16 60 16 16 42 16 42 66 16 46 16 25 36 35 Fat, Gm. li» 150 95 95 160 95 130 95 135 156 95 110 95 135 186 236 Car- i bohy- Calories drate, Gm. 10 1,000 10 1,700 10 960 150 10 1,500 205 10 2,000 10 12 16 10 16 16 L0 10 10 14 20 25 1,700 1,400 960 1,300 1,700 960 1,200 950 1,400 1,900 2,400 25 gm. bread added later caused glyco¬ suria TABLE 2.—Response to Treatment of Blood Sugar in Diabetics with Marked Nephritis The seven cases presented in Table 2 are separated from the rest because of the well known fact that chronic nephritis in diabetics tends to keep the blood sugar at an abnormally high level.2 These patients all had a severe nephritis as shown by decreased output of phenolsul- phonephthalein, hypertension, high blood urea and the persistence of albumin and casts in the urine days after the disappearance of the glycosuria. It is of special interest to note that the blood sugar of each of these individuals is brought to a point well within normal limits. g p The six cases in Table 3 show well the occurrence of hyperglycemia resulting from diets high in protein and the reduction of the blood sugar TABLE 3.—Patients Treated by Varying Regimes No. 38 Case 18-382 Male 118 lbs. 18-613 Female 35 117 lbs. 18-667 Female 46 19-165 Male 75 180 lbs. 19-56 Female 53 168 lbs. Day 14 17 18 20 30 1 9 16 12 13 16 16 12 IS 16 1 3 4 7 » 15 11 13 14 15 16 IS 19 23 24 Blood Sugar per Cent. 11.275 0.215 0.155 0.113 0.400 0.230 0.Ï66 0.214 0.130 0.200 0.220 0.273 0.145 0.180 0.160 0.140 0.135 0.195 0.130 0.190 0.170 0.185 Pro¬ tein, Gm. 16 52 260 16 30 200 16 12 206 16 45 70 16 22 34 50 56 Fat, Gm. 160 220 315 135 100 185 135 106 155 160 166 106 116 13(1 136 126 120 Car¬ bohy¬ drate, Gm. THE USE OF A HIGH FAT DIET IN THE TREATMENT OF DIABETES MELLITUS 10 16 10 16 16 16 10 1(1 12 12 10 10 10 23 23 22 35 Calories 2,075 1,000 2,225 2,075 1,000 1,400 2.075 1,000 1,600 2,075 1,700 1,800 960 1,025 1,150 1,400 1,375 1,400 1,375 1,400 Rem arts "Von Noorden" diet High fat diet "Von Noorden" diet High fat diet "Von Noorden" diet High fat diet 'Von Noorden" diet The excess of pro¬ tein caused a hy¬ perglycemia Urine sugar free after the fifth day TABLE 3.—Patients Treated by Varying Regimes to within normal limits subsequent to the use of a diet low in protein and high in fat. Case 40 is especially instructive in this respect. After four days on a diet containing 37 gm. protein and 1,400 calories, the blood sugar was 0.135 per cent.; after an increase of the protein to 50 gm., with a slight decrease in carbohydrate and total calories, a hyper¬ glycemia of 0.195 per cent, is noted. A return to the former diet Downloaded From: http://archinte.jamanetwork.com/ by a Michigan State University User on 06/09/2015 nloaded From: http://archinte.jamanetwork.com/ by a Michigan State University User on 06/09/2015 brought the blood sugar down to 0.130 per cent, while the substitution of the second diet again produced a hyperglycemia of 0.190 per cent. g p yp g y per The five cases in Table 4 are those in which response to treatment was not satisfactory. Two of these (Cases 42 and 45) had severe TABLE 4.—Patients Not Responding Satisfactorily to Treatment Case Day Blood Sugar per Cent. Pro¬ tein, Gm. Car- Fat, ! bohy- Gm. drate, Gm. 19-440 Male 18 90 lbs. 19-229 Female 54 120 lbs. 20-423 Male 63 81 lbs. 19-205 Muir 61 149 lbs. 20-311 Male 40 124 lbs. THE USE OF A HIGH FAT DIET IN THE TREATMENT OF DIABETES MELLITUS 1 4 6 8 11 12 13 18 21 20 28 41 50 51 57 01 64 1 5 6 7 1 4 9 15 23 1 6 7 13 15 21 27 29 24 1 3 7 15 18 20 29 0.52 0.36 0.29 0.21 0.2O 97 0.42 0.83 0.15 0.16 0.15 0.18 0.15 0.18 0.15 0.15 0.15 0.375 0.187 0.166 0.215 0.20 0.16 0.15 0.16 0.400 0.120 0.120 0.275 0.225 0.150 0.200 0.400 0.135 0.160 0.125 0.140 0.170 0.160 25 37 37 28 2S 34 16 Hi 10 21 48 140 190 105 [00 100 14(1 160 95 130 200 155 100 1.56 240 10 10 Hi 10 10 20 25 10 10 21 31 10 11 1.3 1,675 1,600 1,400 1,700 960 1.300 2,000 ,ß 1,000 1,500 2,500 Broke diet Patient in bal¬ ance; left the hos¬ pital in excellent condition Far advanced squa- mous cell carci¬ noma of uterus Discharged against advice Urine sugar free Urine sugar free Left hosp. against advice Cerebrospinal syph¬ ilis TABLE 4.—Patients Not Responding Satisfactorily to Treatment complicating diseases. We suspected but could not prove that one patient (Case 44) was not adhering to his diet; we can.give no other explanation for the rise in his blood sugar from 0.120 to 0.275 per cent, between the eleventh and thirteenth days, in the absence of any change in diet on our part. Downloaded From: http://archinte.jamanetwork.com/ by a Michigan State University User on 06/09/2015
https://openalex.org/W4390275302	https://revista.fmc.br/ojs/index.php/RCFMC/article/download/821/424	Portuguese	null	Linfoma cutâneo de células T – Diagnóstico e prognóstico	Revista Científica da Faculdade de Medicina de Campos	2,023	cc-by-sa	2,519	Recebido em: 14/02/2023 Recebido em: 14/02/2023 Linfoma cutâneo de Células T – Diagnóstico e prognóstico Cutaneous T-cell lymphoma - diagnosis and prognosis Caio Freire Benjamim Vianna1, Gabriel Maciel de Sales Sardinha2, Júlia Couto Villar3, Ana Luiza de Oliveira Marins3, Alex Batista Paulo4 1 Médico pela Faculdade de Medicina de Campos (FMC), Campos dos Goytacazes, RJ, Brazil. d Clí i édi H it l E l Ál Al i (HEAA) C d G t RJ B il p p ( ), p y , , 2 Residente de Clínica médica no Hospital Escola Álvaro Alvim (HEAA), Campos dos Goytacazes, RJ, Brazil. 3 Acadêmicas no Curso de Graduação em Medicina, Faculdade de Medicina de Campos (FMC), Campos dos Goytacazes, RJ, Brazil. 4 Professor no Curso de Graduação em Medicina, Faculdade de Medicina de Campos (FMC), Campos dos Goytacazes, RJ, Brazil. Autor correspondente: Caio Freire Benjamim Vianna Contato: caiofbenjamim@gmail.com ISSN 2965-6575 ISSN 2965-6575 Rev. Cient. Fac. Med. Campos, v. 18, n. 2, p. 49-52, jul./dez. 2023 doi: 10.29184/1980-7813.rcfmc.821.vol.18.n2.2023 Faculdade de Medicina de Campos https://revista.fmc.br/ojs/index.php/RCFMC Relato de caso Linfoma cutâneo de Células T – Diagnóstico e prognóstico Cutaneous T-cell lymphoma - diagnosis and prognosis RESUMO Este artigo apresenta o relato de um caso clínico de uma paciente do sexo feminino, com 59 anos de idade, que foi diagnosticada com Linfoma Cutâneo de Células T. O principal objetivo deste estudo é discutir a incidência dessa doença e destacar a importância do diagnóstico precoce, bem como descrever suas principais manifestações clínicas e complicações associadas. A coleta de dados para o caso foi realizada por meio da análise minuciosa dos prontuários médicos, exames laboratoriais e de imagem arquivados no Serviço de Arquivo Médico e Estatística do Hospital Escola Álvaro Alvim. O diagnóstico definitivo do Linfoma Cutâneo de Células T foi estabelecido por meio de exame imuno-histoquímico, um procedimento essencial que permite direcionar de forma rápida e precisa a terapia adequada ao paciente. ABSTRACT Palavras-chave: Linfoma Cutâneo de Células T. Linfócitos T. Síndrome de Sézary. Keywords: Cutaneous. Lymphoma. Sezary Syndrome. T-Cell. T-cells. Palavras-chave: Linfoma Cutâneo de Células T. Linfócitos T. Síndrome de Sézary. Keywords: Cutaneous. Lymphoma. Sezary Syndrome. T-Cell. T-cells. Esta obra esta licenciada sob uma Licença Creative Commons. Os usuários têm permissão para copiar redistribuir os trabalhos por qualquer meio ou formato, e também para, tendo como base o seu conteúdo, reutilizar, transformar ou criar, com, propositos legais, até comerciais, desde que citada a fonte. ABSTRACT This article presents the report of a clinical case of a 59-year-old female patient diagnosed with cutaneous T-cell lymphoma. The main objective of this study is to discuss the incidence of this disease and emphasize the importance of early diagnosis, as well as to describe its main clinical manifestations and associated complications. Data collection for the case was conducted through meticulous analysis of medical records, laboratory tests, and imaging studies archived at the Medical and Statistical Archive Service of Hospital Escola Álvaro Alvim. The definitive diagnosis of cutaneous T-cell lymphoma was established through immunohistochemical examination, an essential procedure that allows for the rapid and accurate direction of appropriate therapy for the patient. INTRODUÇÃO proliferação exacerbada, levando à formação de linfomas. Esses tumores são subdivididos em tipos B e T, resultantes de um contato com antígenos que provocam alterações morfológicas e distor ções fenotípicas2. Embora alguns pacientes apre sentem linfomas restritos a um único local, outros podem manifestar-se de maneira agressiva, es pecialmente em casos de doença avançada, afe De acordo com o consenso da Organi� zação Mundial de Saúde, os Linfomas Cutâneos de Células T/NK são um grupo de doenças lin foproliferativas extranodais classificadas e sub divididas com base no seu comportamento clíni co1. Os linfócitos periféricos podem passar por 49 Rev. Cient. Fac. Med. Campos, v. 18, n. 2, p. 49-52, jul./dez. 2023 Linfoma cutâneo de Células T – Diagnóstico e prognóstico neoplásicas, o que orienta o tratamento mais ade quado, juntamente com a determinação do estágio e do grau de envolvimento sistêmico4, 5. Assim, além dos tratamentos direcionados à pele, muitas vezes é necessário administrar tratamento sistê mico com quimioterapia e agentes moduladores da resposta biológica6. tando múltiplos sítios, incluindo locais incomuns, como cérebro, pulmão, ovário e glândula tireoide1. Além dos linfonodos, sítios extranodais como a pele também são comumente afetados. Em tecido cutâneo, alguns Linfomas de Células T se mani festam de maneira evidente e característica por meio de um infiltrado linfocítico. Vale ressaltar que as manifestações clínicas cutâneas são ini cialmente restritas à pele, tratando-se de uma característica dos linfomas cutâneos1-3. Embora as manifestações cutâneas sejam mais comuns em homens, as mulheres também podem apresentar essas manifestações. Geral mente, os linfomas cutâneos são mais prevalentes em idosos entre a quinta e a sétima décadas de vida, sendo raramente observados em indivíduos com menos de 50 anos3, 4. Os Linfomas de Células T são divididos em dois grandes grupos de acordo com seu compor tamento: os de comportamento indolente (micose fungoide clássica, micose fungoide foliculotrópica, reticulose pagetoide, cútis laxa granulomatosa, linfoma cutâneo primário de grande célula anaplá sica, papulose linfomatoide, linfoma subcutâneo de célula T paniculite-símile e linfoma cutâneo primário de pequena e média célula T CD4+ pleo mórfica) e os de comportamento agressivo (Sín drome de Sézary, linfoma extranodal de célula T/ NK tipo nasal, linfoma cutâneo primário agressivo de célula T CD8+ epidermotrópica, linfoma cutâneo de célula T e linfoma cutâneo primário de célula T periférica não especificado)1,4. INTRODUÇÃO A micose fungoi de e a Síndrome de Sézary são responsáveis por cerca de 50% dos casos de linfomas cutâneos pri mários3. O diagnóstico é realizado por meio de exames histológicos e fenotipagem das células DESCRIÇÃO DO CASO Paciente, feminina, preta, 59 anos, ca sada, cozinheira, previamente hígida, sem an tecedentes ou internações prévias, apresentou eritema pruriginoso difuso no abdômen, região genital e membros inferiores, levando-a a pro curar atendimento médico ambulatorial. Inicial mente, foi prescrita terapia sintomática com de xametasona tópica. Ao longo de um período de três meses, observou-se uma progressão das le sões, resultando em múltiplas ulcerações e cros tas disseminadas, com envolvimento adicional de tórax, ombro, pescoço e face conforme retratado antemáticas com ulcerações e crostas difusas em regiões B Figura 1. Comprovação icogonográfica do caso relatado. (A). Lesões exantemáticas com ulcerações e crostas difusas em regiões faciais, cervicais, torácicas e membros superiores. Figura (B). Lesões cutâneas no tórax e membro superior em estágios variados de cicatrização, com áreas hipocrômicas, crostas e descamações. A B B Figura 1. Comprovação icogonográfica do caso relatado. (A). Lesões exantemáticas com ulcerações e crostas difusas em regiões faciais, cervicais, torácicas e membros superiores. Figura (B). Lesões cutâneas no tórax e membro superior em estágios variados de cicatrização, com áreas hipocrômicas, crostas e descamações. 50 Rev. Cient. Fac. Med. Campos, v. 18, n. 2, p. 49-52, jul./dez. 2023 VIANNA C. F. B. et al. com linfonodos maiores medindo 1,59 cm à direi ta e 1,56 cm à esquerda, no menor eixo axial. nas Figuras 1A e 1B onde a Figura 1A exibe “Lesões exantemáticas com ulcerações e crostas difusas em regiões faciais, cervicais, torácicas e mem bros superiores” e a Figura 1B apresenta “Lesões cutâneas no tórax e membro superior em está gios variados de cicatrização, com áreas hipocrô micas, crostas e descamações”. Devido ao desen volvimento de dor associada às lesões, a paciente procurou atendimento de emergência, resultan do em sua internação e transferência para enfer maria clínica. No nono dia de internação hospitalar, diante da progressão contínua do quadro clíni co, foram prescritos três ciclos de quimioterapia seguindo o protocolo CHOEP (ciclofosfamida, doxorrubicina, vincristina, etoposídeo e predni sona). Além disso, foi administrado aciclovir na dose de 200mg a cada 12 horas. O esquema an tibiótico anterior foi substituído por meropenem na dose de 500mg a cada 8 horas e vancomicina na dose de 500mg a cada 6 horas, visando a uma cobertura mais ampla, considerando a suspeita de infecção por microrganismos multirresisten tes. DESCRIÇÃO DO CASO O resultado do estudo imuno-histoquímico estava pendente de liberação, aguardando os procedimentos laboratoriais necessários para a obtenção e interpretação do perfil imunofenotí pico da paciente. Após a realização de uma única sessão de quimioterapia, a paciente desenvolveu pancitopenia e alteração do nível de consciência, levando à necessidade de transfusão de concen trado de hemácias. No entanto, ocorreu uma pio ra progressiva do quadro, evoluindo para choque séptico e resultando em óbito. Durante o período de internação, a pa ciente apresentou agravamento da dor, acom panhado de descamações e infecção secundária. No momento da admissão, foi iniciada antibioti coterapia intravenosa empírica com oxacilina na dose de 1g a cada 4 horas, além de analgesia com tramadol na dose de 50mg a cada 8 horas. Foram solicitados exames laboratoriais, incluindo o teste anti-HIV, que resultou negativo para a presença de anticorpos específicos contra o vírus. O exame laboratorial revelou um quadro de processo infec cioso, caracterizado por leucocitose neutrofílica com contagem de 35.000 leucócitos. Após três dias de internação, não houve melhora clínica sa tisfatória, nem normalização dos marcadores in flamatórios, como a proteína C reativa (PCR) ape sar do esquema antibiótico adotado. Diante desse cenário, optou-se pela substituição da oxacilina por amoxicilina com clavulanato na dose de 1,2g a cada 8 horas, associada a pulsos de dexametasona na dose de 40mg por 4 dias, administração única de ivermectina na dose de 18 mg e morfina para analgesia de alta potência. Os resultados do estudo imuno-histoquí mico e imunofenotipagem revelaram imunoex pressão positiva para o antígeno CD3 e negativa para os antígenos CD20 e CD30, consolidando, dessa forma, o diagnóstico de Linfoma de Células T. Adicionalmente, a característica imunofeno típica apresentada em conjunto com a natureza agressiva do quadro clínico sugere fortemente a presença da Síndrome de Sézary. Em virtude da suspeita clínica de linfoma cutâneo, foi solicitada a realização de uma bióp sia de pele para a obtenção de amostra para estu do imuno-histoquímico. Simultaneamente, foram requisitadas tomografias computadorizadas do tórax, abdômen e pelve, com o objetivo de ava liar a possível disseminação sistêmica da doença. Os exames de imagem revelaram a presença de adenomegalias axilares bilaterais, com linfonodos maiores medindo 1,50 cm à direita e 1,69 cm à es querda, no menor eixo axial. DESCRIÇÃO DO CASO Além disso, foram identificadas adenomegalias inguinais bilaterais, O presente estudo foi conduzido de acor do com as diretrizes estabelecidas na Resolução CSN nº466/12 e recebeu aprovação do Comitê de Ética, com base no parecer de nº 5.792.25 e CAAE 64024922.4.0000.5244. Todos os requisitos éti cos e regulatórios foram rigorosamente seguidos para garantir a validade e integridade da pesquisa. Rev. Cient. Fac. Med. Campos, v. 18, n. 1, p. 49-52, jul./dez. 2023 REFERÊNCIAS Em estágios avançados da Síndrome de Sézary, a corrente sanguínea do paciente irá conter células T pleomórficas em forma e tamanho com núcleo convoluto de aspecto cerebriforme, citoplasma abundante, azurófilo e sem grânulos, que são as chamadas células de Sézary3, 5, 7. O estudo imuno -histoquímico, por sua vez, apresentou positivi dade para os antígenos CD2, CD3 e CD5, no caso relatado, apresentou-se positivo para CD3.i 1. Sanches J, Melotti C, Festa Neto C. Processos lin foproliferativos da pele: part 2 - cutaneous T-cell and NK-cell lymphomas. Anais Brasileiros de Der matologia. 2006;81:7-25. 2. Oliveira PD, Farre L, Bittencourt AL. Adult T-cell leukemia/lymphoma. Rev Assoc Med Bras (1992). 2016;62(7):691-700. 2. Oliveira PD, Farre L, Bittencourt AL. Adult T-cell leukemia/lymphoma. Rev Assoc Med Bras (1992). 2016;62(7):691-700. Para um diagnóstico definitivo da Síndro me de Sézary, é necessário que haja uma popula ção monoclonal na pele e no sangue, com altera ções fenotípicas (CD4/CD8 ≥ 10 e/ou CD4 + CD7 ≥ 40% e/ou CD4+CD26 ≥ 30%), ou a presença de células de Sézary com ≥ 1.000 células/μL 5. 3. Miyashiro DR, Sanches Junior JA. Micose fungoide e síndrome de Sézary: caracterização demográfi ca, clínica, histopatológica, imunopatológica, mo lecular, laboratorial e evolutiva de coorte observa da no ambulatório de linfomas cutâneos da divisão de clínica dermatológica do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo entre os anos de 1989 e 2018. 2021. 3. Miyashiro DR, Sanches Junior JA. Micose fungoide e síndrome de Sézary: caracterização demográfi ca, clínica, histopatológica, imunopatológica, mo lecular, laboratorial e evolutiva de coorte observa da no ambulatório de linfomas cutâneos da divisão de clínica dermatológica do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo entre os anos de 1989 e 2018. 2021. O tratamento pode ser dividido em tera pias relacionadas à pele e terapias sistêmicas. As terapias direcionadas à pele incluem agentes tó picos, fototerapia e radioterapia. As terapias sis têmicas incluem modificadores da resposta bioló gica, imunoterapias e agentes quimioterápicos. A escolha do tratamento é baseada no estágio atual da doença, sendo favoráveis tratamentos direcio nados à pele em estágios iniciais, com o objetivo de melhorar os sintomas e a qualidade de vida do paciente. Entre os esquemas quimioterápicos mais comuns estão o CHOP, CHOEP, CVP e CVP com metotrexato (MTX) 6. 4. Eickhoff CM, Almeida ASd, Fontana M, Almeida EAPd, Ughini M, Rodriguez R. DISCUSSÃO Os Linfomas Cutâneos de Células T inte gram uma variedade de doenças relacionadas à 51 Rev. Cient. Fac. Med. Campos, v. 18, n. 1, p. 49-52, jul./dez. 2023 Linfoma cutâneo de Células T – Diagnóstico e prognóstico pele no momento da apresentação clínica, aumen tando a complexidade de um diagnóstico precoce, como foi o caso dessa paciente. Diante da suspeita clínica de um linfoma, considerou-se a realização de um estudo anatomopatológico. No entanto, muitas vezes a histologia dessas doenças se as semelha entre si em estágios iniciais da doença, tornando-o inconclusivo para fins terapêuticos. Sendo assim, a realização do estudo imuno-histo químico, como foi feito, é mais assertiva. -se observar que os sintomas iniciais do linfoma cutaneo de células T geralmente são lesões exan temáticas pruriginosas inespecíficas, que possuem alto grau de progressão e disseminação na pele. O diagnóstico é feito correlacionando a clínica com exames histopatológicos e imuno-histoquímicos, e o tratamento é direcionado pelo contexto clínico do paciente, ajustando-o de acordo com a evolu ção diária. REFERÊNCIAS Linfoma cutáneo de células T. Rev méd Hosp Säo Vicente de Paulo 1999;11(25):56-8. 5. Araújo M, Dantas AFA, Nobre CF, Theodoro FCM, Silva GA, Machado MLL, et al. Avaliação imuno fenotípica por citometria de fluxo no diagnóstico da síndrome de sézary no Rio Grande do Nor te. Hematology, Transfusion and Cell Therapy. 2022;44:S560. 6. Sanches JA, Cury‐Martins J, Abreu RM, Miyashi ro D, Pereira J. Micose fungoide e síndrome de Sézary: foco no cenário atual de tratamento. Anais Brasileiros de Dermatologia (Portuguese). 2021;96(4):458-71. Quando restritos à pele, os linfomas cutâ neos apresentam uma sobrevida de até 97% nos primeiros 5 anos em pacientes com cerca de 10% de superfície corporal afetada. No entanto, em ca sos de doença com disseminação sistêmica, devi do ao comportamento mais agressivo, a mortali dade varia de 50% a 60% nos primeiros 5 anos 3. 7. Rabelo KA, Silva VEG, Almeida JF, Pereira KG, Nogueres MIC, Barbalho SMM, et al. Síndro me de sézary: relato de caso. Hematology, Transfusion and Cell Therapy. 2021;43:S104-S5. Com base nos dados apresentados, pode Com base nos dados apresentados, pode 52 Rev. Cient. Fac. Med. Campos, v. 18, n. 2, p. 49-52, jul./dez. 2023
https://openalex.org/W2006216887	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0035220&type=printable	English	null	Associations between XPD Asp312Asn Polymorphism and Risk of Head and Neck Cancer: A Meta-Analysis Based on 7,122 Subjects	PloS one	2,012	cc-by	4,233	Received October 26, 2011; Accepted March 12, 2012; Published April 20, 2012 Received October 26, 2011; Accepted March 12, 2012; Published April 20, 2012 Copyright: 2012 Hu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was partly supported by grants from the Medical Development Foundation of Health Department of Jiangsu Province (H200811) and Natural Science Foundation of Jiangsu Higher Education Institutions (08KJB320008). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: niuyuming@yeah.net excision repair (NER) pathway have been implicated in cancer. Associations between polymorphisms in several DNA repair genes and the risks of several types of cancer have been extensively examined. Many epidemiologic cancer studies have focused on single nucleotide polymorphisms (SNPs) in genes in the NER pathway such as XPD, ERCC1, and XPC [5]. The XPD protein is a DNA helicase and is an essential part of the TFIIH transcription factor complex. Some studies have suggested that XPD polymorphisms may be associated with reduced DNA repair because of a possible reduction in helicase activity [6,7]. One of the common XPD polymorphisms in the coding regions is Asp312Asn in exon 10. The functional significance is not yet completely clear, although the amino acid mutations in exon 10 give rise to a loss of an acidic residue and a complete change in the electronic configuration of the amino acid [8,9]. Yuan Yuan Hu1, Hua Yuan2, Guang Bing Jiang3, Ning Chen2, Li Wen4, Wei Dong Leng1, Xian Tao Zeng1, Yu Ming Niu1,2* 1 Department of Stomatology, Taihe Hospital, Hubei University of Medicine, Shiyan, People’s Republic of China, 2 Institute of Dental Research, Nanjing Medical University, Nanjing, People’s Republic of China, 3 Department of Radiology, Taihe Hospital, Hubei University of Medicine, Shiyan, People’s Republic of China, 4 Department of Dermatology, Taihe Hospital, Hubei University of Medicine, Shiyan, People’s Republic of China PLoS ONE \| www.plosone.org Abstract Background: To investigate the association between XPD Asp312Asn polymorphism and head and neck cancer risk through this meta-analysis. Methods: We performed a meta-analysis of 9 published case-control studies including 2,670 patients with head and neck cancer and 4,452 controls. An odds ratio (OR) with a 95% confidence interval (CI) was applied to assess the association between XPD Asp312Asn polymorphism and head and neck cancer risk. Results: Overall, no significant association between XPD Asp312Asn polymorphism and head and neck cancer risk was found in this meta-analysis (Asn/Asn vs. Asp/Asp: OR = 0.95, 95%CI = 0.80–1.13, P = 0.550, Pheterogeneity = 0.126; Asp/Asn vs. Asp/Asp: OR = 1.11, 95%CI = 0.99–1.24, P = 0.065, Pheterogeneity = 0.663; Asn/Asn+Asp/Asn vs. Asp/Asp: OR = 1.07, 95%CI = 0.97–1.19, P = 0.189, Pheterogeneity = 0.627; Asn/Asn vs. Asp/Asp+Asp/Asn: OR = 0.87, 95%CI = 0.68–1.10, P = 0.243, Pheterogeneity = 0.089). In the subgroup analysis by HWE, ethnicity, and study design, there was still no significant association detected in all genetic models. Conclusions: This meta-analysis demonstrates that XPD Asp312Asn polymorphism may not be a risk factor for developing head and neck cancer. Citation: Hu YY, Yuan H, Jiang GB, Chen N, Wen L, et al. (2012) Associations between XPD Asp312Asn Polymorphism and Risk of Head and Neck Cancer: A Meta- Analysis Based on 7,122 Subjects. PLoS ONE 7(4): e35220. doi:10.1371/journal.pone.0035220 Citation: Hu YY, Yuan H, Jiang GB, Chen N, Wen L, et al. (2012) Associations between XPD Asp312Asn Polymorphism and Risk of Head and Neck Cancer: A Meta- Analysis Based on 7,122 Subjects. PLoS ONE 7(4): e35220. doi:10.1371/journal.pone.0035220 Editor: Brock C. Christensen, Dartmouth College, United States of America Editor: Brock C. Christensen, Dartmouth College, United States of America Study characteristic This meta-analysis is guided by the PRISMA statement (Checklist S1). A total of 49 relevant studies were identified (Figure 1). After carefully review, nine eligible case-control studies on the relationship between XPD Asp312Asn polymorphism and HNC risk were included in this meta-analysis [10–18]. Table 1 presents the main characteristics of these studies. Seven studies involved Caucasian populations [10–12,14–16,18], whereas two studies involved Asians [13,17]. Diverse genotyping methods were used, including PCR-SSCP, PCR-RFLP, Taqman, Real-time PCR and SEB PCR. All studies indicated that the genotypic distribution of the controls was consistent with HWE except one [17]. Introduction Head and neck cancers (HNC) constitute about 5% of all cancers recorded in the US, and the incidence is increasing in most developed and developing countries. These cancers have been estimated to be about six times more common among smokers than non-smokers and are most common in males over 50 years old [1,2], which increases to about 15 times if the smokers are also heavy drinkers [3,4]. Although many measures had been done to improve the diagnosis and treatments, the prognosis was still poor. Many environmental factors, such as radiation, diet, smoking, and endogenous or exogenous estrogens, are associated with DNA damage. Unrepaired or misrepaired DNA results in gene mutations, chromosomal alterations, and genomic instability. Several studies have suggested that genes involved in DNA repair play a crucial role in protecting against mutations. Patients with certain cancers have reduced capac- ities for DNA repair. Similarly, the enzymes of the nucleotide The first study on the relationship between HNC and XPD Asp312Asn polymorphism was conducted by Sturgis et al. [10]. April 2012 \| Volume 7 \| Issue 4 \| e35220 1 XPD Asp312Asn Polymorphism and HNC Risk XPD Asp312Asn Polymorphism and HNC Risk They found a borderline significant association between XPD Asp312Asn polymorphism and HNC. Since then, a lot of studies have confirmed or refuted this finding [11–18]. In 2010, a recent meta-analysis was conducted by Flores-Obando et al. [19] demonstrated that increased HNC risk is associated with XPD Asp312Asn polymorphism. Worthy of note, that meta-analysis included five studies were conducted in Caucasian populations and one in an Asian population [10–15]. Today, nine case-control studies on XPD Asp312Asn polymorphism and HNC risk have been published. A comprehensive meta-analysis is needed to provide an updated approach on the overall relationship. Subgroup analyses were also performed on Caucasian and Asian populations to investigate ethnicity-specific effects. Search strategy y The PubMed database was searched with terms ‘‘head and neck cancer’’, ‘‘oral cancer’’, ‘‘oropharyngeal cancer’’, ‘‘laryngeal cancer’’, ‘‘pharyngeal cancer’’, ‘‘XPD’’, ‘‘excision repair cross- complementing group 2’’, ‘‘polymorphism’’, and the combined phrases for all genetic studies on the relationship between XPD polymorphism and HNC risk from 2000, when the first study of the association between XPD Asp312Asn polymorphism and HNC risk was reported, to October 2011. We also used the ‘‘Related Articles’’ option in PubMed to identify additional studies on the same topic. Reference lists in retrieved articles were also screened for. All selected studies complied with the following three criteria: (a) case–control study on the XPD Asp312Asn polymorphism and HNC risk; (b) sufficient published data for estimating the odds ratio (OR) with 95% confidence interval (CI); (c) For multiple publications reporting on the same data or overlapping data, the largest or most recent publication was selected [20]. The main results of this meta-analysis and the heterogeneity test are shown in Table 2. Overall, no significant relationship was observed between XPD Asp312Asn polymorphism and HNC risk in the total populations (for Asn/Asn vs. Asp/Asp: OR = 0.95, 95%CI = 0.80–1.13, P = 0.550, Pheterogeneity = 0.126; Asp/Asn vs. Asp/Asp: OR = 1.11, 95%CI = 0.99–1.24, P = 0.065, Pheterogeneity =0.663; Asn/Asn+Asp/Asn vs. Asp/Asp: OR=1.07, 95%CI=0.97– 1.19, P=0.189, Pheterogeneity=0.627; Asn/Asn vs. Asp/Asp+Asp/Asn: OR=0.87, 95%CI=0.68–1.10, P=0.243, Pheterogeneity=0.089). Similarly, in the succeeding analysis of HWE studies, no significant association was found between XPD Asp312Asn polymorphism and Figure 1. Flow diagram of the study selection process. doi:10.1371/journal.pone.0035220.g001 Data extraction Two investigators (Hu and Yuan) independently extracted the following data from each included publication: the first author’s name, publication data, sources of controls, racial descent of the study population (categorized as either Asian or Caucasian), genotyping method, number of cases, cases and controls with different genotypes, and Hardy-Weinberg equilibrium(HWE). Statistical analysis Figure 3 shows the shapes of the funnel plots of codominant model (Asp/Asn vs. Asp/Asp), used in the studies for examining all populations. The result was further supported by analysis via modified Egger’s tests. No significant publication bias was found in this meta-analysis (P = 0.093 for Asn/Asn vs. Asp/ Asp; P = 0.370 for Asp/Asn vs. Asp/Asp; P = 0.173 for Asn/ Asn+Asp/Asn vs. Asp/Asp; P = 0.215 for Asn/Asn vs. Asp/ Asp+Asp/Asn). plots in all genetic models did not reveal any evidence of obvious asymmetry. Figure 3 shows the shapes of the funnel plots of codominant model (Asp/Asn vs. Asp/Asp), used in the studies for examining all populations. The result was further supported by analysis via modified Egger’s tests. No significant publication bias was found in this meta-analysis (P = 0.093 for Asn/Asn vs. Asp/ Asp; P = 0.370 for Asp/Asn vs. Asp/Asp; P = 0.173 for Asn/ Asn+Asp/Asn vs. Asp/Asp; P = 0.215 for Asn/Asn vs. Asp/ Asp+Asp/Asn). HNC risk(for Asn/Asn vs. Asp/Asp: OR = 0.95, 95%CI = 0.80– 1.14, P = 0.593, Pheterogeneity = 0.120; Asp/Asn vs. Asp/Asp: OR = 1.11, 95%CI = 0.99–1.24, P = 0.089, Pheterogeneity = 0.586; Asn/Asn+Asp/Asn vs. Asp/Asp: OR = 1.07, 95%CI = 0.96–1.19, P = 0.219, Pheterogeneity = 0.528; Asn/Asn vs. Asp/Asp+Asp/Asn: OR = 0.82, 95%CI = 0.69–1.11, P = 0.278, Pheterogeneity = 0.082). Finally, in the stratified analysis of ethnicity and study design, we also did not find any significant association between XPD Asp312Asn polymorphism and HNC. Sensitivity analysis A single study involved in the meta-analysis was deleted each time to reflect the influence of the individual dataset to the pooled ORs. The analysis results demonstrate a borderline increased risk after excluding the studies that in Asp/Asn vs. Asp/Asp model [14,16,18] (Figure 2). The other corresponding pooled ORs were not materially altered (data not shown), indicating that our results are statistically robust. Today, genetic susceptibility to cancer has attracted growing attention to the study of gene polymorphisms involved in tumorigenesis. The XPD gene has been mapped to chromosome 19q13.3 and it is composed of 23 exons. Germline mutations in the XPD gene can result in xeroderma pigmentosum and other diseases. The XPD protein is involved in transcription-coupled NER and is an integral member of the basal transcription factor BTF2/TFIIH complex. Statistical analysis Crude ORs with 95% CIs were computed to assess the strength of the correlation between the XPD Asp312Asn polymorphism and HNC risk. The pooled ORs were performed for codominant model (Asn/Asn vs. Asp/Asp,Asp/Asn vs. Asp/Asp), dominant model (Asn/Asn+Asp/Asn vs. Asp/Asp), and recessive model (Asn/Asn vs. Asp/Asp+Asp/Asn), respectively. In the subgroup analysis, statistical analysis was conducted on Asians and Caucasians. Heterogeneity assumption was assessed by the chi- square based Q-test [21]. The pooled OR estimation of each study was calculated by the fixed-effects model (the Mantel–Haenszel method) when P.0.10. Otherwise, the random-effects model (the DerSimonian and Laird method) was used [22]. The potential publication bias was estimated by the modified Egger’s linear regression test, which proposed by Harbord et al. [23]. Statistical analysis was performed using STATA version 11.0 (Stata Corporation, College Station, TX, USA) and Review Manage (v.4.2; Oxford, England), using two-sided P-values, with P,0.05 considered statistically significant. Figure 1. Flow diagram of the study selection process. doi:10.1371/journal.pone.0035220.g001 April 2012 \| Volume 7 \| Issue 4 \| e35220 April 2012 \| Volume 7 \| Issue 4 \| e35220 PLoS ONE \| www.plosone.org 2 XPD Asp312Asn Polymorphism and HNC Risk Table 1. Characteristics of case-control studies on XPD Asp312Asn polymorphism and HNC risk included in the meta-analysis. Table 1. Characteristics of case-control studies on XPD Asp312Asn polymorphism and HNC risk includ y y F rst author Year Racial descent Source of controls Case Control Genotype distribution Genotying type P for HWE{ Case Control Asp/Asp Asp/Asn Asn/Asn Asp/Asp Asp/Asn Asn/Asn Sturgis 2002 Caucasian Hospital-based 313 313 123 165 25 142 135 36 PCR-SSCP 0.650 Matullo 2006 Caucasian Population-based 82 1094 32 46 4 418 506 170 TaqMan 0.411 An 2007 Caucasian Hospital-based 829 854 330 395 104 370 386 98 PCR-RFLP 0.860 Majumder 2007 Asian Hospital-based 305 387 152 119 34 205 146 36 PCR-RFLP 0.183 Harth 2008 Caucasian Hospital-based 311 298 113 158 40 101 145 52 Real-time PCR 0.997 Abbasi 2009 Caucasian Population-based 246 644 93 119 34 258 304 82 Real-time PCR 0.606 Jelonek 2010 Caucasian Hospital-based 29 58 10 14 5 14 36 8 PCR-RFLP 0.052 Ji 2010 Asian Hospital-based 264 342 235 29 0 309 30 3 SBE PCR 0.026 Gugatschka 2011 Caucasian Population-based 291 462 116 133 42 171 208 83 TaqMan 0.158 doi:10.1371/journal.pone.0035220.t001 i doi:10.1371/journal.pone.0035220.t001 plots in all genetic models did not reveal any evidence of obvious asymmetry. Publication bias The Asp to Asn change at position 312 of XPD changes the electronic configuration of amino acid and alters the interaction between XPD protein and its helicase activator [6]. Wolfe et al. Funnel plot and modified Egger’s test were performed to estimate the publication bias of literature. The shapes of the funnel Table 2. Summary ORs and 95% CI of XPD Asp312Asn polymorphism and HNC risk. Asn/Asn vs. Asp/Asp Asp/Asn vs. Asp/Asp Asn/Asn+Asp/Asn vs. Asp/Asp Asn/Asn vs. Asp/Asp+Asp/Asn OR 95% CI P P* OR 95% CI P P* OR 95% CI P P* OR 95% CI P P* Total 0.95 0.80–1.13 0.550 0.126 1.11 0.99–1.24 0.065 0.663 1.07 0.97–1.19 0.189 0.627 0.87 0.68–1.10 0.243 0.089{ HWE 0.95 0.80–1.14 0.593 0.120 1.11 0.99–1.24 0.089 0.586 1.07 0.96–1.19 0.219 0.528 0.82 0.69–1.11 0.278 0.082{ Ethnicity Asian 1.20 0.73–2.00 0.470 0.213 1.14 0.87–1.50 0.345 0.649 1.14 0.88–1.48 0.328 0.951 1.16 0.71–1.89 0.546 0.216 Caucasian 0.92 0.76–1.11 0.366 0.121 1.11 0.98–1.25 0.110 0.468 1.06 0.94–1.19 0.317 0.430 0.83 0.64–1.08 0.165 0.085{ Design Hospital based 1.01 0.81–1.25 0.938 0.296 1.14 1.00–1.31 0.052 0.468 1.12 0.98–1.27 0.092 0.543 0.95 0.78–1.16 0.631 0.224 Population based 0.75 0.43–1.32 0.248 0.063{ 1.04 0.85–1.28 0.684 0.692 0.98 0.81–1.19 0.865 0.611 0.73 0.42–1.28 0.271 0.045{ *Test for heterogeneity. {Estimates for random effects model. doi:10.1371/journal.pone.0035220.t002 Table 2. Summary ORs and 95% CI of XPD Asp312Asn polymorphism and HNC risk. Table 2. Summary ORs and 95% CI of XPD Asp312Asn polymorphism and HNC risk. Table 2. Summary ORs and 95% CI of XPD Asp312Asn polymorphism and HNC risk. April 2012 \| Volume 7 \| Issue 4 \| e35220 3 XPD Asp312Asn Polymorphism and HNC Risk Figure 2. Sensitivity analysis through deletion of one study at a time to reflect the influence of the individual dataset to the pooled ORs in Asp/Asn vs. Asp/Asp model. doi:10.1371/journal.pone.0035220.g002 Figure 2. Sensitivity analysis through deletion of one study at a time to reflect the influence of the individual dataset to the pooled ORs in Asp/Asn vs. Asp/Asp model. doi:10.1371/journal.pone.0035220.g002 Asp312Asn polymorphism has been shown to increase the risk of bladder cancer and lung cancer, but it is not associated with breast cancer [27–29]. demonstrated that the 312 codon polymorphisms significantly decrease the constitutive ERCC2 mRNA levels, especially in smokers [24]. Hou et al. reported that the XPD 312 variant allele may be associated with the reduced repair of aromatic DNA adducts [25]. Matullo et al. References Flores-Obando RE, Gollin SM, Ragin CC (2010) Polymorphisms in DNA damage response genes and head and neck cancer risk. Biomarkers 15: 379–399. 7. Winkler GS, Araujo SJ, Fiedler U, Vermeulen W, Coin F, et al. (2000) TFIIH with inactive XPD helicase functions in transcription initiation but is defective in DNA repair. J Biol Chem 275: 4258–4266. 20. 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(2009) Laryngeal cancer risk associated with smoking and alcohol consumption is modified by genetic polymorphisms in ERCC5, ERCC6 and RAD23B but not by polymorphisms in five other nucleotide excision repair genes. Int J Cancer 125: 1431–1439. 3. Blot WJ, McLaughlin JK, Winn DM, Austin DF, Greenberg RS, et al. (1988) Smoking and drinking in relation to oral and pharyngeal cancer. Cancer Res 48: 3282–3287. 4. Lichtenstein P, Holm NV, Verkasalo PK, Iliadou A, Kaprio J, et al. (2000) Environmental and heritable factors in the causation of cancer–analyses of cohorts of twins from Sweden, Denmark, and Finland. N Engl J Med 343: 78–85. 16. Jelonek K, Gdowicz-Klosok A, Pietrowska M, Borkowska M, Korfanty J, et al. (2010) Association between single-nucleotide polymorphisms of selected genes involved in the response to DNA damage and risk of colon, head and neck, and breast cancers in a Polish population. J Appl Genet 51: 343–352. 5. Goode EL, Ulrich CM, Potter JD (2002) Polymorphisms in DNA repair genes and associations with cancer risk. Cancer Epidemiol Biomarkers Prev 11: 1513–1530. 17. Ji YB, Tae K, Lee YS, Lee SH, Kim KR, et al. (2010) XPD Polymorphisms and Risk of Squamous Cell Carcinoma of the Head and Neck in a Korean Sample. Clin Exp Otorhinolaryngol 3: 42–47. 6. Coin F, Marinoni JC, Rodolfo C, Fribourg S, Pedrini AM, et al. (1998) Mutations in the XPD helicase gene result in XP and TTD phenotypes, preventing interaction between XPD and the p44 subunit of TFIIH. Nat Genet 20: 184–188. 18. Gugatschka M, Dehchamani D, Wascher TC, Friedrich G, Renner W (2011) DNA repair gene ERCC2 polymorphisms and risk of squamous cell carcinoma of the head and neck. Exp Mol Pathol 91: 331–334. 19. Acknowledgments Despite the considerable efforts to test for possible association between XPD Asp312Asn polymorphism and HNC risk, some limitations should be addressed. First, these results are based on unadjusted estimates that lack the original data from the eligible studies, which limits the evaluation of the effects of the gene-gene and gene-environment interactions during HNC development. Second, the sample size is still relatively small. Thus, we could not have enough statistical data to find the true relationship between XPD Asp312Asn polymorphism and HNC risk. Finally, each gene We thank everyone who helped with this study. Author Contributions Conceived and designed the experiments: YYH HY YMN. Performed the experiments: YYH HY GBJ NC. Analyzed the data: LW WDL XTZ. Wrote the paper: YYH HY. Critical review of manuscript: YMN. Conceived and designed the experiments: YYH HY YMN. Performed the experiments: YYH HY GBJ NC. Analyzed the data: LW WDL XTZ. Wrote the paper: YYH HY. Critical review of manuscript: YMN. Publication bias proposed that exposure to environ- mental carcinogens, such as polycyclic aromatic hydrocarbons (PAHs), also accelerate cancer development through the codon 312 variant allele of XPD [26]. The first study, published in 2002, revealed a borderline correlation between XPD Asp312Asn polymorphism and HNC risk in codominant model (for Asn/Asn vs. Asp/Asp: OR, 1.41; 95% CI: 1.01–1.97) [10]. To date, no consensus has been reached on the correlation between XPD Asp312Asn polymorphism and HNC risk. Majumder et al. [13] found that variant genotype (Asn/ Asn) at codon 312 of XPD is associated with increased risk of Correlations between the polymorphisms and some cancer risks have been studied, but the results remain controversial. The XPD Figure 3. Funnel plot analysis to detect publication bias for Asp/Asn vs. Asp/Asp genotype. Each point represents a separate study for the indicated association. doi:10.1371/journal.pone.0035220.g003 Figure 3. Funnel plot analysis to detect publication bias for Asp/Asn vs. Asp/Asp genotype. Each point represents a separate study for the indicated association. doi:10.1371/journal.pone.0035220.g003 PLoS ONE \| www.plosone.org April 2012 \| Volume 7 \| Issue 4 \| e35220 XPD Asp312Asn Polymorphism and HNC Risk XPD Asp312Asn Polymorphism and HNC Risk cancer among rapid and intermediate acetylators (OR = 1.9, 95% CI = 1.2–2.9). However, other studies showed that HNC risk is not significantly related to XPD Asp312Asn polymorphism. Ji et al. [17] found that the OR of the Asp312Asn polymorphism genotype Asp/Asn is 1.94 (95% CI = 0.92–4.08) relative to the Asp/Asp genotype. Matullo et al. [11], An et al. [12], Harth et al. [14], Abbasi et al. [15], and Jelonek et al. [16] also reported similar risks of HNC. is known to have a moderate effect on HNC development. The combinations of certain genotypes may be more discriminating as risk factors than a single locus genotype. In our meta-analysis, linkage disequilibrium (LD) and haplotype analysis were not performed. In spite of these limitations, no publication bias was observed, and a large number of subjects still significantly guarantee the statistical power of the analysis. In conclusion, despite these limitations,our meta-analysis suggests that XPD Asp312Asn polymorphism may not be associated with HNC development. In the future, large-scale case-control and population-based association studies are neces- sary to validate the risks identified in the present meta-analysis and to investigate the potential gene-gene and gene-environment interactions between XPD Asp312Asn polymorphism and HNC cancer. The present meta-analysis of nine eligible studies, including 2670 cases and 4452 controls focused on XPD Asp312Asn polymorphism and HNC risk, was performed to derive a more precise estimate of the association, but no significant association was found in the total population when all the studies were pooled. Similarly, no significant association was detected in all genetic models during the satisfied analysis based on the HWE, ethnicity and study design. Our finding is not in accordance with that previously published by Flores-Obando et al [19]. A marginally significant association was observed between the XPD Asp312Asn heterozygous and combined variants and HNC in their study. The considerably larger sample size of our study may account for this difference relative to the previous study. 29. Wang F, Chang D, Hu FL, Sui H, Han B, et al. (2008) DNA repair gene XPD polymorphisms and cancer risk: a meta-analysis based on 56 case-control studies. Cancer Epidemiol Biomarkers Prev 17: 507–517. 27. Pabalan N, Francisco-Pabalan O, Sung L, Jarjanazi H, Ozcelik H (2010) Meta- analysis of two ERCC2 (XPD) polymorphisms, Asp312Asn and Lys751Gln, in breast cancer. Breast Cancer Res Treat 124: 531–541. 28. Zhang J, Qiu LX, Leaw SJ, Hu XC, Chang JH (2011) The association between XPD Asp312Asn polymorphism and lung cancer risk: a meta-analysis including 16,949 subjects. Med Oncol 28: 655–660. XPD Asp312Asn Polymorphism and HNC Risk 27. Pabalan N, Francisco-Pabalan O, Sung L, Jarjanazi H, Ozcelik H (2010) Meta- analysis of two ERCC2 (XPD) polymorphisms, Asp312Asn and Lys751Gln, in breast cancer. Breast Cancer Res Treat 124: 531–541. 28. Zhang J, Qiu LX, Leaw SJ, Hu XC, Chang JH (2011) The association between XPD Asp312Asn polymorphism and lung cancer risk: a meta-analysis including 16,949 subjects. Med Oncol 28: 655–660. PLoS ONE \| www.plosone.org References Majumder M, Sikdar N, Ghosh S, Roy B (2007) Polymorphisms at XPD and XRCC1 DNA repair loci and increased risk of oral leukoplakia and cancer among NAT2 slow acetylators. Int J Cancer 120: 2148–2156. PLoS ONE \| www.plosone.org 5 April 2012 \| Volume 7 \| Issue 4 \| e35220 April 2012 \| Volume 7 \| Issue 4 \| e35220 PLoS ONE \| www.plosone.org April 2012 \| Volume 7 \| Issue 4 \| e35220 6
https://openalex.org/W1850995982	https://europepmc.org/articles/pmc4937655?pdf=render	English	null	Effect of the CYP3A inhibitors, diltiazem and ketoconazole, on ticagrelor pharmacokinetics in healthy volunteers	Journal of drug assessment	2,013	cc-by	7,436	Journal of Drug Assessment Vol. 2, 2013, 30–39 Journal of Drug Assessment Vol. 2, 2013, 30–39 2155-6660 doi:10.3109/21556660.2013.785413 Article 0011.R1/785413 All rights reserved: reproduction in whole or part not permitted Article 0011.R1/785413 doi:10.3109/21556660.2013.785413 lay Conclusions: la These results suggest that ticagrelor can be co-administered with moderate CYP3A inhibitors. However, co-administration of strong CYP3A inhibitors with ticagrelor is not recommended. Objectives: orma U al Distr ers can do rsonal us Objectives: Two open-label, two-period, crossover studies in healthy volunteers were designed to determine the pharmacokinetic interactions between ticagrelor, a P2Y12 receptor antagonist, and a moderate (diltiazem) and a strong (ketoconazole) cytochrome P450 (CYP) 3A inhibitor. orma U al Distr ers can do rsonal us Objectives: Two open-label, two-period, crossover studies in healthy volunteers were designed to determine the pharmacokinetic interactions between ticagrelor, a P2Y12 receptor antagonist, and a moderate (diltiazem) and a strong (ketoconazole) cytochrome P450 (CYP) 3A inhibitor. ight © s a e eca , g o , e a a e, US Address for correspondence: Renli Teng, Clinical Pharmacology, AstraZeneca LP, FOC W1-677, 1800 Concord Pike, P.O. Box 15437, Wilmington, DE 19850-5437, USA. Tel.: þ1 302 885 9472; Fax: þ1 302 886 4872; renli.teng@astrazeneca.com Keywords: Antiplatelet therapy – Diltiazem – Ketoconazole – Pharmacokinetics – Ticagrelor Accepted: 11 March 2013; published online: 2 April 2013 Citation: J Drug Assess 2013; 2:30–39 Address for correspondence: Renli Teng, Clinical Pharmacology, AstraZeneca LP, FOC W1-677, 1800 Concord Pike, P.O. Box 15437, Wilmington, DE 19850-5437, USA. Tel.: þ1 302 885 9472; Fax: þ1 302 886 4872; renli.teng@astrazeneca.com Address for correspondence: Renli Teng, Clinical Pharmacology, AstraZeneca LP, FOC W1-677, 1800 Concord Pike, P.O. Box 15437, Wilmington, DE 19850-5437, USA. T l þ1 302 885 9472 F þ1 302 886 4872 © Address for correspondence: Renli Teng, Clinical Pharmacology, AstraZeneca LP, FOC W1-677, 1800 Concord Pike, P.O. Box 15437, Wilmington, DE 19850-5437, USA. Tel.: þ1 302 885 9472; Fax: þ1 302 886 4872; renli.teng@astrazeneca.com Keywords: Antiplatelet therapy Diltiazem Ketoconazole © or C Results: igh Accepted: 11 March 2013; published online: 2 April 2013 Citation: J Drug Assess 2013; 2:30–39 le or ed use prohibit view and print a Compared with ticagrelor alone, diltiazem co-administration significantly increased the mean maximum concentration (Cmax) and mean area under the plasma concentration–time curve (AUC) for ticagrelor by 69% and 174%, respectively. Diltiazem co-administration reduced Cmax by 38% but had no significant effect on AUC for AR-C124910XX. Cmax and AUC for ticagrelor were increased by 135% and 632%, respectively, by ketoconazole co-administration, whereas these parameters were reduced by 89% and 56%, respectively, for AR-C124910XX. Diltiazem and ketoconazole pharmacokinetic parameters were not significantly affected by the presence of ticagrelor. Methods: © 2013 Inf Commerc d Authorised u ngle copy for p Seventeen volunteers received diltiazem (240 mg once daily) for 14 days. In the second study, ketoconazole (n ¼ 14) 200 mg twice daily was given for 10 days. A single oral 90-mg ticagrelor dose was administered on day 8 (diltiazem) or day 4 (ketoconazole). In each study, volunteers received a single 90-mg oral dose of ticagrelor before or after washout (14 days). Pharmacokinetic parameters for ticagrelor, AR-C124910XX (primary metabolite), diltiazem, and ketoconazole were assessed. Abstract Renli Teng Kathleen Butler AstraZeneca LP, Wilmington, Delaware, USA Renli Teng Kathleen Butler AstraZeneca LP, Wilmington, Delaware, USA 30 Effect of CYP3A inhibitors on ticagrelor pharmacokinetics Teng & Butler igh Accepted: 11 March 2013; published online: 2 April 2013 Citation: J Drug Assess 2013; 2:30–39 www.informahealthcare.com/JDA ! 2013 Informa UK Ltd Study populations Key inclusion criteria for both studies were: males or females (post-menopausal or surgically sterile); age 18–45 years; body weight 50 kg; and a body mass index (BMI) of 18–30 kg/m2. For both studies, the key exclusion criteria included: smoking/tobacco use in the past 6 months; use of drugs known to increase propensity for bleeding within 2 weeks of study start; a history or presence of any condition known to interfere with drug absorption, distribution, metabolism, or excretion; alcohol or sub- stance abuse in the previous 12 months; consumption of Seville oranges, grapefruit-containing products, alcohol, medicines, or nutritional supplements within 1 week of study start. The complexity of ACS necessitates the use of mul- tiple drugs to manage the disease and a wide variety of co-morbidities7,8. Consequently, drug–drug interactions may occur which could result in altered exposure to a co-administered drug, thereby affecting efficacy and safety9,10. The most abundant drug-metabolizing enzyme in the liver is cytochrome P450 (CYP) 3A11,12, and clin- ically important interactions involving this enzyme are well documented12. Although ticagrelor is a direct- acting antiplatelet agent, it is metabolized to at least ten metabolites13. The major metabolite, AR-C124910XX, which is present at approximately 30–40% of the levels of ticagrelor13–17, is approximately equipotent in inhibiting platelet aggregation (AstraZeneca, data on file). Experiments with human liver microsomes demonstrated that ticagrelor is principally metabolized by CYP3A18. For both studies, all volunteers provided informed con- sent in writing. The protocols were approved by an insti- tutional review board (diltiazem study: Southern Institutional Review Board, Miami, FL, USA; ketocon- azole study: Research Consultants’ Review Committee, West Austin, TX, USA). The studies were conducted according to AstraZeneca bioethics policy, applicable regulatory requirements, and in accordance with the Declaration of Helsinki and in line with good clinical practice. AR-C124910XX is further metabolized by UDP- glucuronosyltransferase to a highly polar glucuronidated metabolite, or via hydroxylation by an unknown isoenzyme to a minor hydroxylated derivative. Given that the glucuronidated metabolite is highly polar and expected to be excreted rapidly in the urine, and the hydroxylated metabolite is minor, the pharmacological relevance of these biotransformations is likely to be minimal13. Introduction Ticagrelor is an oral P2Y12 receptor antagonist that inhibits adenosine dipho- sphate-induced platelet aggregation1. Results from the PLATelet inhibition and patient Outcomes (PLATO) phase III trial showed that, compared with clopidogrel þ aspirin, ticagrelor þ aspirin significantly reduced the rate of myocardial infarction/stroke/death from vascular causes in patients with acute coronary syndromes (ACS)2. Subsequently, ticagrelor has been approved for use in the prevention of atherothrombotic events in adult ACS patients in more than 70 countries, including the EU3 and the United States4. Recently updated European guidelines recommend ticagrelor combined with aspirin as one of several antiplatelet therapies for managing 30 Effect of CYP3A inhibitors on ticagrelor pharmacokinetics Teng & Butler www.informahealthcare.com/JDA ! 2013 Informa UK Ltd Journal of Drug Assessment Volume 2 April 2013 Patients and methods Study populations ACS patients with non-ST-segment elevation5. Furthermore, updated European guidelines also gave ticagrelor a Class I recommendation for patients present- ing with persistent ST-segment elevation6. Effect of CYP3A inhibitors on ticagrelor pharmacokinetics Teng & Butler 31 ! 2013 Informa UK Ltd www.informahealthcare.com/JDA Pharmacokinetic sample collection In both studies, blood samples (2 or 3 mL) were collected for the evaluation of ticagrelor and AR-C124910XX plasma concentrations at the following times: 0, 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 16, 20, 24, 36, 48, 72, 96, 120, 144, and 168 hours post-ticagrelor dosing on day 1 (i.e., follow- ing ticagrelor alone in both studies), day 4 (i.e., after tica- grelor co-administration with ketoconazole), and day 8 (i.e., after ticagrelor co-administration with diltiazem). The exceptions to these sampling times, in the All plasma samples were stored at 20C until assayed. Study designs and treatment Both studies were single-center, randomized, open-label studies with a two-period, in-patient, crossover design. For both studies, a low non-loading dose of ticagrelor (i.e., a single oral dose of 90 mg) was selected, so if an interaction occurred between ticagrelor and the CYP3A4 inhibitor, then the increase in ticagrelor plasma concentrations would minimize any safety issues. The maximum tolerated single dose of ticagrelor is 900 mg26,27. The approved dosing regimen of ticagrelor is 180 mg loading dose followed by 90 mg twice daily there- after3,4. The daily dose (240 mg) of diltiazem selected for the ticagrelor-diltiazem study was within the approved clinical range (180–480 mg daily19), and was expected to inhibit CYP3A421. The selected dose of ketoconazole (200 mg twice daily) is the maximum clinically approved oral dose23 and is commonly used in phase I drug–drug interaction studies10,25 to inhibit CYP3A4 activity. The evaluation of potential drug–drug interactions is important in drug development. Known inhibitors of CYP3A are commonly used as model compounds to inves- tigate such interactions10. Diltiazem is a calcium channel blocker used to treat angina pectoris and mild-to-moderate arterial hypertension19. This compound is an inhibitor of CYP3A activity20,21 and is classified as a moderate inhibi- tor of this enzyme10. The antifungal agent, ketocon- azole22,23, also inhibits CYP3A24 and is considered to be a strong CYP3A inhibitor10. Both of these agents are com- monly used in evaluating the effects of CYP3A inhibition on the pharmacokinetic parameters of co-administered drugs10,21,25. Given the key role of CYP3A in drug metabolism, including that of ticagrelor, and multidrug use in ACS, two drug–drug interaction studies in healthy volunteers were conducted. The primary objectives of these studies were to assess the effects of co-administration of a moder- ate (diltiazem) and a strong (ketoconazole) CYP3A inhibi- tor on ticagrelor pharmacokinetic parameters. Secondary objectives included: assessment of AR-C124910XX phar- macokinetic parameters; effect of ticagrelor on pharmaco- kinetic parameters of diltiazem and ketoconazole; safety and tolerability. Figure 1 depicts the overall study design for both stu- dies. At the start of each study, volunteers were rando- mized to one of two treatments (A or B). After the washout period, volunteers received the alternate dosing regimen. On the scheduled day, ticagrelor was co-adminis- tered with the CYP3A4 inhibitor in the morning after fasting for 10 hours followed by a further fast for 4 hours. Study designs and treatment (a) Volunteers admitted on day –1 and discharged on day 15. †Volunteers admitted on day –1 and discharged on day 8. Volunteers admitted on day –1 and discharged on day 15. †Volunteers admitted on day –1 and discharged on day 8. Screening and Enrolment (days −21 to −1) Volunteers admitted on day –1 and discharged on day 11. †Volunteers admitted on day –1 and discharged on day 8. Treatment A (n=7) Twice daily dose (200 mg each dose) of ketoconazole for 10 days. One 90 mg oral dose of ticagrelor on the morning of day 4. Treatment B† (n=8) Single 90 mg oral dose of ticagrelor in the morning of day 1 only. Washout (minimum 14 days) Randomization on day 1 Period 2 Period 1 Treatment A* (n=7) Twice daily dose (200 mg each dose) of ketoconazole for 10 days. One 90 mg oral dose of ticagrelor on the morning of day 4. Treatment B† (n=7) Single 90 mg oral dose of ticagrelor in the morning of day 1 only. (b) Figure 1. Study design. (a). Diltiazem study (n ¼ 17). (b). Ketokonazole study (n ¼ 14). Volunteers admitted on day –1 and discharged on day 11. †Volunteers admitted on day –1 and discharged on day 8. Figure 1. Study design. (a). Diltiazem study (n ¼ 17). (b). Ketokonazole study (n ¼ 14). period except for 2 hours before and after ticagrelor administration. ticagrelor-ketoconazole study, were: on days 1 and 4 when a sample was collected at 18 hours (instead of at 16 and 20 hours) post-ticagrelor dosing; on day 4 add- itional samples were collected at 60, 84, 108, 132, and 156 hours post-ticagrelor dosing. For each study, the volunteers were restricted from con- suming alcohol, caffeine-containing products, Seville oranges or grapefruit-containing foods, over-the-counter preparations (including herbal remedies), and any drug known to increase bleeding. Blood samples for analysis of diltiazem were collected pre-dose on days 1–6 and 10–14 for analysis of trough con- centrations, and at 0, 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 16, 20, and 24 hours post-diltiazem dosing on days 7 and 8. For analysis of ketoconazole, blood samples were collected pre-dosing on days 3 and 4, and at 12 hours post-ketoconazole dosing on day 4. 32 Effect of CYP3A inhibitors on ticagrelor pharmacokinetics Teng & Butler www.informahealthcare.com/JDA ! 2013 Informa UK Ltd Study designs and treatment Water was permitted ad libitum throughout each Journal of Drug Assessment Volume 2 April 2013 Volunteers admitted on day –1 and discharged on day 15. †Volunteers admitted on day –1 and discharged on day 8. Treatment A* (n=9) Once daily dose (240 mg) of diltiazem for 14 days. One 90 mg oral dose of ticagrelor on the morning of day 8. Treatment B† (n=9) Single 90 mg oral dose of ticagrelor in the morning of day 1 only. Washout (minimum 14 days) Screening and Enrolment (days −21 to −1) Screening and Enrolment (days −21 to −1) Randomization on day 1 Period 2 Period 1 Treatment A* (n=9) Once daily dose (240 mg) of diltiazem for 14 days. One 90 mg oral dose of ticagrelor on the morning of day 8. Treatment B† (n=8) Single 90 mg oral dose of ticagrelor in the morning of day 1 only. Volunteers admitted on day –1 and discharged on day 11. †Volunteers admitted on day –1 and discharged on day 8. Treatment A (n=7) Twice daily dose (200 mg each dose) of ketoconazole for 10 days. One 90 mg oral dose of ticagrelor on the morning of day 4. Treatment B† (n=8) Single 90 mg oral dose of ticagrelor in the morning of day 1 only. Washout (minimum 14 days) Randomization on day 1 Period 2 Period 1 Treatment A* (n=7) Twice daily dose (200 mg each dose) of ketoconazole for 10 days. One 90 mg oral dose of ticagrelor on the morning of day 4. Treatment B† (n=7) Single 90 mg oral dose of ticagrelor in the morning of day 1 only. (a) (b) Figure 1. Study design. (a). Diltiazem study (n ¼ 17). (b). Ketokonazole study (n ¼ 14). Treatment A* (n=9) Once daily dose (240 mg) of diltiazem for 14 days. One 90 mg oral dose of ticagrelor on the morning of day 8. Treatment B† (n=9) Single 90 mg oral dose of ticagrelor in the morning of day 1 only. Washout (minimum 14 days) Screening and Enrolment (days −21 to −1) Randomization on day 1 Period 2 Period 1 Treatment A* (n=9) Once daily dose (240 mg) of diltiazem for 14 days. One 90 mg oral dose of ticagrelor on the morning of day 8. Treatment B† (n=8) Single 90 mg oral dose of ticagrelor in the morning of day 1 only. Baseline demographics and characteristics, and disposition Eighteen volunteers were enrolled in the diltiazem study to ensure that at least 12 volunteers were evaluable. The majority were male (14/18, 78%), and all were Hispanic. The mean (range) age was 33 (18–44) years, and the mean (range) BMI was 27 (22–30) kg/m2. In the ketoconazole study, 16 volunteers were randomized to ensure that at least 12 volunteers were evaluable. Thirteen (81%) were male, and the majority (12/16, 75%) were Caucasian. The mean (range) age was 30 (20–45) years, and the mean (range) BMI was 25 (19–31) kg/m2. Pharmacokinetic parameters for ticagrelor, AR- C124910XX, and diltiazem were estimated by standard non-compartmental analyses (WinNonlin Professional, Pharsight Corporation, Mountain View, California, USA). Plasma concentration–time data were used to cal- culate Cmax, time to maximum concentration (tmax), AUC, and the terminal elimination half-life (t½). The latter was calculated as 0.693/z, where z is the terminal phase elimination rate constant, derived by least-squares regression analysis of the plasma concentration–time data obtained over the terminal log-linear phase. AUC was calculated using the linear trapezoidal method and extra- polated to infinity. AR-C124910XX:ticagrelor ratios for Cmax and AUC were calculated. Diltiazem pharmacoki- netic parameters were estimated based on steady-state concentrations. Ketoconazole steady-state plasma concen- trations were expressed as mean, CV, and range. One volunteer in the diltiazem study was withdrawn from the study at the start of the second period due to a positive drug screen. In the ketoconazole study, two volunteers discontinued the study. One volunteer was withdrawn following ticagrelor þ ketoconazole adminis- tration in the first period due to a right bundle branch block. The other volunteer was withdrawn during ticagrelor þ ketoconazole administration in the second period due to non-compliance. Analytical methods Ticagrelor, AR-C124910XX, diltiazem, and ketoconazole were analyzed using fully-validated, reversed-phase liquid chromatography with tandem mass spectrometry methods. www.informahealthcare.com/JDA ! 2013 Informa UK Ltd Journal of Drug Assessment Volume 2 April 2013 The limits of quantification (LOQ) for ticagrelor and AR-C124910XX were 5 and 2.5 ng/L, respectively28. The LOQ for diltiazem was 1 ng/mL, and 10 ng/mL for ketoconazole (AstraZeneca, data on file). The limits of quantification (LOQ) for ticagrelor and AR-C124910XX were 5 and 2.5 ng/L, respectively28. The LOQ for diltiazem was 1 ng/mL, and 10 ng/mL for ketoconazole (AstraZeneca, data on file). either CYP3A4 inhibitor were 0.7–1.43. For the potential effect of ticagrelor on diltiazem parameters, the pre- specified limits for 90% CI were 0.8–1.25. Data analyses Safety and tolerability of ticagrelor alone and in the presence of diltiazem or ketoconazole were evaluated by assessment of adverse events (AEs), vital signs, electrocar- diographic (ECG), physical examination, and laboratory parameters (clinical chemistry, hematology, and urinaly- sis) throughout the studies. In a previous healthy volunteer study (n ¼ 8) with a single oral dose of ticagrelor (100 mg; D5130C05266, AstraZeneca data on file), the inter-subject coefficients of variation (CV) were 40% for the maximum plasma con- centration (Cmax) and 42% for the area under the plasma concentration–time curve (AUC). Based on this variabil- ity and assuming an intra-subject correlation of 0.65, a two one-sided testing procedure (a-level ¼ 0.05; true ratio ¼ 1) estimated that a sample size of 12 healthy volunteers would provide a statistical power of 90% for the 90% confidence intervals (CI) for Cmax and AUC to be contained within a pre-specified no-effect range of 0.70–1.43 for the ticagre- lor/ketoconazole study. Using the variability data observed in the ticagrelor/ketoconazole study (CV 38.5% for Cmax and 38.9% for AUC) and the same assumptions, power requirements, and no-effect range, the sample size for the ticagrelor/diltiazem study was also estimated to be 12 healthy volunteers. ! 2013 Informa UK Ltd www.informahealthcare.com/JDA Effect of diltiazem on ticagrelor pharmacokinetic parameters Statistical analyses were conducted using SAS version 8 (SAS Institute, Cary, North Carolina, USA). Pharmacokinetic parameters were summarized by descrip- tive statistics. AUC and Cmax values for ticagrelor and AR-C124910XX were analyzed, following log-transforma- tion, by analysis of variance with terms for treatment, period, and sequence, and the volunteer within sequence was included as a random effect. After exponentiation, geometric least square mean point estimates and 90% CIs for the ratio ticagrelor þ CYP3A4 inhibitor/ticagrelor alone were calculated. The pre-specified limits for the 90% CI values indicating no interaction between ticagrelor and Co-administration of diltiazem with ticagrelor increased plasma concentrations of ticagrelor (Figure 2a). Table 1 shows the pharmacokinetic parameters of ticagrelor in the presence and absence of diltiazem. Ticagrelor was rapidly absorbed; the median tmax was 2 hours following adminis- tration of ticagrelor alone, which was unaffected by diltia- zem. The half-life of ticagrelor was increased by 96% by co-administration of diltiazem. For ticagrelor, Cmax was increased by 69% and AUC by 174% by the presence of diltiazem. The 90% CIs of the geometric least-square (GLS) mean ratios for both Cmax and AUC for Effect of CYP3A inhibitors on ticagrelor pharmacokinetics Teng & Butler 33 Journal of Drug Assessment Volume 2 April 2013 Figure 2. Mean ( standard deviation) plasma concentration–time profiles of ticagrelor (a) and AR-C124910XX (b) following a single 90-mg oral dose of ticagrelor in the presence and absence of diltiazem (240 mg once daily) (n ¼ 17). Figure 2. Mean ( standard deviation) plasma concentration–time profiles of ticagrelor (a) and AR-C124910XX (b) following a single 90-mg oral dose of ticagrelor in the presence and absence of diltiazem (240 mg once daily) (n ¼ 17). Table 1. Pharmacokinetic parameters and statistical analyses after a single oral 90-mg dose of ticagrelor in the presence and absence of diltiazem. www.informahealthcare.com/JDA ! 2013 Informa UK Ltd Effect of diltiazem on ticagrelor pharmacokinetic parameters Parameter* Ticagrelor (90 mg) alone n ¼ 17 Ticagrelor (90 mg) plus diltiazem (240 mg qd) n ¼ 17 GLS mean ratio: point estimate (90% CI) Ticagrelor Cmax (ng/mL) 519 (34) 878 (30) 1.69 (1.47–1.95) AUC (ngh/mL) 3701 (37) 10,099 (24) 2.74 (2.40–3.13) t½ (h) 8.3 (2.3) 16.3 (4.4) – tmax (h) 2.0 (1.0–4.0) 2.0 (2.0–4.0) – AR-C124910XX Cmax (ng/mL) 173 (38) 109 (36) 0.62 (0.57–0.68) AUC (ngh/mL) 1630 (29) 1424 (29) 0.87 (0.83–0.92) t½ (h) 8.4 (1.3) 12.5 (4.7) – tmax (h) 3.0 (2.0–4.0) 3.0 (2.0–4.0) – Metabolite:parent ratios Cmax ratio 0.3 (36) 0.1 (24) – AUC ratio 0.4 (39) 0.1 (28) – Values are: geometric mean (% CV) for Cmax, AUC and metabolite:parent ratios; mean (standard deviation) for t½; median (range) for tmax. qd, once daily; GLS, geometric least-square; CI, confidence interval; Cmax, maximum plasma concentration; AUC, area under the plasma concentration–time curve from time 0 to infinity; t½, terminal elimination half-life; tmax, time to Cmax. Values are: geometric mean (% CV) for Cmax, AUC and metabolite:parent ratios; mean (standard deviation) for t½; median (range) for tmax. qd, once daily; GLS, geometric least-square; CI, confidence interval; Cmax, maximum plasma concentration; AUC, area under the plasma concentration–time curve from time 0 to infinity; t½, terminal elimination half-life; tmax, time to Cmax. 34 Effect of CYP3A inhibitors on ticagrelor pharmacokinetics Teng & Butler www.informahealthcare.com/JDA ! 2013 Informa UK Ltd Journal of Drug Assessment Volume 2 April 2013 ketoconazole with ticagrelor (Figure 3b). Pharmacokinetic parameters for AR-C124910XX are summarized in Table 2. The half-life and tmax of AR-C124910XX were both prolonged in the presence of ketoconazole. The Cmax and AUC for AR-C124910XX were decreased by 89% and 56%, respectively, by the co-administration of ketoconazole. The 90% CIs of the GLS mean ratio for both parameters were outside the limits of 0.70–1.43 (Table 2). Ketoconazole co-administered with ticagrelor markedly decreased the metabolite:parent ratios Cmax and AUC by 97% and 94%, respectively (Table 2). ticagrelor þ diltiazem/ticagrelor were outside the prespeci- fied no-effect limits of 0.70–1.43 (Table 1). Plasma concentrations of AR-C124910XX were decreased following administration of ticagrelor with dil- tiazem compared with ticagrelor alone (Figure 2b). Pharmacokinetic parameters for AR-C124910XX are sum- marized in Table 1. This metabolite was rapidly formed; the median tmax was 3 hours after ticagrelor alone, which was unaffected by diltiazem. The half-life of AR- C124910XX was increased by 49% by co-administration of diltiazem with ticagrelor. Safety and tolerability Ticagrelor was well tolerated in the absence or presence of diltiazem. No AEs were reported following a single oral dose of ticagrelor. Overall, 14 mild AEs were reported in eight volunteers receiving ticagrelor þ diltiazem, all of which resolved by the end of the study; 13 of these AEs were considered to be treatment related. Headache, the most common AE, was reported as a single AE in six vol- unteers and occurred four times in another volunteer. The other AEs were dizziness (n ¼ 1), pharyngolaryngeal pain (n ¼ 1), somnolence (n ¼ 1), and pruritus (n ¼ 1). Co-administration of ticagrelor with ketoconazole was well tolerated. Overall, six volunteers had seven AEs during the co-administration of ticagrelor and ketocon- azole, and four volunteers had five AEs with ticagrelor alone. Of these 12 AEs, seven were considered to be related to treatment. Eleven AEs were mild and resolved without intervention. One volunteer had a right bundle branch block which was present after the 14-day washout period following ticagrelor þ ketoconazole co-administra- tion, and discontinued the study; this event was considered mild and not to be treatment-related. Two bleeding- related AEs occurred with ticagrelor þ ketoconazole: one event of hematoma and one event of hemorrhoidal hem- orrhage. The other AEs in the ticagrelor þ ketoconazole treatment were: flatulence, attention disturbance, headache, and dysmenorrhea. During administration of ticagrelor alone, the five AEs recorded in four volunteers Effect of diltiazem on ticagrelor pharmacokinetic parameters The Cmax for AR-C124910XX was decreased by 38% by diltiazem and the 90% CIs of the GLS mean ratio for ticagrelor þ diltiazem/ticagrelor were outside the limits of 0.70–1.43. The AUC was slightly reduced (13%) by the co-administration of diltiazem, although the 90% CIs of the GLS mean ratio were within the prespecified limits (Table 1). The metabol- ite:parent ratios were decreased by 66% and 75% for Cmax and AUC, respectively, by the presence of diltiazem (Table 1). Effect of ticagrelor on ketoconazole plasma concentrations Co-administration of ticagrelor with ketoconazole did not affect the steady-state plasma concentrations of ketocon- azole. The mean (% CV; range) plasma concentrations of ketoconazole pre-dosing on day 4 (i.e., in the absence of ticagrelor) were 1410 (73; 396–3540) ng/mL, and at 12 hours post-dosing with ticagrelor on day 4 were 1271 (84;334–4150) ng/mL. Effect of ticagrelor on diltiazem pharmacokinetic parameters Co-administration of ticagrelor had no effect on the pharmacokinetic parameters of diltiazem. At steady-state diltiazem, the mean (% CV) Cmax and AUC were 194 (41) ng/mL and 2998 (41) ngh/mL, respectively. In the presence of ticagrelor, Cmax and AUC were 198 (47) ng/mL and 2880 (53) ngh/mL, respectively. The point estimates (90% CIs) of the GLS mean ratios were 1.02 (0.89–1.17) for Cmax and 0.96 (0.87–1.06) for AUC, which were both within the prespecified limits of 0.80–1.25. Median tmax (range) of diltiazem was not greatly affected by co-administration of ticagrelor, i.e. 8 (0–16) hours (diltiazem alone) and 10 (4–16) hours (diltiazem þ ticagrelor). ! 2013 Informa UK Ltd www.informahealthcare.com/JDA Effect of CYP3A inhibitors on ticagrelor pharmacokinetics Teng & Butler 35 Effect of ketoconazole on ticagrelor pharmacokinetic parameters The plasma concentration–time profile of ticagrelor co-administered with ketoconazole was markedly increased compared with ticagrelor alone (Figure 3a). The median tmax of ticagrelor was similar (2 hours) in the presence and absence of ketoconazole (Table 2). The half-life of ticagrelor was increased to approximately 362% in the presence of ketoconazole. The Cmax and AUC for ticagrelor were increased by 135% and 632%, respectively, by the co-administration of ketoconazole. The 90% CIs of the GLS mean ratio for both parameters were above the upper boundary of the limits of 0.70–1.43 (Table 2). The AR-C124910XX plasma concentration–time profile was markedly decreased by co-administration of Effect of CYP3A inhibitors on ticagrelor pharmacokinetics Teng & Butler 35 Journal of Drug Assessment Volume 2 April 2013 Figure 3. Mean ( standard deviation) plasma concentration–time profiles of ticagrelor (a) and AR-C124910XX (b) following a single 90-mg oral dose of ticagrelor in the presence and absence of ketoconazole (200 mg twice daily) (n ¼ 17). Figure 3. Mean ( standard deviation) plasma concentration–time profiles of ticagrelor (a) and AR-C124910XX (b) following a single 90-mg oral dose of ticagrelor in the presence and absence of ketoconazole (200 mg twice daily) (n ¼ 17). Table 2. Pharmacokinetic parameters and statistical analyses after a single oral 90-mg dose of ticagrelor in the presence and absence of ketoconazole (200 mg twice daily). Parameter* Ticagrelor (90 mg) alone n ¼ 14 Ticagrelor (90 mg) plus ketoconazole (200 mg bid) n ¼ 14 GLS mean ratio: point estimate (90% CI) Ticagrelor Cmax (ng/mL) 654 (33) 1537 (30) 2.35 (2.13–2.60) AUC (ngh/mL) 3640 (35) 26,640 (36) 7.32 (6.43–8.34) t½ (h) 7.1 (4.9–9.8) 25.7 (16.6–31.9) – tmax (h) 2.0 (1.0–3.0) 2.0 (2.0–4.1) – AR-C124910XX Cmax (ng/mL) 207 (29) 23 (33) 0.11 (0.09–0.14) AUC (ngh/mL) 1769 (28) 782 (35) 0.44 (0.38–0.51) t½ (h) 8.0 (6.3–16.7) 20.0 (11.1–37.2) – tmax (h) 2.0 (2.0–4.0) 6.0 (4.0–12.0) – Metabolite:parent ratios Cmax ratio 0.32 (22) 0.01 (36) – AUC ratio 0.49 (24) 0.03 (33) – Values are: geometric mean (% CV) for Cmax, AUC and metabolite:parent ratios; median (range) for t½, and tmax. bid, twice daily; GLS, geometric least-square; CI, confidence interval; Cmax, maximum plasma concentration; AUC, area under the plasma concentration–time curve from time 0 to infinity; t½, terminal elimination half-life; tmax, time to Cmax. Table 2. www.informahealthcare.com/JDA ! 2013 Informa UK Ltd Effect of ketoconazole on ticagrelor pharmacokinetic parameters Pharmacokinetic parameters and statistical analyses after a single oral 90-mg dose of ticagrelor in the presence and absence of ketoconazole (200 mg twice daily). Values are: geometric mean (% CV) for Cmax, AUC and metabolite:parent ratios; median (range) for t½, and tmax. bid, twice daily; GLS, geometric least-square; CI, confidence interval; Cmax, maximum plasma concentration; AUC, area under the plasma concentration–time curve from time 0 to infinity; t½, terminal elimination half-life; tmax, time to Cmax. 36 Effect of CYP3A inhibitors on ticagrelor pharmacokinetics Teng & Butler www.informahealthcare.com/JDA ! 2013 Informa UK Ltd Journal of Drug Assessment Volume 2 April 2013 were: flatulence, photophobia, viral conjunctivitis, increased appetite, and joint swelling. reduction in Cmax and the slight decrease in AUC for AR-C124910XX, such that the metabolite:parent ratio was reduced from approximately 30% (without diltiazem) to 10% (with diltiazem). These results demonstrate that CYP3A is involved in the conversion of ticagrelor to AR-C124910XX and confirm the in vitro findings18. No deaths or serious AEs occurred in either study. In both studies, no clinically meaningful changes in hematol- ogy, clinical chemistry, urinanalysis, vital signs (including blood pressure and pulse rate), physical findings, or ECG ketoconazole study data were observed. As expected with diltiazem, which has an approved indication for the treat- ment of hypertension, there was a marginal decrease in systolic and diastolic blood pressure compared to baseline. This decrease was not prolonged. Additionally, in the diltiazem study, two volunteers had abnormal ECG evalu- ations of first-degree atria-ventricular block during administration of diltiazem/ticagrelor, which resolved by the end of the study. This finding is consistent with the pharmacology of diltiazem. It is well-recognized that there is a wide overlap of sub- strates between CYP3A and P-glycoprotein29, and studies have shown that ticagrelor is a substrate for both proteins18 (AstraZeneca, data on file). Given that diltiazem is also an inhibitor of P-glycoprotein10, its action in increasing ticagrelor exposure may result from inhibition of both CYP3A and P-glycoprotein. It is well-recognized that there is a wide overlap of sub- strates between CYP3A and P-glycoprotein29, and studies have shown that ticagrelor is a substrate for both proteins18 (AstraZeneca, data on file). Given that diltiazem is also an inhibitor of P-glycoprotein10, its action in increasing ticagrelor exposure may result from inhibition of both CYP3A and P-glycoprotein. Discussion CYP3A is abundant in the intestine and liver29, and is a key enzyme involved in the metabolism of many xeno- biotics11,12. Extensive investigations have established that drugs can act as substrates, inhibitors, or inducers of CYP3A29. Consequently, clinically significant drug–drug interactions can occur which affect efficacy and/or safety9,10,12. In vitro assessments have demonstrated that ticagrelor is a substrate and a weak inhibitor of CYP3A18. Thus, co-administration of CYP3A inhibitors would be expected to increase exposure to ticagrelor and to reduce its metabolism to AR-C124910XX. Similar proportional increases in exposure to ticagrelor have been reported in patients with atherosclerosis receiv- ing 200 mg ticagrelor twice daily (mean AUC 15,104 ngh/ mL, CV 39%) compared with those receiving 100 mg tica- grelor twice daily (5337 ngh/mL, CV 45%), at 28 days, i.e. a 283% increase in exposure compared with a dose similar to the recommended dose of ticagrelor16. This almost 3-fold increase in exposure to ticagrelor was only asso- ciated with a slight increase in minor bleeding events, i.e. 17/39 (44%) and 19/37 (51%) of patients in the twice-daily 100 and 200 mg ticagrelor groups, respect- ively16. In patients with ACS, a similar 277% increase in ticagrelor exposure at week 4 was reported in those receiving 180 mg ticagrelor twice daily compared with 90 mg twice daily (mean AUC SD, 90 mg twice daily: 4762 2443 ngh/mL; 180 mg twice daily: 13,198 4982 ngh/mL), which was associated with a dose-dependent increase in IPA17. Total bleeding events in these patients with ACS were comparable between the two treatment groups: number of events (Kaplan–Meier event rates) 90 mg twice daily: 32 (9.8) and 180 mg twice daily: 25 (8.0) through week 4; and 90 mg twice daily: 34 (10.9) and 180 mg twice daily: 33 (11.4) through week 1231. Collectively, these data indicate that the mag- nitude of increase in ticagrelor exposure seen with a mod- erate CYP3A inhibitor is unlikely to result in significant The results of the present studies using a moderate (dil- tiazem) and a strong (ketoconazole) CYP3A inhibitor con- firm in vitro data that ticagrelor is a substrate for CYP3A. Pharmacokinetic parameters of ticagrelor and its primary metabolite, AR-C124910XX, were used to assess the effects of CYP3A inhibitors. The Cmax and AUC values for ticagrelor and AR-C124910XX following a single oral dose of ticagrelor alone were consistent between the cur- rent interaction studies. Effect of ketoconazole on ticagrelor pharmacokinetic parameters Increased exposure to ticagrelor and reduced metabol- ism to its active metabolite (AR-C124910XX) resulting from diltiazem co-administration are unlikely to affect the efficacy of ticagrelor. Both the parent compound and AR-C124910XX directly inhibit platelet aggregation30 and are approximately equipotent in this respect (AstraZeneca, data on file). Therefore, the decreased exposure to AR-C124910XX may offset some of the effect of the increased exposure to the parent compound. However, as increased exposure to ticagrelor may result in prolonged inhibition of platelet aggregation (IPA), the incidence of bleeding events may also increase. No such events were reported in the ticagrelor-diltiazem study, albeit with a single 90-mg ticagrelor dose in healthy volunteers (who may not have the same propensity for bleeding as ACS patients). ! 2013 Informa UK Ltd www.informahealthcare.com/JDA Effect of CYP3A inhibitors on ticagrelor pharmacokinetics Teng & Butler 37 Acknowledgments The authors thank the Principal Investigators, Dr K. Lasseter (SFBC International, Miami, FL, USA; diltiazem study) and Dr D. Hoelscher (PPD Development Inc., West Austin, TX, USA) and their teams. Colleagues at Clinical Pharmacology & DMPK including Helen Winter (Pharmacokineticist), and those at York Bioanalytical Solutions are acknowledged for their contribution to the pharmacokinetic analyses. Statistical support was provided by Patrick Mitchell and Jennifer Hamer-Maansson (both employees of AstraZeneca). We also thank Jackie Phillipson (Gardiner-Caldwell Communications) who provided medical writing support funded by AstraZeneca. It should be noted that all volunteers in the diltiazem study were Hispanic, whereas the majority of volunteers in the ketoconazole study were Caucasian. As some differences in CYP3A activity have been observed between different races36, some caution should be used in applying these data more broadly. Conclusion Of the 14 healthy volunteers in the ticagrelor- ketoconazole study, two bleeding events were recorded, possibly due to the higher exposure to ticagrelor. In con- trast, the maximum tolerated single dose of ticagrelor (900 mg) in healthy volunteers (n ¼ 6) resulted in a much higher exposure to ticagrelor (mean [% CV], Cmax 5153 [42] ng/mL; AUC 39,153 [38] ngh/mL) and no bleeding-related AEs were recorded26,27. However, both of these healthy volunteer studies were of short duration and only investigated a single ticagrelor dose. As the co-administration of the strong CYP3A inhibitor, keto- conazole, resulted in a 5-fold increase in ticagrelor AUC, this finding indicates a significant drug–drug inter- action10. Thus, long-term co-administration of ticagrelor with drugs classified as strong CYP3A inhibitors is not recommended3,4. In conclusion, co-administration of ticagrelor with CYP3A inhibitors resulted in higher exposure to ticagrelor and lower exposure to its metabolite, AR-C124910XX. The magnitude of these effects suggests that moderate CYP3A inhibitors can be co-administered with ticagrelor without the need to modify the dose of ticagrelor. In con- trast, co-administration of ticagrelor with strong CYP3A inhibitors is not recommended. Discussion Moreover, these results were also comparable with findings from studies in healthy volunteers14. For example, following a single oral dose of ticagrelor (100 mg) in healthy volunteers (n ¼ 9), mean standard deviation (SD) for AUC was 3683 753 and 1460 408 ngh/mL for ticagrelor and AR-C124910XX, respectively14. The consistency of phar- macokinetic parameters for ticagrelor and AR- C124910XX in the present studies with other results con- firms their validity in investigating drug–drug interactions with model CYP3A inhibitors. Our findings demonstrated that moderate inhibition of CYP3A by diltiazem resulted in a near doubling of tica- grelor’s half-life, and significantly increased Cmax (69%) and AUC (174%) for ticagrelor. The metabolism of tica- grelor was also reduced as shown by the significant Effect of CYP3A inhibitors on ticagrelor pharmacokinetics Teng & Butler 37 Journal of Drug Assessment Volume 2 April 2013 bleeding events. Indeed, the use of moderate CYP3A inhibitors was permitted with ticagrelor in PLATO2,32. Ticagrelor can be co-administered with drugs classified as moderate CYP3A inhibitors, with no dose adjustment3,4. bleeding events. Indeed, the use of moderate CYP3A inhibitors was permitted with ticagrelor in PLATO2,32. Ticagrelor can be co-administered with drugs classified as moderate CYP3A inhibitors, with no dose adjustment3,4. Patients with ACS have a wide variety of co-morbidities and often receive multiple drugs7,8. The results of the present studies indicate that clinically signifi- cant drug–drug interactions with ticagrelor are likely to occur with co-administration of strong, but not moderate, CYP3A inhibitors. Examples of drugs which are strong CYP3A inhibitors include certain antifungal agents (e.g., itraconazole), antibiotics (e.g., clarithromycin, telithro- mycin), antiretroviral drugs (e.g., ritonavir, atazanavir) and antidepressants (e.g., nefazodone)10. Thus, co-admin- istration of such agents is contraindicated with ticagrelor, and alternative drugs should be considered3,4. However, ticagrelor can be co-administered with moderate CYP3A inhibitors and no dose adjustment is required3,4. Strong inhibition of CYP3A activity by ketoconazole co-administration markedly increased Cmax (135%) and AUC (632%) of ticagrelor, versus ticagrelor alone. Furthermore, these parameters for AR-C124910XX were significantly decreased by 89% and 56%, respectively. As with diltiazem, ketoconazole is also an inhibitor of P-glycoprotein10. As discussed above, the action of keto- conazole in increasing ticagrelor exposure may result from inhibition of both CYP3A and P-glycoprotein. Further studies are needed to assess the respective contributions of these pathways to increased ticagrelor exposure. Transparency Declaration of funding This study was funded by AstraZeneca. Declarations of financial relationships All of the authors are employees of AstraZeneca. In addition to inhibiting CYP3A activity, diltiazem and ketoconazole are also substrates for CYP3A33,34. Ticagrelor has been shown to be a weak-to-moderate inhibitor of CYP3A activity in vitro18. However, in the current studies, a single dose of ticagrelor did not affect the pharmacokinetics of diltiazem or the ability to achieve steady-state ketoconazole plasma levels. In contrast, while the current data do not apparently support in vitro obser- vations, other healthy volunteer studies have demon- strated that ticagrelor can have a clinically significant effect by increasing exposure to other CYP3A substrates, such as atorvastatin and simvastatin. A recent study demonstrated that ticagrelor increased mean atorvastatin Cmax and AUC by 23% and 36%, respectively35. Similarly, simvastatin Cmax and AUC were increased by 81% and 56% with ticagrelor35. R.T. is a member of the American College of Clinical Pharmacology. 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https://openalex.org/W4253342530	https://www.qeios.com/read/ORGXM3/pdf	English	null	Mebi	Definitions	2,020	cc-by	61	Mebi National Cancer Institute Qeios · Definition, February 7, 2020 Open Peer Review on Qeios Qeios ID: ORGXM3 · https://doi.org/10.32388/ORGXM3 Source National Cancer Institute. Mebi. NCI Thesaurus. Code C68570. A prefix used in binary numeral system for denoting a quantity of two raised to the power of 20, which is equal to 1 048 576. Qeios ID: ORGXM3 · https://doi.org/10.32388/ORGXM3 1/1
https://openalex.org/W2921436153	https://europepmc.org/articles/pmc6402163?pdf=render	English	null	Orthopedic surgeons’ views on the osteoporosis care gap and potential solutions: survey results	Journal of orthopaedic surgery and research	2,019	cc-by	4,660	© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. (2019) 14:72 (2019) 14:72 Barton et al. Journal of Orthopaedic Surgery and Research (2019) 14:72 https://doi.org/10.1186/s13018-019-1103-3 Open Access Abstract Introduction: Osteoporosis is often not recognized until one or more fractures occur, yet post-fracture screening remains uncommon. Orthopedic surgeons are well situated to address this care gap. Both a protocol-based approach and fracture liaison services (FLS) have been proposed. The present surveys assess orthopedists’ attitudes to these alternative models for addressing this care gap. Methods: Two digital surveys were sent to all orthopedic surgeons and orthopedic midlevel providers at a large level 1 trauma center 1.5 years apart. Results: Thirty-six of 47 survey recipients (77%) responded to the first survey; all 55 recipients (100%) responded to the second. Respondents recognized the importance of osteoporosis care, the inadequacy of current measures, and the potential of orthopedic surgeons to help address this gap. Respondents reported regular encounters with fragility fracture patients but limited familiarity with core aspects of osteoporosis screening and treatment, especially pharmacotherapy. While some respondents (40%) reported willingness to attempt a protocol-based approach to addressing this care gap, many others expressed reservations (60%) and support for a FLS-based approach was much higher (95%). Conclusions: A fracture liaison service model best fits the observed attitudes of orthopedic surgeons at this level 1 trauma center relative to a protocol-based approach. Protocol-based approaches may be preferable in alternate settings. Keywords: Fracture prevention, Fragility fracture, Osteoporosis, Quality improvement, Protocol, Fracture liaison service, FLS, Survey Keywords: Fracture prevention, Fragility fracture, Osteoporosis, Quality improvement, Protocol, Fracture liaison service, FLS, Survey Orthopedic surgeons’ views on the osteoporosis care gap and potential solutions: survey results David W. Barton1* , Daniel C. Griffin1 and Jonathan J. Carmouche1,2 Introduction patients and to initiate appropriate screening, fracture care, and definitive osteoporosis treatment in those individuals. Osteoporosis is a major cause of disability worldwide through its association with fractures. Up to 50% of women and 20% of men will experience a fragility frac- ture at some point in their lives [1]. Fragility fractures, also known as minimal trauma fractures (MTF), are de- fined as fractures resulting from a fall from standing or an equivalent low-energy mechanism. Osteoporosis pa- tients are frequently not recognized until they have ex- perienced one or more fractures. Orthopedic surgeons are positioned to diagnose osteoporosis in fracture US Medicare data indicate that 65% of women be- tween 65 and 85 years of age who sustain a fracture are neither worked up nor treated for osteoporosis within 6 months of this fracture [2]. Two models of systematic interventions have been proposed to improve secondary prevention of osteoporotic fractures. In the first model, a protocol is introduced to encour- age orthopedic surgeons to assess patients for osteopor- osis following fracture and initiate treatment as indicated [5, 6]. This model benefits from relatively low entry barriers as the orthopedist(s) may introduce the protocol with relative ease and without initially hiring * Correspondence: DaBarton@VT.edu 1Virginia Tech Carilion School of Medicine, 2 Riverside Circle, Roanoke, VA 24016, USA Full list of author information is available at the end of the article Methods Two electronic surveys were sent to all orthopedic sur- geons and dedicated orthopedic midlevel providers at a busy level 1 trauma center in the USA. The first survey was sent via email in late October 2015, the second in April 2017. Responses to each survey were allowed over a 3-week period. Two weekly reminder emails were sent in each case. The other model relies on the use of dedicated health- care professional to evaluate patients for osteoporosis following fracture and initiate treatment as indicated. This model is commonly known as the fracture liaison service (FLS). The FLS model has shown excellent re- sults at many institutions but has significant barriers to implementation [10–12]. The first survey consisted of seven questions, the second 21. Responses to each survey were assessed using descrip- tive statistics and figures. The text of each survey is in- cluded in Additional file 1. Survey results were collected and tabulated through a commercial survey service [13]. The available data make clear that osteoporosis screening and treatment post-fracture represent a major gap in care within the orthopedic scope of practice and some sort of systematic intervention is necessary to manage it. The present surveys sought to determine the relative suitability of a protocol-based versus FLS ap- proach to address this care gap at a large tertiary care center. No surveys have previously been published examining orthopedic surgeons’ attitudes towards these alternative models for osteoporosis management. Barton et al. Journal of Orthopaedic Surgery and Research (2019) 14:72 Page 2 of 7 Barton et al. Journal of Orthopaedic Surgery and Research (2019) 14:72 Page 2 of 7 Barton et al. Journal of Orthopaedic Surgery and Research (2019) 14:72 Page 2 of 7 Table 1 Recipient and respondent characteristics for both surveys Survey #1 (respondent/recipient) Survey #2 (respondent/recipient) Respondents 36/47 55/55 Physician 28/34 38/38 MD/DO orthopedic surgeon 24/28 35/35 Orthopedic traumatologist 4/4 4/4 Podiatrist (DPM) 4/4 3/3 Non-operative orthopedist 1/2 2/2 Midlevel providers (orthopedic) 8/13 17/17 PA (orthopedic) 8/12 16/16 NP (non-operative orthopedic) 0/1 1/1 additional personnel. Several articles have been pub- lished in the orthopedic literature suggesting strategies and protocols for orthopedic surgeons to use to improve their screening and initial treatment of osteoporosis in fracture patients [7–9]. Results Thirty-six of the 47 survey recipients (77%) responded to the first survey (survey #1). All 55 responded (100%) to the second survey (survey #2). Table 1 reports recipi- ent and respondent characteristics for both surveys. Survey #2 includes several questions on respondent demographics including area of specialization, years in practice, and frequency of call. Respondents’ areas of Fig. 1 Respondents’ areas of specialization based on responses to the second survey. Information on subspecialty area was not collected in the first survey. Three respondents selected multiple areas of specialization Fig. 1 Respondents’ areas of specialization based on responses to the second survey. Information on subspecialty area was not collected in the first survey. Three respondents selected multiple areas of specialization Barton et al. Journal of Orthopaedic Surgery and Research (2019) 14:72 Barton et al. Journal of Orthopaedic Surgery and Research (2019) 14:72 Page 3 of 7 specialization are depicted in Fig. 1. Forty percent of re- spondents had been in practice for 5 years or less, 31% for 6 to 15 years, and 29% for more than 15 years. Thirty-five percent of respondents did not take call. Fifty-one percent of respondents took call once per month or more, and 14% took call less than once per month. Survey #2 included two questions about respon- dents’ exposure to minimal trauma fracture patients. Most respondents (33, 60%) believed they saw five or fewer minimal trauma fracture (MTF) patients per week, 13 respondents (24%) thought they saw 6–10 MTF pa- tients, 8 thought they saw 11–20 (15%), and 1 respond- ent saw more than 20. Most respondents (43, 78%) believed they spent less than 1 h per week on MTF pa- tients, while 11 respondents (20%) spent 1–3 h, and 1 re- spondent spent more than 3 h. a b Fig. 3 a Depiction of whether respondents knew what the FRAX score is. b Depiction of the frequency of FRAX score use in respondents’ clinical practice a b Fig. 3 a Depiction of whether respondents knew what the FRAX score is. b Depiction of the frequency of FRAX score use in respondents’ clinical practice b Figure 4a shows providers’ self-reported comfort with providing patient guidance on various aspects of osteo- porosis care. Figure 4b shows self-reported frequency of osteoporosis management steps in MTF patients. Results 5 a A summary of prescriber comfort with and self-reported use of different osteoporosis medications when summed across all providers in the department. b Respondents’ self-reported reasons why survey respondents felt uncomfortable prescribing medications Figure 7c shows free-text response themes to survey #1. Free-text responses to survey #2 were less com- mon (7/55 vs 13/36). They tended to focus on general statements of support and specific suggestions for im- provements to the FLS plan. Recipients of survey #1 were asked, “If an established protocol was given to you, how likely are you to follow the protocol if it includes ordering DXA scans and writ- ing the initial prescriptions for osteoporosis medica- tions?” Responses are displayed in Fig. 6a. Forty percent of respondents either refused (11%) or expressed some reluctance (29%) to follow a protocol intended to help orthopedic surgeons better recognize and initiate man- agement of osteoporosis. In contrast, 53 respondents (95%) to survey #2 supported initiation of a fracture li- aison service to evaluate and manage low bone mass in MTF patients. Two others (4%) support the initiative with reservations, and only one person was indifferent. No one opposed the establishment of the FLS (Fig. 6b). Respondents were significantly more likely to support an FLS versus a protocol-based approach (OR 11.56, 95% CI 3.01–44.40). Results Fig- ure 5a shows a summary of prescriber comfort with and self-reported use of different osteoporosis medications when summed across all providers in the department. Figure 5b shows self-reported reasons why survey re- spondents felt uncomfortable prescribing medications. It revealed the greatest indication for not prescribing a therapy was limited experience with that therapy. Both surveys assessed respondents’ views on the im- portance of osteoporosis care, adequacy of currently pro- vided care, and sense of ownership over osteoporosis management, as displayed in Fig. 2. Survey #2 evaluated respondents’ familiarity with vari- ous aspects of osteoporosis management. Respondents’ familiarity with and use of the Fracture Risk Assessment Tool (FRAX) score is displayed in Fig. 3. a b c Fig. 2 a Respondents’ perceptions of the importance of osteoporosis care. b The perceived adequacy of osteoporosis care by setting. c How responsible respondents felt for initiating osteoporosis care a b Fig. 4 a Respondents’ self-reported comfort with providing patient guidance on various aspects of osteoporosis care. b Respondents’ self- reported frequency of osteoporosis management steps in MTF patients a a b b b Fig 4 a Respondents’ self reported comfort with providing patient b c Fig. 2 a Respondents’ perceptions of the importance of osteoporosis care. b The perceived adequacy of osteoporosis care by setting. c How responsible respondents felt for initiating osteoporosis care Fig. 2 a Respondents’ perceptions of the importance of osteoporosis care. b The perceived adequacy of osteoporosis care by setting. c How responsible respondents felt for initiating osteoporosis care Fig. 4 a Respondents’ self-reported comfort with providing patient guidance on various aspects of osteoporosis care. b Respondents’ self- reported frequency of osteoporosis management steps in MTF patients Barton et al. Journal of Orthopaedic Surgery and Research (2019) 14:72 Page 4 of 7 Barton et al. Journal of Orthopaedic Surgery and Research (2019) 14:72 Barton et al. Journal of Orthopaedic Surgery and Research Page 4 of 7 a b Fig. 5 a A summary of prescriber comfort with and self-reported use of different osteoporosis medications when summed across all providers in the department. b Respondents’ self-reported reasons why survey respondents felt uncomfortable prescribing medications a b Fi 5 A f ib f t ith d lf t d f diff t t i di ti h d ll id i a b b Fig. Discussion Orthopedic surgeons at a large level 1 trauma center with a high volume of osteoporotic fracture care over- whelmingly recognized the importance of osteoporosis. This is reassuring as it suggests the surveyed orthopedic surgeons take osteoporosis seriously as a disease entity. They recognized that post-fracture osteoporosis care is imperfect in both the orthopedic and primary care set- ting, suggesting that they recognize the post-fracture osteoporosis care gap. Because orthopedic surgeons rec- ognized the magnitude of the post-fracture osteoporosis care gap, we were able to assess orthopedic surgeons’ willingness to commit to either a protocol-based or FLS-based approach without concern that lack of com- prehension might hinder their commitment to either solution. Figure 7a and b depict respondents’ perceptions about the impact of having a dedicated advanced care provider (ACP), such as an NP or PA, facilitating osteoporosis care in the orthopedic setting following MTF on patient care and respondents’ workloads. Page 5 of 7 Barton et al. Journal of Orthopaedic Surgery and Research (2019) 14:72 (2019) 14:72 Page 5 of 7 Barton et al. Journal of Orthopaedic Surgery and Research of osteoporosis evaluation demonstrates the knowledge gap they profess. Further, when asked about their con- cerns regarding starting osteoporosis medications, they commonly cited atypical femur fractures and osteo- necrosis of the jaw. These side-effects, while real, are heavily outweighed through fracture prevention. This finding is consistent with a larger pattern of hyper-awareness of these side-effects with regard to bis- phosphonate use in the osteoporosis population. While these side-effects do occasionally occur with oral bispho- sphonates and may also occur with denosumab, they are rare at osteoporosis doses, occurring more often when used as part of chemotherapy. This misperception is thought to have contributed to a decline in the treat- ment rate of osteoporosis [14]. The potential for pill esophagitis with oral bisphosphonates was also com- monly cited and is a legitimate concern in the local population, as gastroesophageal reflux disease is highly prevalent locally and increases the risk of this relatively prevalent side-effect. The most commonly cited reason was lack of familiarity, which is a good reason in the short term given the potential for drug-drug interactions and adverse effects. While it is possible to address some of these knowledge gaps through educational interven- tions, such interventions should be targeted to most effi- ciently improve clinical care. Discussion Given the knowledge gaps documented here, it is likely more efficient to target such educational interventions to focus on identifying which patients need further work up, initial orders for work up, and where to refer them. Twenty-seven percent of respondents said one of their leading reasons for not providing better osteoporosis care was because doing so was not their responsibility. Their rejection of the role in which a post-fracture osteoporosis protocol would place them and general discomfort with the critical task of ini- tiating pharmacotherapy for osteoporosis within such a protocol suggested that implementing a protocol to ad- dress the post-fracture osteoporosis care gap might not represent the best solution at this level 1 trauma center. When asked directly whether they supported a protocol-based approach to addressing the post-fracture osteoporosis care gap, 40% of respondents were either unsure of whether they would follow it or openly rejected it. Based in part on these results, the Department of a b Fig. 6 a, b Respondents’ attitudes towards either a protocol-based or FLS-based approach to osteoporosis management a a b Fig. 6 a, b Respondents’ attitudes towards either a protocol-based or FLS-based approach to osteoporosis management Fig. 6 a, b Respondents’ attitudes towards either a protocol-based or FLS-based approach to osteoporosis management The surveyed orthopedic surgeons endorsed feeling moderately responsible for osteoporosis care. That find- ing fits with our later observations that in the free-text responses, orthopedic surgeons generally endorsed tak- ing an increased role in osteoporosis screening, but most were uncomfortable assuming a more active role in pharmacotherapy for osteoporosis. On the ranked choice scale, many orthopedists expressed unfamiliarity with how to approach osteoporosis management and most said they were uncomfortable prescribing medications for osteoporosis, further reinforcing the observation that many orthopedists are uncomfortable assuming direct responsibility for initiating osteoporosis care. This is echoed by the finding that only 22% of respondents knew what the FRAX score is, although 38% thought it sounded familiar, and 80% never use it in clinical prac- tice. Although the FRAX score is used more for primary screening than evaluation after fracture, where ortho- pedic surgeons are more commonly involved, respon- dents’ lack of familiarity with this common component Based in part on these results, the Department of Orthopaedic Surgery opted to pursue the objective of improving osteoporosis recognition and management post-fracture by using a fracture liaison service (FLS) model instead of a protocol-based approach. Discussion The FLS model also bene- fits from economies of scale in that the nurse practi- tioner running it is dedicated to bone health assessments and management in the post-fracture period and thus quickly becomes experienced in this domain. Other FLS models rely on a nurse coordinator to or- chestrate dual-energy X-ray absorptiometry (DXA) or- ders and referral to an outside provider for evaluation and management. We selected a model with an in-house nurse practitioner over a purely coordinator- based approach in part because of the scarcity of en- docrinologists, rheumatologists, and primary care pro- viders with an interest in osteoporosis in our area. Given all these factors, most of which we believe generalize, we encourage implementation of FLS pro- grams wherever feasible. Our conclusion is consistent with the recommenda- tions of professional bodies including the International Osteoporosis Foundation (IOF), American Orthopaedic Association (AOA), and American Society of Bone Min- eral Research (ASBMR), which all strongly endorse the FLS model as the preferred approach to secondary pre- vention of osteoporotic fractures [3, 4, 15]. observed in our survey since it calls on orthopedic sur- geons to refer patients with suspected fragility fractures to our fracture liaison service, where a nurse practitioner formally evaluates them for osteoporosis and initiates treatment as indicated. Because this concentrates the workup and management in the hands of a non-surgeon, it alleviates the burden of initiating medical management for osteoporosis that the orthopedic surgeons would have had under the protocol. The FLS model also bene- fits from economies of scale in that the nurse practi- tioner running it is dedicated to bone health assessments and management in the post-fracture period and thus quickly becomes experienced in this domain. Other FLS models rely on a nurse coordinator to or- chestrate dual-energy X-ray absorptiometry (DXA) or- ders and referral to an outside provider for evaluation and management. We selected a model with an in-house nurse practitioner over a purely coordinator- based approach in part because of the scarcity of en- docrinologists, rheumatologists, and primary care pro- viders with an interest in osteoporosis in our area. Given all these factors, most of which we believe generalize, we encourage implementation of FLS pro- grams wherever feasible. However, the FLS model is not feasible in all settings. The logistical and institutional political barriers to imple- menting an FLS can be extensive. A major impediment is the need to hire dedicated personnel. Discussion As ex- pected, this approach was significantly more time con- suming to implement at 20 months from initial plan to first day of clinic versus an expected 6 months for a protocol-based approach with paired lecture series. The FLS conforms better to the physician preferences Barton et al. Journal of Orthopaedic Surgery and Research (2019) 14:72 Page 6 of 7 Barton et al. Journal of Orthopaedic Surgery and Research (2019) 14:72 Page 6 of 7 a b c Fig. 7 a Depiction of respondents’ perceptions of whether having access to a dedicated bone health ACP (NP, PA) would improve patient care. b Depiction of the anticipated impact of adding a bone health ACP on respondents’ workloads. c Free-text response themes to survey #1 (n = 13) a b a c Fig. 7 a Depiction of respondents’ perceptions of whether having access to a dedicated bone health ACP (NP, PA) would improve patient care. b Depiction of the anticipated impact of adding a bone health ACP on respondents’ workloads. c Free-text response themes to survey #1 (n = 13) c Fig. 7 a Depiction of respondents’ perceptions of whether having access to a dedicated bone health ACP (NP, PA) would improve patient care. b Depiction of the anticipated impact of adding a bone health ACP on respondents’ workloads. c Free-text response themes to survey #1 (n = 13) n of respondents’ perceptions of whether having access to a dedicated bone health ACP (NP, PA) would improve patient ca e anticipated impact of adding a bone health ACP on respondents’ workloads. c Free-text response themes to survey #1 (n Fig. 7 a Depiction of respondents’ perceptions of whether having access to a dedicated bone health ACP (NP, PA) w b Depiction of the anticipated impact of adding a bone health ACP on respondents’ workloads. c Free-text response observed in our survey since it calls on orthopedic sur- geons to refer patients with suspected fragility fractures to our fracture liaison service, where a nurse practitioner formally evaluates them for osteoporosis and initiates treatment as indicated. Because this concentrates the workup and management in the hands of a non-surgeon, it alleviates the burden of initiating medical management for osteoporosis that the orthopedic surgeons would have had under the protocol. Competing interests The authors declare that they have no competing interests. The first and senior authors have a complementary interest as they were involved in the planning and implementation of the fracture liaison service at our institution. References 1. Lippuner K, Johansson H, Kanis JA, Rizzoli R. Remaining lifetime and absolute 10-year probabilities of osteoporotic fracture in swiss men and women. Osteoporos Int. 2009;20(7):1131–40. 1. Lippuner K, Johansson H, Kanis JA, Rizzoli R. Remaining lifetime and absolute 10-year probabilities of osteoporotic fracture in swiss men and women. Osteoporos Int. 2009;20(7):1131–40. p ; ( ) 2. NCQA. Osteoporosis testing and management in older women. Washington, D.C: National Center for Quality Assurance; 2017. 3. AOA. Own the bone. http://www.ownthebone.org/. Accessed 27 Feb 2018. 4. Capture the fracture. http://www.capturethefracture.org/. Accessed 27 Feb 2018. 5. Collinge C, LeBus G, Gardner MJ, Gehrig L. Osteoporosis in orthopaedic trauma patients: a diagnosis and treatment protocol. J Orthop Trauma. 2008;22(8):541–7 discussion 548-549. 2. NCQA. Osteoporosis testing and management in older women. Washington, D.C: National Center for Quality Assurance; 2017. 3. AOA. Own the bone. http://www.ownthebone.org/. Accessed 27 Feb 2018. 4. Capture the fracture. http://www.capturethefracture.org/. Accessed 27 Feb 2018. g y 3. AOA. Own the bone. http://www.ownthebone.org/. Accessed 27 Feb 2018. 4. Capture the fracture. http://www.capturethefracture.org/. Accessed 27 Feb 2018. 5. Collinge C, LeBus G, Gardner MJ, Gehrig L. Osteoporosis in orthopaedic trauma patients: a diagnosis and treatment protocol. J Orthop Trauma. 2008;22(8):541–7 discussion 548-549. 6. Scottish Intercollegiate Guidelines Network (SIGN). Management of osteoporosis and the prevention of fragility fractures. Edinburgh: SIGN; 2015. (SIGN publication no. 142). [March 2015]. Available from: http://www.sign.ac.uk 7. Dell RM, Greene D, Anderson D, Williams K. Osteoporosis disease management: what every orthopaedic surgeon should know. J Bone Joint Surg Am. 2009;91(Suppl 6):79–86. Author details 1 1Virginia Tech Carilion School of Medicine, 2 Riverside Circle, Roanoke, VA 24016, USA. 2Department of Orthopaedic Surgery, Carilion Clinic, 2331 Franklin Rd SW, Roanoke, VA 24014, USA. 1Virginia Tech Carilion School of Medicine, 2 Riverside Circle, Roanoke, VA 24016, USA. 2Department of Orthopaedic Surgery, Carilion Clinic, 2331 Franklin Rd SW, Roanoke, VA 24014, USA. This study demonstrates that orthopedic surgeons at this institution recognize the importance of osteoporosis care and the existence of a care gap whereby patients present- ing with fragility fractures are often not evaluated or treated for osteoporosis. They wish to provide better care. They support an increasing role for orthopedic surgeons in screening for osteoporosis, but many expressed reserva- tions about taking responsibility for initiating osteoporosis treatment and endorsed related knowledge gaps. These re- sponses helped guide us to establish a fracture liaison ser- vice rather than pursue a protocol-based approach to addressing the osteoporosis care gap at our institution. For orthopedists without ready access to the resources to hire personnel to establish a fracture liaison service, protocol-based approaches remain useful interventions. Abbreviations 9. Gosch M, Kammerlander C, Roth T, Doshi HK, Gasser RW, Blauth M. Surgeons save bones: an algorithm for orthopedic surgeons managing secondary fracture prevention. Arch Orthop Trauma Surg. 2013;133(8):1101–8. FLS: Fracture liaison service; MTF: Minimal trauma fracture; DXA: Dual-energy X-ray absorptiometry; MD: Medical doctor; DO: Doctor of osteopathy; DPM: Doctor of podiatric medicine FLS: Fracture liaison service; MTF: Minimal trauma fracture; DXA: Dual-energy X-ray absorptiometry; MD: Medical doctor; DO: Doctor of osteopathy; DPM: Doctor of podiatric medicine 10. Huntjens KM, van Geel TA, van den Bergh JP, et al. Fracture liaison service: impact on subsequent nonvertebral fracture incidence and mortality. J Bone Joint Surg Am. 2014;96(4):e29. Discussion New organizational structures and billing models may also need to be com- pleted prior to implementation. These steps take time which may delay introduction of an intervention. Resource-limited settings may have difficulty marshaling the initial resources to establish an FLS, despite their ten- dency to be financially self-sustaining in the long term. Smaller private practices and independent surgeons are unlikely to have the resources to establish even a minimal, nurse coordinator-based FLS. These factors may help explain why FLS centers are still relatively sparse, despite widespread calls to imple- mentation by professional bodies and in the literature. The distribution of FLS centers can be visualized using the map Barton et al. Journal of Orthopaedic Surgery and Research (2019) 14:72 Barton et al. Journal of Orthopaedic Surgery and Research Page 7 of 7 (2019) 14:72 Page 7 of 7 of IOF Capture the Fracture certified centers [4]. The large gaps in that map demonstrate the scarcity of official FLS programs throughout much of the world, even in relatively affluent nations such as the USA and Canada. Given these barriers, some institutions may opt to pursue a protocol-based approach to address this care gap. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Ethics approval and consent to participate This study was ruled exempt by the Carilion Clinic Institutional Review Board. Participation in the survey was completely voluntary. Received: 13 October 2018 Accepted: 15 February 2019 Received: 13 October 2018 Accepted: 15 February 2019 Funding This project was supported by student research funds through Virginia Tech Carilion School of Medicine. No outside funding was obtained for this project. 13. Survey monkey. https://www.surveymonkey.com/. Accessed 27 Feb 2018. 14. Bauer DC. Osteoporosis treatment after hip fracture: bad news and getting worse osteoporosis treatment after hip fracture. Invited commentary. JAMA Network Open. 2018;1(3):e180844. Authors’ contributions JC and DB drafted the surveys and distributed them. DB and DG collated and interpreted the data. DB and DG drafted the manuscript. JC and DB contributed to the conceptual design. All authors contributed to manuscript editing and approved the final draft. Additional file 1: Survey text. (DOCX 20 kb) 8. Farmer RP, Herbert B, Cuellar DO, et al. Osteoporosis and the orthopaedic surgeon: basic concepts for successful co-management of patients’ bone health. Int Orthop. 2014;38(8):1731–8. Acknowledgements h k ll 11. Huntjens KM, van Geel TC, Geusens PP, et al. Impact of guideline implementation by a fracture nurse on subsequent fractures and mortality in patients presenting with non-vertebral fractures. Injury. 2011;42(Suppl 4):S39–43. Thank you to our colleagues in the Department of Orthopaedic Surgery and the Musculoskeletal Education and Research Center at Carilion Clinic for their consistent support and encouragement of our work on this topic. 12. Nakayama A, Major G, Holliday E, Attia J, Bogduk N. Evidence of effectiveness of a fracture liaison service to reduce the re-fracture rate. Osteoporos Int. 2016;27(3):873–9. Availability of data and materials S i d il bl 15. Eisman JA, Bogoch ER, Dell R, et al. Making the first fracture the last fracture: ASBMR task force report on secondary fracture prevention. J Bone Miner Res. 2012;27(10):2039–46. Supporting data available on request. Consent for publication The hospital IRB ruled that informed consent was not necessary for this study as it did not meet the criteria for human subjects research given its focus on expert opinions. As such, specific participant consent for publication is not required.
https://openalex.org/W4386449408	https://www.researchsquare.com/article/rs-3220730/latest.pdf	English	null	Elevated serum B-cell activator factor levels predict rapid progressive interstitial lung disease in anti-melanoma differentiation associated protein 5 antibody positive dermatomyositis	Research Square (Research Square)	2,023	cc-by	5,788	Elevated serum B-cell activator factor levels predict rapid progressive interstitial lung disease in anti- melanoma differentiation associated protein 5 antibody positive dermatomyositis Jiangsu Province Hospital and Nanjing Medical University First Affiliated Hospital https://orcid.org/0000-0002-5160-2023 Jiangsu Province Hospital and Nanjing Medical University First Affiliated Hospital https://orcid.org/0000-0002-5160-2023 Research Article Keywords: dermatomyositis, melanoma differentiation-associated protein 5 antibody, serum B cell activating factor, rapidly progressive interstitial lung disease, biomarker. Posted Date: September 6th, 2023 Page 1/18 DOI: https://doi.org/10.21203/rs.3.rs-3220730/v1 Conclusions Serum BAFF could be a useful prognostic biomarker for early detecting RP-ILD risk in anti-MDA5+DM patients Serum BAFF could be a useful prognostic biomarker for early detecting RP-ILD risk in anti-MDA5+DM patients. DOI: https://doi.org/10.21203/rs.3.rs-3220730/v1 DOI: https://doi.org/10.21203/rs.3.rs-3220730/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Orphanet Journal of Rare Diseases on April 19th, 2024. See the published version at https://doi.org/10.1186/s13023-024-03153-6. Page 2/18 Results Serum BAFF level was higher in anti-MDA5+DM patients than those in ASS patients and HC (3882.32 ± 1880.09 vs. 2540.89 ± 1403.04 and 2486.28 ± 767.97 pg/mL, p = 0.0056 and 0.0038, respectively). Within anti-MDA5+DM groups, RP-ILD patients exhibited higher BAFF concentration than non-RP-ILD group (4549.78 ± 1839.97 vs. 3297.28 ± 1794.69 pg/mL, p = 0.04). The BAFF concentration was positively correlated with levels of C-reactive protein (CRP), dehydrogenase (LDH) and cytokeratin (CK) in anti-MDA5+DM patients (r = 0.350, p = 0.035; r = 0.393, p = 0.016; r = 0.518, p = 0.001; respectively). The best cut-off value of BAFF concentration was 2971.5 pg/mL by ROC curve (AUC area = 0.690, p = 0.045) and BAFF > 2971.5 pg/mL was an independent risk factor for RP-ILD using multivariate analysis (OR = 9.389, 95% CI = 1.609 - 54.769; p = 0.013). Background Rapid progressive interstitial lung disease (RP-ILD) is the leading cause of anti-melanoma differentiation associated protein 5 antibody positive dermatomyositis (anti-MDA5+DM) related death. Elevated serum B-cell activating factor (BAFF) levels have been implicated in connective tissue diseases associated ILD. Here, we evaluate whether BAFF could be a prognostic biomarker for predicting RP-ILD in anti-MDA5+DM patients. Methods Serums were collected from 39 patients with anti-MDA5+DM (20 with RP-ILD and 19 with non-RP-ILD), 20 antisynthase syndrome (ASS) patients and 20 healthy controls (HC). BAFF concentration was measured by an enzyme-linked immunosorbent assay. Study population Patients enrolled in this study include 39 anti-MDA5+DM patients (27 women and 12 men; mean age, 53.94 ± 13.46 years), 20 ASS patients (14 women and 6 men; mean age, 54.7 ± 11.87 years), and 20 age and sex matched healthy controls (HC) (11 women and 9 men; mean age, 48.95 ± 11.71 years). The diagnosis of myositis in all patients met the European NeuroMusclar Center (ENMC) criteria or Sontheimer criteria. The study protocol was approved by the First Affiliated Hospital of Nanjing Medical University Committee on Ethics (ID: 2020-SR-265). Informed consent was obtained from each study participant. . Introduction Anti-melanoma differentiation-associated gene 5 antibody positive (anti-MDA5+) dermatomyositis (DM) is a specific subtype of DM and is also known as clinically amyopathic dermatomyositis (CADM). Anti- MDA5+DM is characterized with subtle or no muscle involvement, but it is associated with a high incidence of interstitial lung disease (ILD). Importantly, approximately 30% of anti-MDA5+DM related ILD will develop life-threaten rapidly progressive interstitial lung disease (RP-ILD) [1–5]. Anti-MDA5+DM associated RP-ILD is often resistant to glucocorticoids and immunosuppressive therapy. Despite aggressive management, the mortality rate of RP-ILD patients is still as high as 50–70%. Therefore, it is Page 3/18 Page 3/18 imperative to find novel biomarkers for predicting RP-ILD prognosis and to seek new therapeutic targets for preventing progression. B cell activating factor (BAFF), also known as B lymphocyte stimulator (BLyS), is produced by various immune cells, including monocytes, macrophages, dendritic cells, and T cells [6]. BAFF binds to receptors on the surface of B cells and is crucial for B-cell differentiation, maturation and survival [7]. BAFF transgenic mice could develop the features of systemic lupus erythematosus (SLE) and Sjögren syndrome (SS) [8]. Elevated levels of BAFF have been observed in various autoimmune diseases, including ANCA-associated renal vasculitis, SLE, SS, systemic sclerosis (SSc) and idiopathic inflammatory myopathy (IIM) [7,9−10]. Consequently, targeting BAFF signaling has been considered as a promising therapeutic strategy for these diseases. Recent studies further suggested that serum BAFF levels are associated with connective tissue disease (CTD) associated ILD. Serum levels of BAFF were significantly higher in CTD-ILD patients compared to healthy subjects, and it was inversely correlated with pulmonary function [11, 12]. Overexpression of BAFF was also found in alveolar macrophages and lymphocytes in CTD-ILD patients [11]. SSc patients with elevated serum BAFF levels had decreased vital capacity more frequently [13]. Similarly, DM patients with elevated serum BAFF levels had ILD more frequently [14]. Despite the previously studies have observed the elevated serum levels of BAFF in DM patients, to the best of our knowledge, the relationship between serum BAFF levels and RP-ILD development in anti- MDA5+DM has never been reported. In the current study, we investigated the serum BAFF levels in patients with anti-MDA5+DM and explored the clinical correlation between serum BAFF level and ILD severity and progression. Measurement of BAFF The blood samples were centrifuged at 3000 rpm for 10 min, then the collected serum samples were stored at -80℃. Serum BAFF concentration (pg/mL) was measured using an enzyme-linked immunosorbent assay (ELISA) test according to the manufacturer's instructions (BOSTER, Wuhan, China). Diagnosis of RP-ILD and non-RP-ILD Anti-MDA5+DM patients and ASS patients are divided into RP-ILD and non-RP-ILD subgroups according to the presence of any of the following four conditions within one month of the onset of respiratory Page 4/18 symptoms: 1) acute and progressive worsening of dyspnea requiring hospitalization or supplementary oxygen; 2) lung function including forced vital capacity (FVC) decreases by more than 10%, or diffusion capacity for carbon monoxide of the Lung (DLCO) falls over 15% with the decreased FVC; 3) high resolution CT (HRCT) of chest demonstrates that the extent of interstitial abnormalities increased more than 20%; 4) arterial blood gas analysis suggests respiratory failure or the oxygen partial pressure reduction is greater than 10 mmHg. In the anti-MDA5+ DM group, 20 RP-ILD and 19 non-RP-ILD patients were included. In the ASS group, 6 patients had RP-ILD and 14 patients with non-RP-ILD. Statistical analysis Normal distribution measurement data were expressed as mean ± standard deviation (SD), whereas the skewed distribution measurement data were expressed as median (range). The clinical characteristics and lab data of the participants were compared with t test or analysis of variance (ANOVA) for continuous variables and chi-square tests for categorical variables. Univariate and multivariate logistic regression modeling was performed to analyze the relationship between serum BAFF concentration and the risk of RP-ILD as well as other clinical and laboratory parameters. Receiver-operating characteristic (ROC) curve was used to identify the optimal cutoff value of BAFF. Parameters in univariate logistic analysis based on statistical trend with p value less than 0.1 were included in the subsequent multivariate analysis. The p value below 0.05 was considered as statistically significant. Statistically analyzed and graph drawn using IBM SPSS Statistics 23.0 and GraphPad Prism 8, respectively. Participants characteristics The clinical characteristics of the participants are shown in Table 1. There was no significant difference in age and gender between the Anti-MDA5+DM patients and ASS patients. The proportion of myasthenia, Gottron’s sign, Heliotrope rash, V sign and periungual erythema was higher in anti-MDA5+DM groups than those in ASS patients (35.89% vs. 5%, p = 0.01 for myasthenia; 58.9% vs. 20%, p = 0.004 for Gottron’s sign; 28.2% vs. 5%, p = 0.044 for Heliotrope rash; 38.46% vs. 5%, p = 0.006 for V sign; 25.64% vs. 0%, p = 0.012 for periungual erythema, respectively). Moreover, serum ferritin (SF) was markedly increased in anti- MDA5+DM group (n = 39) as compared with those in ASS group (n = 20) (916.94 ± 771.48 vs. 258.20 ± 156.77 ng/mL, p = 0.001) (Table 1). When anti-MDA5+DM patients were divided into RP-ILD (n = 20) and non-RP-ILD groups (n = 19), patients with RP-ILD exhibited significantly higher serum CRP levels than those non-RP-ILD patients [8.81 (2.93–31.88) vs. 3.96 (2.17–7.99) mg/L, p = 0.021] (Table 2) at baseline. Page 5/18 Page 5/18 All-cause mortality rate of all anti-MDA5+DM patients was 25.64%. All deaths occurred in RP-ILD group due to respiratory failure. There were no statistically significant differences in clinical manifestations between anti-MDA5+DM patients with RP-ILD and with non-RP-ILD. All-cause mortality rate of all anti-MDA5+DM patients was 25.64%. All deaths occurred in RP-ILD group due to respiratory failure. There were no statistically significant differences in clinical manifestations between anti-MDA5+DM patients with RP-ILD and with non-RP-ILD. All-cause mortality rate of all anti-MDA5+DM patients was 25.64%. All deaths occurred in RP-ILD group due to respiratory failure. There were no statistically significant differences in clinical manifestations between anti-MDA5+DM patients with RP-ILD and with non-RP-ILD. Page 6/18 Table 1 Clinical manifestations and laboratory features. Parameters NC ASS MDA5+ p- value Case number 20 20 39 Gender, female, no. (%) 11(55%) 14(70%) 27(69.2%) 0.704 Age, mean ± SD, years 48.95 ± 11.71 54.70 ± 11.87 53.94 ± 13.46 0.834 Medical history, months 10.90 ± 16.35 3.00 ± 2.75 0.051 RPILD, no. (%) 6 (30%) 20 (51.28%) 0.119 Myasthenia, no. (%) 1 (5%) 14 (35.89%) 0.010 Gottron’s sign, no. (%) 4 (20%) 23 (58.9%) 0.004 Heliotrope rash, no. (%) 1 (5%) 11 (28.2%) 0.044 V sign, no. (%) 1 (5%) 15 (38.46%) 0.006 Shawl sign, no. (%) 1 (5%) 10 (25.64%) 0.079 Periungual erythema, no. Participants characteristics (%) 0 (0%) 10 (25.64%) 0.012 Skin ulcers, no. (%) 0 (0%) 7 (17.95%) 0.083 Mechanic’s hands, no. (%) 4 (20%) 13 (33.33%) 0.284 Arthritis, no. (%) 1 (5%) 10 (25.64%) 0.079 CK, median (range), IU/L 64.00 (39.75– 92.50) 47.00 (29.00- 105.00) 0.659 LDH, median (range), U/L 268.50 (210.25– 367.00) 319.00 (276.00- 418.00) 0.070 ESR, median (range), mm/H 36.00 (7.00–45.00) 36.00 (25.00- 48.75) 0.761 SF, mean ± SD, ng/mL 258.20 ± 156.77 916.94 ± 771.48 0.001 CRP, median (range), mg/L 6.30 (1.62–27.82) 4.94 (2.30–11.40) 0.692 BAFF, mean ± SD, pg/mL 2486.28 ± 767.97 2540.89 ± 1403.04 3882.32 ± 1880.09 0.001 Data are presented as mean ± SD or median (range) or case number (percentage); Student’s t-test, Pearson’s Chi square test and analysis of variance (ANOVA) were used to analysis. All of the clinical and laboratory parameters were obtained at the first evaluation. Anti-MDA5+DM: anti-melanoma differentiation-associated protein 5 antibody positive dermatomyositis; RP-ILD: rapidly progressive interstitial lung disease; CK: cytokeratin; CRP: C-reactive protein; LDH: dehydrogenase; ESR: erythrocyte sedimentation rate; SF: serum ferritin; BAFF: B-cell activating factor. Table 1 Clinical manifestations and laboratory features. Data are presented as mean ± SD or median (range) or case number (percentage); Student’s t-test, Pearson’s Chi square test and analysis of variance (ANOVA) were used to analysis. All of the clinical and laboratory parameters were obtained at the first evaluation. Anti-MDA5+DM: anti-melanoma differentiation-associated protein 5 antibody positive dermatomyositis; RP-ILD: rapidly progressive interstitial lung disease; CK: cytokeratin; CRP: C-reactive protein; LDH: dehydrogenase; ESR: erythrocyte sedimentation rate; SF: serum ferritin; BAFF: B-cell activating factor. Data are presented as mean ± SD or median (range) or case number (percentage); Student’s t-test, Pearson’s Chi square test and analysis of variance (ANOVA) were used to analysis. All of the clinical and laboratory parameters were obtained at the first evaluation. Anti-MDA5+DM: anti-melanoma differentiation associated protein 5 antibody positive dermatomyositis; RP ILD: rapidly progressive and laboratory parameters were obtained at the first evaluation. Anti-MDA5+DM: anti-melanoma differentiation-associated protein 5 antibody positive dermatomyositis; RP-ILD: rapidly progressive interstitial lung disease; CK: cytokeratin; CRP: C-reactive protein; LDH: dehydrogenase; ESR: erythrocyte sedimentation rate; SF: serum ferritin; BAFF: B-cell activating factor. Page 7/18 Table 2 Table 2 Clinical manifestations and laboratory features. Parameters non-RP-ILD RP-ILD p-value Case number 19 20 Gender, female, no. Participants characteristics (%) 13 (68.42%) 14 (70%) 0.915 Age, mean ± SD, years 51.05 ± 16.05 56.70 ± 10.10 0.194 Medical history, months 3.73 ± 2.90 2.35 ± 2.07 0.103 Myasthenia, no. (%) 5 (26.31%) 9 (45%) 0.224 Gottron’s sign, no. (%) 13 (68.42%) 10 (50%) 0.242 Heliotrope rash, no. (%) 5 (26.31%) 6 (30%) 0.798 V sign, no. (%) 9 (47.36%) 6 (30%) 0.265 Shawl sign, no. (%) 6 (31.57%) 4 (20%) 0.408 Periungual erythema, no. (%) 5 (26.31%) 5 (25%) 0.925 Skin ulcers, no. (%) 5 (26.31%) 2 (10%) 0.184 Mechanic’s hands, no. (%) 6 (27.27%) 7 (35%) 0.821 Arthritis, no. (%) 6 (21.21%) 4 (20%) 0.408 CK, median (range), IU/L 47.00 (35.00-122.00) 51.00 (25.50-98.25) 0.561 LDH, median (range), U/L 317.00 (253.00-418.00) 319.00 (277.25–423.00) 0.853 ESR, median (range), mm/H 33.50 (22.00-46.50) 41.00 (21.00–54.00) 0.348 SF, median (range), ng/mL 586.10 (453.25–876.70) 916.40 (641.75-1121.93) 0.692 CRP, median (range), mg/L 3.96 (2.17–7.99) 8.81 (2.93–31.88) 0.021 BAFF, mean ± SD, pg/mL 3297.28 ± 1794.69 4549.78 ± 1839.97 0.040 Data are presented as mean ± SD or median (range) or case number (percentage). Student’s t-test, Pearson’s Chi square test and analysis of variance (ANOVA) were used to analysis. All of the clinical and laboratory parameters were obtained at the first evaluation. Anti-MDA5+DM: anti-melanoma differentiation-associated protein 5 antibody positive dermatomyositis; RP-ILD: rapidly progressive interstitial lung disease; CK: cytokeratin; LDH: dehydrogenase; ESR: erythrocyte sedimentation rate; SF: serum ferritin; CRP: C-reactive protein; BAFF: B-cell activating factor. Elevated serum BAFF levels in anti-MDA5+ DM Serum levels of BAFF was significantly higher in patients with anti-MDA5+ DM than those in patients with ASS (3882.32 ± 1880.09 vs. 2540.89 ± 1403.04, p = 0.0056) or HC (3882.32 ± 1880.09 vs. 2486.28 ± 767.97 pg/mL, p = 0.003) (Fig. 1 and Table 1). No significant difference in BAFF levels was found Page 8/18 between ASS patients and HC. When patients were divided into RP-ILD and non-RP-ILD groups, the RP-ILD patients had the significantly higher BAFF levels as compared to non-RP-ILD patients (4549.78 ± 1839.97 vs. 3297.28 ± 1794.69 pg/mL, p = 0.04) (Fig. 1 and Table 2). Clinical correlation of serum BAFF levels in anti-MDA5+ DM To explore the characteristics of patients with high serum BAFF levels, we evaluated the correlation between serum BAFF levels and clinical parameters. The results showed that BAFF levels were correlated positively with the serum levels of CK, LDH and CRP in anti-MDA5+DM patients (r = 0.518, p = 0.001 for CK; r = 0.393, p = 0.016 for LDH; r = 0.350, p = 0.035 for CRP; respectively) (Fig. 2A-C). However, neither serum levels of serum ferritin (SF) nor erythrocyte sedimentation rate (ESR) level was significantly correlated with serum BAFF levels (p = 0.183 and p = 0.658, respectively) (Fig. 2D-E). Independent predictability of BAFF to RP-ILD in anti- y g g y g q g 2971.5 pg/mL were put into the multivariate regression analysis. Anti-MDA5+DM: anti-melanoma differentiation-associated protein 5 antibody positive dermatomyositis; RP-ILD: rapidly progressive interstitial lung disease; CK: cytokeratin; LDH: lactate dehydrogenase; ESR: erythrocyte sedimentation rate; CRP: C-reactive protein; SF: serum ferritin; BAFF: B-cell activating factor. Independent predictability of BAFF to RP-ILD in anti- In order to predict the occurrence of RP-ILD in anti-MDA5+DM, ROC curve was used to determine the cut- off value of serum BAFF concentration. The data showed that 2971.5 pg/mL has the highest diagnostic efficiency to distinguish the patients with or without RP-ILD (AUC area = 0.690, 95% CI = 0.514–0.867, sensitivity 90% and specificity 55.6%; p = 0.045) (Fig. 3). In order to further clarify the application efficiency of the above cut-off value in anti-MDA5+DM patients with RP-ILD, we next performed the logistic regression analysis. After continuous variables were transformed into dichotomies, the results of univariate analysis showed that CRP > 8 mg/L and BAFF > 2971.5 pg/mL were candidate risk factors for anti-MDA5+DM with RP-ILD. When they were included into the multivariate equation, BAFF > 2971.5 pg/mL was an independent risk factor for RP-ILD in anti-MDA5+DM patients (OR = 9.389, 95% CI = 1.609– 54.769; p = 0.013) (Table 3). Page 9/18 Table 3 Table 3 Logistic analysis of RP-ILD influenced by characteristics of DM patients. RP-ILD Unadjusted Adjusted OR (95% CI) p-value OR (95% CI) p-value Gender, Female 1.077 (0.276–4.197) 0.915 Age 1.034 (0.988–1.078) 0.198 Myasthenia 2.291 (0.595–8.825) 0.228 Gottron’s sign 0.462 (0.125–1.703) 0.246 Heliotrope rash 1.350 (0.379–4.811) 0.643 V sign 0.690 (0.213–2.237) 0.536 shawl signs 0.675 (0.174–2.614) 0.570 Periungual erythema 1.333 (0.341–5.208) 0.679 arthritis 0.674 (0.174–2.614) 0.570 mechanic hands 1.185 (0.382–3.675) 0.769 skin ulcers 0.467(0.083–2.627) 0.387 CK, > 200 U/L 0.593 (0.088–4.009) 0.592 LDH, > 227 IU/L 2.235 (0.186–26.908) 0.526 ESR, > 21 mm/h 1.133(0.198–6.486) 0.888 CRP, > 8 mg/L 3.750 (0.917–15.342) 0.066 2.337 (0.487–11.228) 0.289 SF, > 336.2 ng/ml 1.373 (0.462–4.073) 0.568 BAFF, > 2971.5 pg/ml 11.250 (1.991–63.560) 0.006 9.389 (1.609–54.769) 0.013 Binary logistical regression analysis was used in regression equation. CRP > 8 mg/L and BAFF > 2971.5 pg/mL were put into the multivariate regression analysis. Anti-MDA5+DM: anti-melanoma differentiation-associated protein 5 antibody positive dermatomyositis; RP-ILD: rapidly progressive interstitial lung disease; CK: cytokeratin; LDH: lactate dehydrogenase; ESR: erythrocyte sedimentation rate; CRP: C-reactive protein; SF: serum ferritin; BAFF: B-cell activating factor. Binary logistical regression analysis was used in regression equation. CRP > 8 mg/L and BAFF > 2971.5 pg/mL were put into the multivariate regression analysis. Anti-MDA5+DM: anti-melanoma differentiation-associated protein 5 antibody positive dermatomyositis; RP-ILD: rapidly progressive interstitial lung disease; CK: cytokeratin; LDH: lactate dehydrogenase; ESR: erythrocyte sedimentation rate; CRP: C-reactive protein; SF: serum ferritin; BAFF: B-cell activating factor. Discussion In the present study, we measured serum BAFF level and explored its clinical implication in patients with anti-MDA5+DM. We found that serum BAFF levels were significantly increased in anti-MDA5+DM patients compared with those in ASS patients and HC. Moreover, serum BAFF level was associated with disease severity of ILD, and BAFF levels > 2971.5 pg/mL was an independent risk factor for RP-ILD in anti- Page 10/18 Page 10/18 MDA5+DM patient. These findings indicated that BAFF participates in the pathological process of ILD and might serve as a biomarker for RP-ILD risk prediction in anti-MDA5+DM patients. MDA5+DM patient. These findings indicated that BAFF participates in the pathological process of ILD and might serve as a biomarker for RP-ILD risk prediction in anti-MDA5+DM patients. Previous studies have revealed that serum BAFF level was elevated in DM, and associated with the prevalence of ILD [15, 16]. The major finding of current study is that serum BAFF level was significant positive correlation with RP-ILD in anti-MDA5+DM. These findings have at least 2 important clinical implications. First, our present results suggest that serum BAFF had certain predictive value for RP-ILD and poor prognosis in patients with anti-MDA5+DM. As a life-threatening complication of anti-MDA5+DM, early recognition and early treatment of RP-ILD is the key to improve prognosis. Although several serum risk factors, including anti-MDA5 antibody titer, CRP, LDH, and SF [17–19], are thought to be associated with the development of ILD in MDA5+ patients, however, they are not a good predictor of the occurrence of RP- ILD. In current study, with a cut-off value of 2971.5 pg/ml of serum BAFF can help distinguish RP-ILD patients from anti-MDA5+DM patients (Fig. 3). Multivariate regression further suggested that BAFF was an independent risk factor for RP-ILD in anti-MDA5+DM. Second, our finding of an association of RP-ILD with elevated serum imply BAFF-blocking therapy could be an attractive novel treatment for anti-MDA5+DM patients, especially patients with a tendency toward RP-ILD. Elevated CD19+ B cells have been found in anti-MDA5+DM patients compared with ASS, and are associated with poor outcomes [20]. As a salvage therapy strategy, Rituximab is usually selected for the treatment of RP-ILD with a resistance to conventional therapy or with a life-threatening condition [21]. However, the uncertain efficacy and high risk of infection limits the widespread adoption of B-cell depletion in clinical practice. BAFF plays an important role in the activation and homeostasis of B cell. Discussion The increased serum BAFF level is significantly correlated with disease-specific antibodies level in some autoimmune diseases, such as anti-SSA in SS, anti-dsDNA in SLE, anti-histone in SSc [22–25] and anti-Jo1 in IIM [26–28]. It is thought that BAFF may contribute to the development of RP-ILD in anti-MDA5+DM by promoting the survival and activation of autoreactive B cells, and then enhance the production of a variety of autoantibodies, including anti-MDA5 autoantibodies. Besides, BAFF contributes to progression of ILD by impairing apoptosis of naive B cells via BAFF receptor [29]. Additionally, BAFF also promotes pulmonary interstitial fibrosis by acting as a potent inducer of TIMP-1, α-SMA, CCL2, and IL-6 [30]. Therefore, more research is needed to determine the long-term safety and efficacy of BAFF inhibition in the treatment of anti- MDA5+DM associated RP-ILD. We also found that BAFF levels were correlated positively with the serum level of CRP, CK and LDH in anti- MDA5+DM patients. We previously reported elevated serum CRP and LDH levels represented the high inflammation condition in anti-MDA5+ DM patients and linked to RP-ILD and poor outcomes [31]. Consistent with these findings, the current data indicate that excessive BAFF level is involved in the Page 11/18 Page 11/18 Page 11/18 inflammatory response or autoimmune reaction in anti-MDA5+DM patients. Combined serum BAFF levels with other serum markers including CRP and LDH may reflect severity of lung injury and can help early identify RP-ILD patients in anti-MDA5+DM patients. This study had several limitations. Due to the low incidence of MDA5, we included a limited number of cases in this study. Besides, lung function tests were not performed in all patients and there was a lack of correlation between BAFF levels and FVC or DLCO values, which have been reported as risk factors for RP-ILD combined with anti-MDA5+DM [32]. In addition, we did not obtain the dynamic change of BAFF level with treatment response and the number of effector B cells at the matched time point. These results are need to further validation in a large and prospective cohort. Consent for publication Not applicable. Conclusions The current study revealed that elevated serum BAFF levels are associated with an increased risk of developing RP-ILD. Therefore, combining serum BAFF levels with imaging and clinical features can aid the early detection of high-risk RP-ILD patients. Furthermore, BAFF may be a potential target for preventing RP-ILD in anti-MDA5+DM patients. Further research is needed to fully understand the role of BAFF in RP-ILD development, and explore the potential benefits and risks of BAFF inhibition in anti- MDA5+DM patients. Ethics approval and consent to participate The study protocol was approved by the First Affiliated Hospital of Nanjing Medical University Committee on Ethics (ID: 2020-SR-265). Informed consent was obtained from each study participant. . Availability of data and materials All data generated or analysed during this study are included in this published article . Competing interests The authors declare that they have no competing interests. and Education (JSDW202203) of China. and Education (JSDW202203) of China. Acknowledgements We thank all authors for their continuous and excellent support with patients’ data collection, data analysis, statistical analysis, and valuable suggestions for the article. Authors’ contributions WT designed the study. YS and MZ contributed to the study design. HY, CL, YZ, LX recruited patients and obtained consent for the study. YS, CL and YQ performed the experimental work. YS and CL analyzed and interpreted the data. YS, FW and WT wrote the manuscript. All authors read, revised, and approved the final version of the manuscript. Funding This work was supported by the National Natural Science Foundation of China (NSFC): 82171794, 81971532 and Jiangsu Province Capability Improvement Project through Science, Technology Page 12/18 and Education (JSDW202203) of China. Authors’ information Department of Rheumatology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, China. Yumeng Shi, Hanxiao You, Chang Liu, Yulu Qiu, Chengyin Lv, Yujing Zhu, Lingxiao Xu, Miaojia Zhang, Wenfeng Tan Yumeng Shi, Hanxiao You, Chang Liu, Yulu Qiu, Chengyin Lv, Yujing Zhu, Lingxiao Xu, Miaojia Zhang, Wenfeng Tan Department of Cardiology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, China. Department of Cardiology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, China. Fang wang References Circulating and skin biopsy-present cytokines related to the pathogenesis of cutaneous lupus erythematosus. Autoimmun Rev. 2023;22(2):103262. 10. Kumar G, Axtell RC. Dual Role of B Cells in Multiple Sclerosis. Int J Mol Sci. 2023;24(3):2336. Axtell RC. Dual Role of B Cells in Multiple Sclerosis. Int J Mol Sci. 2023;24(3 10. Kumar G, Axtell RC. Dual Role of B Cells in Multiple Sclerosis. Int J Mol S 11. Hamada T, Samukawa T, Kumamoto T, Hatanaka K, Tsukuya G, Yamamoto M, et al. 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Elevated serum B-cell activating factor levels in patients with dermatomyositis: Association with interstitial lung disease. J Dermatol. 2019;46(12):1190-6. 15. Kobayashi N, Kobayashi I, Mori M, Sato S, Iwata N, Shigemura T, et al. Increased Serum B Cell Activating Factor and a Proliferation-inducing Ligand Are Associated with Interstitial Lung Disease in Patients with Juvenile Dermatomyositis. J Rheumatol. 2015;42(12):2412-8. 15. Kobayashi N, Kobayashi I, Mori M, Sato S, Iwata N, Shigemura T, et al. Increased Serum B Cell Activating Factor and a Proliferation-inducing Ligand Are Associated with Interstitial Lung Disease in Patients with Juvenile Dermatomyositis. J Rheumatol. 2015;42(12):2412-8. 16. Krystufkova O, Hulejova H, Mann HF, Pecha O, Putova I, Ekholm L, et al. Serum levels of B-cell activating factor of the TNF family (BAFF) correlate with anti-Jo-1 autoantibodies levels and disease activity in patients with anti-Jo-1positive polymyositis and dermatomyositis. Arthritis Res Ther. 2018;20(1):158. 17. Gono T, Masui K, Nishina N, Kawaguchi Y, Kawakami A, Ikeda K, et al. Risk Prediction Modeling Based on a Combination of Initial Serum Biomarker Levels in Polymyositis/Dermatomyositis- Associated Interstitial Lung Disease. Arthritis Rheumatol. 2021;73(4):677-86. 17. References 1. Sato S, Hirakata M, Kuwana M, Suwa A, Inada S, Mimori T, et al. Autoantibodies to a 140-kd polypeptide, CADM-140, in Japanese patients with clinically amyopathic dermatomyositis. Arthritis Rheum. 2005;52(5):1571-6. 1. Sato S, Hirakata M, Kuwana M, Suwa A, Inada S, Mimori T, et al. Autoantibodies to a 140-kd polypeptide, CADM-140, in Japanese patients with clinically amyopathic dermatomyositis. Arthritis Rheum. 2005;52(5):1571-6. 2. Sato S, Hoshino K, Satoh T, Fujita T, Kawakami Y, Fujita T, et al. RNA helicase encoded by melanoma differentiation-associated gene 5 is a major autoantigen in patients with clinically amyopathic dermatomyositis: Association with rapidly progressive interstitial lung disease. Arthritis Rheum. 2009;60(7):2193-200. 2. Sato S, Hoshino K, Satoh T, Fujita T, Kawakami Y, Fujita T, et al. RNA helicase encoded by melanoma differentiation-associated gene 5 is a major autoantigen in patients with clinically amyopathic dermatomyositis: Association with rapidly progressive interstitial lung disease. Arthritis Rheum. 2009;60(7):2193-200. 3. Nakashima R, Imura Y, Kobayashi S, Yukawa N, Yoshifuji H, Nojima T, et al. The RIG-I-like receptor IFIH1/MDA5 is a dermatomyositis-specific autoantigen identified by the anti-CADM-140 antibody. Rheumatology (Oxford). 2010;49(3):433-40. 4. Hamaguchi Y, Kuwana M, Hoshino K, Hasegawa M, Kaji K, Matsushita T, et al. Clinical correlations with dermatomyositis-specific autoantibodies in adult Japanese patients with dermatomyositis: a multicenter cross-sectional study. Arch Dermatol. 2011;147(4):391-8. Page 13/18 Page 13/18 5. Koga T, Fujikawa K, Horai Y, Okada A, Kawashiri SY, Iwamoto N, et al. The diagnostic utility of anti- melanoma differentiation-associated gene 5 antibody testing for predicting the prognosis of Japanese patients with DM. Rheumatology (Oxford). 2012;51(7):1278-84. 6. Kumric M, Zivkovic PM, Ticinovic Kurir T, Vrdoljak J, Vilovic M, Martinovic D, et al. Role of B-Cell Activating Factor (BAFF) in Inflammatory Bowel Disease. Diagnostics (Basel). 2021;12(1):45. 6. Kumric M, Zivkovic PM, Ticinovic Kurir T, Vrdoljak J, Vilovic M, Martinovic D, et al. Role of B-Cell Activating Factor (BAFF) in Inflammatory Bowel Disease. Diagnostics (Basel). 2021;12(1):45. 7. Singh H, Kumar U, Senapati S. Translational implications of humoral and cellular immune dysfunction in granulomatosis with polyangiitis. Cytokine. 2023;164:156154. 7. Singh H, Kumar U, Senapati S. Translational implications of humoral and cellular immune dysfunction in granulomatosis with polyangiitis. Cytokine. 2023;164:156154. 8. Saez Moya M, Gutierrez-Cozar R, Punet-Ortiz J, Rodriguez de la Concepcion ML, Blanco J, Carrillo J, et al. Autoimmune B Cell Repertoire in a Mouse Model of Sjogren's Syndrome. Front Immunol. 2021;12:666545. 9. Erazo-Martinez V, Tobon GJ, Canas CA. References Gono T, Masui K, Nishina N, Kawaguchi Y, Kawakami A, Ikeda K, et al. Risk Prediction Modeling Based on a Combination of Initial Serum Biomarker Levels in Polymyositis/Dermatomyositis- Associated Interstitial Lung Disease. Arthritis Rheumatol. 2021;73(4):677-86. 18. Xu Y, Yang CS, Li YJ, Liu XD, Wang JN, Zhao Q, et al. Predictive factors of rapidly progressive- interstitial lung disease in patients with clinically amyopathic dermatomyositis. Clin Rheumatol. 2016;35(1):113-6. 18. Xu Y, Yang CS, Li YJ, Liu XD, Wang JN, Zhao Q, et al. Predictive factors of rapidly progressive- interstitial lung disease in patients with clinically amyopathic dermatomyositis. Clin Rheumatol. 2016;35(1):113-6. Page 14/18 Page 14/18 19. Feng L, Wang H, Zhao Y, Zhao T, Zhang J, Huang F, et al. Clinical manifestations and outcome of anti-aminoacyl-transfer RNA synthetase antibody and anti-melanoma differentiation-associated gene 5 antibody positive patients with interstitial lung disease. Clin Exp Rheumatol. 2022;40(1):97- 103. 20. Wang Y, Zhu L, Ju B, Luo J, Li Q, Lv X, et al. Alterations of peripheral blood B cell subsets in Chinese patients with adult idiopathic inflammatory myopathies. Clin Exp Rheumatol. 2022;40(2):260-6. 21. He C, Li W, Xie Q, Yin G. Rituximab in the Treatment of Interstitial Lung Diseases Related to Anti- Melanoma Differentiation-Associated Gene 5 Dermatomyositis: A Systematic Review. Front Immunol. 2021;12:820163. 22. Mariette X, Roux S, Zhang J, Bengoufa D, Lavie F, Zhou T, et al. The level of BLyS (BAFF) correlates with the titre of autoantibodies in human Sjogren's syndrome. Ann Rheum Dis. 2003;62(2):168-71. 23. Stohl W, Metyas S, Tan SM, Cheema GS, Oamar B, Roschke V, et al. Inverse association between circulating APRIL levels and serological and clinical disease activity in patients with systemic lupus erythematosus. Ann Rheum Dis. 2004;63(9):1096-103. 24. Matsushita T, Hasegawa M, Yanaba K, Kodera M, Takehara K, Sato S. Elevated serum BAFF levels in patients with systemic sclerosis: enhanced BAFF signaling in systemic sclerosis B lymphocytes. Arthritis Rheum. 2006;54(1):192-201. 25. Vosters JL, Roescher N, Polling EJ, Illei GG, Tak PP. The expression of APRIL in Sjogren's syndrome: aberrant expression of APRIL in the salivary gland. Rheumatology (Oxford). 2012;51(9):1557-62. 26. Krystufkova O, Vallerskog T, Helmers SB, Mann H, Putova I, Belacek J, et al. Increased serum levels of B cell activating factor (BAFF) in subsets of patients with idiopathic inflammatory myopathies. Ann Rheum Dis. 2009;68(6):836-43. 27. Baek A, Park HJ, Na SJ, Shim DS, Moon JS, Yang Y, et al. Figures Figures Figure 1 Serum BAFF concentration in HC, ASS and anti-MDA5+DM patients. A. Serum BAFF levels in HC (n = 20 ASS (n = 20) and anti-MDA5+DM patients (n = 39); B. Serum BAFF concentrations in anti-MDA5+DM patients with (n = 20) and without RP-ILD (n = 19); *p< 0.01; p< 0.05. References The expression of BAFF in the muscles of patients with dermatomyositis. J Neuroimmunol. 2012;249(1-2):96-100. 28. Lopez De Padilla CM, McNallan KT, Crowson CS, Bilgic H, Bram RJ, Hein MS, et al. BAFF expression correlates with idiopathic inflammatory myopathy disease activity measures and autoantibodies. J Rheumatol. 2013;40(3):294-302. 29. Maglione PJ, Gyimesi G, Cols M, Radigan L, Ko HM, Weinberger T, et al. BAFF-driven B cell hyperplasia underlies lung disease in common variable immunodeficiency. JCI Insight. 2019;4(5): e122728. 30. Matson EM, Abyazi ML, Bell KA, Hayes KM, Maglione PJ. B Cell Dysregulation in Common Variable Immunodeficiency Interstitial Lung Disease. Front Immunol. 2020;11:622114. 31. Xu L, You H, Wang L, Lv C, Yuan F, Li J, et al. Identification of Three Different Phenotypes in Anti- Melanoma Differentiation-Associated Gene 5 Antibody-Positive Dermatomyositis Patients: Implications for Prediction of Rapidly Progressive Interstitial Lung Disease. Arthritis Rheumatol. 2023;75(4):609-19. Page 15/18 32. Wu W, Xu W, Sun W, Zhang D, Zhao J, Luo Q, et al. Forced vital capacity predicts the survival of interstitial lung disease in anti-MDA5 positive dermatomyositis: a multi-centre cohort study. Page 15/18 32. Wu W, Xu W, Sun W, Zhang D, Zhao J, Luo Q, et al. Forced vital capacity predicts the survival of interstitial lung disease in anti-MDA5 positive dermatomyositis: a multi-centre cohort study. Page 15/18 Rheumatology (Oxford). 2021;61(1):230-9. Figure 1 Figure 1 Serum BAFF concentration in HC, ASS and anti-MDA5+DM patients. A. Serum BAFF levels in HC (n = 20), ASS (n = 20) and anti-MDA5+DM patients (n = 39); B. Serum BAFF concentrations in anti-MDA5+DM patients with (n = 20) and without RP-ILD (n = 19); *p< 0.01; p< 0.05. Serum BAFF concentration in HC, ASS and anti-MDA5+DM patients. A. Serum BAFF levels in HC (n = 20), ASS (n = 20) and anti-MDA5+DM patients (n = 39); B. Serum BAFF concentrations in anti-MDA5+DM patients with (n = 20) and without RP-ILD (n = 19); *p< 0.01; p< 0.05. Page 16/18 Page 16/18 Figure 2 The correlation of serum BAFF and disease activity in anti-MDA5+DM. Correlation of serum BAFF levels with serum levels of CRP(A)，LDH (B), CK (C), SF (D), ESR (E) in patients with anti-MDA5+DM at the first evaluation. Serum BAFF levels were determined by a specific ELISA. Figure 2 Figure 2 The correlation of serum BAFF and disease activity in anti-MDA5+DM. Correlation of serum BAFF levels with serum levels of CRP(A)，LDH (B), CK (C), SF (D), ESR (E) in patients with anti-MDA5+DM at the first evaluation. Serum BAFF levels were determined by a specific ELISA. The correlation of serum BAFF and disease activity in anti-MDA5+DM. Correlation of serum BAFF levels with serum levels of CRP(A)，LDH (B), CK (C), SF (D), ESR (E) in patients with anti-MDA5+DM at the first evaluation. Serum BAFF levels were determined by a specific ELISA. Page 17/18 Page 17/18 Figure 3 ROC curve of serum BAFF concentration in anti-MDA5+DM patients. ROC curve shows the area under th curve (AUC) of serum BAFF concentration in anti-MDA5+DM patients based on patients whether development of RP-ILD. Figure 3 ROC curve of serum BAFF concentration in anti-MDA5+DM patients. ROC curve shows the area under the curve (AUC) of serum BAFF concentration in anti-MDA5+DM patients based on patients whether development of RP-ILD. ROC curve of serum BAFF concentration in anti-MDA5+DM patients. ROC curve shows the area under the curve (AUC) of serum BAFF concentration in anti-MDA5+DM patients based on patients whether development of RP-ILD. Page 18/18
https://openalex.org/W2336048935	https://europepmc.org/articles/pmc5403848?pdf=render	English	null	Factors related to monitoring during admission of acute patients	Journal of clinical monitoring and computing	2,016	cc-by	6,107	Thomas Schmidt1 • Camilla N. Bech2 • Mikkel Brabrand2,3 • Uffe Kock Wiil1 • Annmarie Lassen2 Received: 17 December 2015 / Accepted: 5 April 2016 / Published online: 12 April 2016 The Author(s) 2016. This article is published with open access at Springerlink.com Abstract Understanding the use of patient monitoring systems in emergency and acute facilities may help to identify reasons for failure to identify risk patients in these settings. Hence, we investigate factors related to the uti- lization of automated monitoring for patients admitted to an acute admission unit by introducing monitor load as the proportion between monitored time and length of stay. A cohort study of patients admitted and registered to patient monitors in the period from 10/10/2013 to 1/10/2014 at the acute admission unit of Odense University Hospital in Denmark. Admissions with at least one measurement were analyzed using quantile regression by looking at the impact of distance from nursing ofﬁce, number of concurrent patients, wing type (medical/surgical), age, sex, comorbidities, and severity conditioned on how much patients were monitored during their admissions. We reg- istered 11,848 admissions, of which we were able to link patient monitor readings to 3149 (26.6 %) with 50 % being monitored \1.4 % of total admission time. Distance from nursing ofﬁce had little inﬂuence on patients monitored \10 % of their admission time. But for other patients, being positioned further away from the ofﬁce reduced the level of monitoring. Higher levels of severity were related to higher degrees of monitoring, but being admitted to the surgical wing reduce how much patients were monitored, and periods with many concurrent patients lead to a small increase in monitoring. We found a signiﬁcant variation concerning how much patients were monitored during admission to an acute admission unit. Our results point to potential patient safety improvements in clinical proce- dures, and advocate an awareness of how patient moni- toring systems are utilized. Electronic supplementary material The online version of this article (doi:10.1007/s10877-016-9876-y) contains supplementary material, which is available to authorized users. & Thomas Schmidt schmidt@mmmi.sdu.dk Camilla N. Bech Camilla.noergaard.bech@rsyd.dk Mikkel Brabrand mikkel.brabrand@rsyd.dk Uffe Kock Wiil ukwiil@mmmi.sdu.dk Annmarie Lassen annmarie.lassen@rsyd.dk 1 The Maersk Mc-Kinney Moeller Institute, University of Southern Denmark, Campusvej 55, 5230 Odense, Denmark 2 Department of Emergency Medicine, Odense University Hospital, Odense, Denmark 3 Department of Emergency Medicine, Hospital of South West Jutland, Esbjerg, Denmark Keywords Emergency departments Computerized decision support Patient monitoring J Clin Monit Comput (2017) 31:641–649 DOI 10.1007/s10877-016-9876-y ORIGINAL RESEARCH Factors related to monitoring during admission of acute patients Thomas Schmidt1 • Camilla N. Bech2 • Mikkel Brabrand2,3 • Uffe Kock Wiil1 • Annmarie Lassen2 1 Background Patients of all sorts and with a wide range of diagnoses are treated in emergency departments (ED) around the world every single day. Keeping track of such a diverse group of patients challenges both clinicians and systems. To cope with this, several health information systems have been developed speciﬁcally for managing the ﬂow and treatment of patients. Still, a substantial number of acutely admitted patients deteriorate during their admission with an increased risk of adverse outcomes [1]. There is wide- spread consensus that the risk of such deterioration can be 1 The Maersk Mc-Kinney Moeller Institute, University of Southern Denmark, Campusvej 55, 5230 Odense, Denmark 2 Department of Emergency Medicine, Odense University Hospital, Odense, Denmark 3 Department of Emergency Medicine, Hospital of South West Jutland, Esbjerg, Denmark 12 3 J Clin Monit Comput (2017) 31:641–649 642 reduced by a more frequent and rigorous approach to monitoring of patient vital signs [2]. However, the decision to continuously monitor a patient’s vital signs can still be a result of multiple causes; e.g., raised patient concern, or to optimize working procedures by not having to attach sen- sors repeatedly on patients requiring frequent registrations. Or perhaps also as a mean for boosting situational aware- ness in high load periods [3]. As such, monitoring can be viewed as an important part of the afferent limb as it provides feedback needed to initiate interventions [4]. The notion that an increased rate of vital sign registrations reduce the risk of adverse events has spurred a surge in quality assurance programs worldwide, despite concerns about the effectiveness of routinely measured vital signs have been raised [5]. Partly because the process of vital sign registrations is associated with both human and machine related errors [6]. Evidently, there exists a gap between the clinical reality and the vital sign registration procedures deﬁned by guidelines [7, 8], and as most research on automated monitoring has been conducted in the settings of intensive care units (ICU) [9] we in this work focus instead on monitoring in acute settings. deemed necessary, an intensive care consultant is contacted and need and relevancy for transfer has to be acknowl- edged by both parties. The ward is structured into three wings, one wing for surgical patients (12 beds), and two wings for medical patients (18 and 16 beds). 2.1 Data description We expect very sick patients to be monitored more than the less sick; and, it has been documented that clinicians are prone to skip vital sign registrations of less severe patients [8]. This can potentially lead to dire consequences for these patients as the risk of deterioration is present across all severity levels [10]. Understanding the utilization of patient monitoring systems in the dispersed and shared working environments of EDs and acute wards may help to identify some of the reasons for failure to rescue patients [11]. All vital signs from all monitors at the ward in the period from 10th of October 2013 to 1st of October 2014 are captured in a research database using a customized appli- cation written in Java. The department relies on Philips IntelliVue MP30/50 monitors in a networked setup as monitoring information from beds are aggregated on Phi- lips IntelliVue Information Centers in each nursing ofﬁce. When a patient is attached to a monitor our system receives a packet containing vital sign information at different intervals. Every minute we register heart rate and respira- tion rate from 3-lead ECG, pulse rate and peripheral oxy- gen saturation (spO2) measured via pulse oximetry. Depending on the clinical assessment of the patient, sys- tolic and diastolic blood pressures are registered in inter- vals from 5 to 60 min using cuffs. In this project, nurses are asked to enter patient identiﬁcation into the Philips moni- tors by personal identiﬁcation number (PIN) and name, thus enabling us to link the vital signs from a given bed location to a speciﬁc patient. Apart from this, the data collection instills no further change to existing clinical practice. We include all patients registered on the monitor with at least one measurement. However, not all patients get their information entered into the monitors, and con- sequently our system holds an amount of vital values which we cannot associate with speciﬁc patients. The character- istics of the not-identiﬁed patients are included in our analysis to enable between-group comparisons. Although increased levels of automated monitoring may improve the detection of patient deterioration, several factors may inﬂuence the extent to which a patient is being monitored. 2.1 Data description The purpose of this paper is to investigate the use of automated monitoring of patients admitted to an acute admission unit by analyzing how much the effects of distance from the nursing ofﬁce, number of concurrently admitted patients, wing type (medical/surgical), age, sex, comorbidities, and severity change conditioned on how much patients are monitored during admission. 1 Background The processing and management of patients in this ED has been documented in an earlier ﬁeld study conducted by the ﬁrst author [12]. In relevance to this paper, the most important aspects are the department’s reliance on a 5-level triage and observation regimen system which deﬁnes a baseline level of clinical alertness for each level (Blue, Green, Yellow, Orange, Red), and that the bedside ward is structured into three distinct wings, with a nurse ofﬁce in the center of each wing. Each bed on every wing is equipped with its own vital signs monitoring unit. The degree of monitoring for each patient is deﬁned by the attending physician based on the observation regimen, and in some cases adjusted by nurses afterwards. The assigned observation regimen is registered in the patient’s electronic medical record. 2.2 Analysis We aim to describe patient and department related factors and their relationship to how much patients are monitored. During their admission, patients will be intermittently attached to bedside vital sign monitors. We use the extent to which a patient is monitored as our point of interest by deﬁning monitor load as the proportion between time attached to monitor and length of stay. A monitor load percentage of 100 % means that the patient is being con- tinuously monitored throughout their entire admission; which in the study settings translates to 1 automatic reading per minute. Between group comparison for distribution of triage categories as severity, and comorbidities between patients registered on the monitors, and not-registered patients are evaluated using Chi squared tests. The preprocessing and regression analysis is conducted in R (version 3.1.1) using the quantreg package [21]. The data is preprocessed by calculating the all the aggregated exposure variables such as distance, comorbidity and concurrent load. Access to the registry of patient data was approved by the Danish Data Protection Agency (Datatilsynet—J.nr. 2013-41-2238), and the Danish Health and Medicines Authority (Sundhedsstyrelsen—J.nr. 3-3013-518/1). The study has been presented to the Research Ethics Committee of Southern Denmark, but as this is a non-interventional study an approval was not needed according to Danish law. p Table 1 provides an overview of the exposure variables used in the model. Categorical variables are automatically converted to dummy variables. Concurrent patient load is calculated based on the number of active beds in the wing during each patient’s admission period. The analysis includes exposures relating speciﬁcally to each individual; age, observation regimen, Charlson comorbidity index [15], and sex. And external factors; distance from nursing ofﬁce, concurrent load, and wing type. The relationship between monitor load and each of the exposure variables are investigated via scatter or box-plots. We focus specif- ically on the relationship between distance from nursing ofﬁce and monitor load using univariate linear regression analysis, and investigate how the relationship between these variables change conditioned on what quantile of monitor load we look at. All variables are combined in a multivariate model to examine the partial effects of each variable when controlling for all others [17–19]. We apply QR for the quantiles s = (0.10, 0.25, 0.50, 0.75, 0.90) and linear multiple Ordinary Least Squares regression. 2 Methods Our work is based on a cohort study conducted at the acute admission unit at Odense University Hospital, a 1000 bed teaching hospital serving as a primary hospital for a local population of 280,000 citizens. After initial assessment in the ED, admitted patients projected for short-term stays of up to 48 h are transferred to the admission unit. Patients can be transferred to intensive care on clinical indication. If Using the PIN, we link the collected data with supple- mental information from population based national patient registries. Arrival, admission, and discharge information 123 643 J Clin Monit Comput (2017) 31:641–649 are retrieved from the Danish National Patient Registry [13, 14]. We correct for multiplicity using the Holm–Bonferroni method, and investigate issues with multicollinarity between exposure variables using the variance inﬂation factor (VIF) [20]. Finally, we test for differences in regression coefﬁcients between the quantiles using the ANOVA method. 3 Results During the data collection period there were 11,848 admissions to the acute ward representing 35,727 days. Of these we are able to link monitor use to 3149 admissions (26.6 %) for 10,844 days (30.4 %), representing 1031 fully monitored days. Patient monitor utilization was also reg- istered for patients who we could not identify on their monitors, equating to 1271 fully monitored days. Patients in our dataset are on average admitted to the ward for 3.3 days, compared to 2.9 days for not-included patients. 115 of the patients admitted to monitors in the dataset Table 1 Overview of exposure variables Independent variable Type Values Description Comorbidity Index (CI) [15] Ordinal A, B, C, D A: CI = 0, B: CI = 1; C: CI = 2; D: CI [ 2 Severity Ordinal Regimen levels (1–5) See [16] Age Ordinal 15 - x Sex Nominal Female/male Distance Ordinal 0 - x Distance in meters from ofﬁce on each wing Wings Nominal MAU1, Surgial, MAU2 MAU1-2: (Medical Admission Unit) wings Concurrent load Ordinal 1 - x Average number of patients admitted to the wing per day during the admission period of the patient Table 1 Overview of exposure variables 123 644 J Clin Monit Comput (2017) 31:641–649 Figure 2 exempliﬁes this by showing the linear regres- sion line of the relationship between distance from nursing ofﬁce and monitor load in Fig. 2a, and quantile regression lines based on the 0.20, 0.50 (the median) and 0.80 quan- tiles in Fig. 2b. From the regression coefﬁcients, we observe that the association between monitor load and distance from nursing ofﬁce grows stronger for the upper quartiles of monitor load. Online Supplement 1 (Figure 4) shows the individual relationships between each exposure variable (age, sex, comorbidity group, triage, wing type, and the number of other patients treated during admission) and monitor load. experienced respiratory distress, seven patients suffered strokes, and one patient had both respiratory and heart related deterioration during admission. Table 2 summa- rizes data for patients identiﬁable from the monitors, and from patients not registered to monitors. While the differences in proportions for both comor- bidities and triage between patients identiﬁable on the monitors, and other patients, are statistically signiﬁcant, there are no substantial clinical differences between these factors. We do however observe that a lower percentage of surgical patients are identiﬁable on the monitors. In Fig. 3 Results 1, we observe the highly skewed distribution of how much patients are monitored. 50 percent of all the admissions have a monitor load of less than 0.014; meaning that half of all the cases are monitored less than 1.4 percent of their admission. Moving upwards, 70 percent of all admissions are monitored less than 28 percent of their total admission length. Thus, as the distribution of monitor load is heavily right skewed, standard Ordinary Least Squares regression cannot provide plausible insight. However, applying a QR approach enables us to analyze the rela- tionship between the different exposure variables and monitor load conditioned on monitor load. The results of the multivariate QR results are shown for all exposures in Fig. 3 as quantile process plots from the 0.10th up to the 0.90th quantile. The solid horizontal line for each variable indicates the Ordinary Least Squares regression coefﬁcient, and the dotted horizontal lines show the conﬁdence interval. Similarly the QR regression results at each quartile are marked with the regression coefﬁcient of the exposure variable, and the conﬁdence interval as the grey band. E.g., we ﬁnd that distance from nursing ofﬁce has the strongest inﬂuence for patients who are monitored a lot (i.e., admissions in the upper quantiles of Fig. 1). For Table 2 Exposure characteristics Admitted to monitor Not admitted to monitor Number of admissions 3149 8699 Number of patients 2815 4104 Male [n (%)] 1526 (48.4) 4314 (49.6) Mean age Male 63.8 years, SD = 18.5 years 60.9 years, SD = 21.1 years Female 66.8 years, SD = 20.9 years 63.8 years, SD = 22.6 years Comorbidity (Charlson Score (CS)) [n (%)] (A) CS = 0 1124 (35.7) 3481 (40.0) (B) CS = 1 641 (20.3) 1643 (18.9) (C) CS = 2 498 (15.8) 1297 (14.9) (D) CS [ 2 886 (28.2) 2278 (26.2) Triage [n (%)] Missing 514 (16.4) 1554 (17.9) Blue 7 (0.2) 27 (0.3) Green 431 (13.7) 1341 (15.4) Yellow 1301 (41.3) 3333 (38.3) Orange 842 (26.7) 2315 (26.6) Red 54 (1.7) 129 (1.5) Average number of registered vital signs/admission 408 registrations, SD = 633 – Wing [n (%)] Surgical 809 (25.7) 3948 (45.4) MAU1 1015 (32,2) 4751 (54.6) [both medical wings] MAU2 1325 (42.1) 12 645 J Clin Monit Comput (2017) 31:641–649 bservation regimens we ﬁnd that Orange classes have a several exposure variables but the ANOVA ﬁnds that all ig. 3 Results 1 Quantile plot for the esponse variable—illustrating he distribution of monitor load y its quantile distribution ig. 2 Univariate regression plot of Distance from nursing ofﬁce and registered Monitor load. a Ordinary Least Squares (mean ased) linear egression. b Mean linear regression, Median (Q50), 20th Quantile and 80th Quantile linear regression Clin Monit Comput (2017) 31:641–649 645 Fig. 1 Quantile plot for the response variable—illustrating the distribution of monitor load by its quantile distribution Fig. 2 Univariate regression plot of Distance from nursing ofﬁce and registered Monitor load. a Ordinary Le regression. b Mean linear regression, Median (Q50), 20th Quantile and 80th Quantile linear regression Fig. 2 Univariate regression plot of Distance from nursing ofﬁce and registered Monitor load. a Ordinary Least Squares (mean ased) linear regression. b Mean linear regression, Median (Q50), 20th Quantile and 80th Quantile linear regression several exposure variables, but the ANOVA ﬁnds that all QR coefﬁcients are signiﬁcantly different from one another. observation regimens, we ﬁnd that Orange classes have a stronger inﬂuence across the quantiles of monitoring load, but also that its impact decreases for highly monitored patients. An example of how to interpret the results from Table 3 in Online Supplement 2 and Fig. 3 is provided in Online Supplement 3. Table 3 in Online Supplement 2 conveys the results of both regression approaches. Our multiple linear regression model has an adjusted R2 of 0.1719, and are thus compa- rable to those of [22], and the model is overall statistically signiﬁcant. The VIF is below 1.62 for all exposure vari- ables, and we thus dismiss issues of multicollinarity. The Holm–Bonferroni adjustment changes the signiﬁcance of 3.1 Sensitivity analysis To address and investigate the potential impact of missing values in the dataset, we reran the analysis with missing 12 3 J Clin Monit Comput (2017) 31:641–649 646 Fig. 3 Quantile regression process plots for exposures—showing the regression coefﬁcients for the quantiles of exposure variables and the intercept when controlling for all factors Fig. 3 Quantile regression process plots for exposures—showing the regression coefﬁcients for the quantiles of exposure variables and the intercept when controlling for all factors values removed. This had little impact on the distribution of the remaining triage coefﬁcients, and did not substan- tially alter the exposure coefﬁcients or their signiﬁcance. decreeing more monitoring of patients, does not necessarily reduce the proportion of patients with adverse events [29]. Vital sign readings are often used to support clinical intu- itive hunches, and less as objective points of Ref. [30]. Even so, little research on what determine frequency of vital sign registrations have been published [22]. Since most assessment systems rely on intermittent or spot-driven observations, continuous monitoring in its current state may simply yield excessive amounts of data which can only be utilized fully through integration into clinical decision support systems. Also, the risk of more monitoring leading to alarm fatigue and habituation has to be factored in by careful consideration of calibrating the alarm thresholds [31, 32]. 123 4 Discussion We ﬁnd that distance from nursing ofﬁce has little inﬂu- ence on patients monitored less than 10 % of their admission time. But for other patients who are monitored more than this, distance from nursing ofﬁce becomes has more impact in reducing the degree of monitoring. We also note that higher levels of observation regimens have a signiﬁcant impact on monitoring load. Being admitted to the surgical wing greatly reduces how much patients are monitored, and periods with a high amount of concurrent patients have little effect on the degree of monitoring. Recent studies have rectiﬁed the assumption that deviance from protocol is solely due to clinical misjudg- ments, and instead taken a more holistic approach to the problem by investigating several factors such as day of week, time of day, and characteristics of both patients and clinicians [33, 34]. In this study, we ﬁnd evidence for adherence to observation regimen protocols through insight into how much patients are actually monitored during admission. Along these lines it is problematic that patients on the surgical wing are monitored much less than medical wing patients given that adverse events are also associated The increased focus on identiﬁcation of deteriorating patients can be seen in the body of published work on Early Warning Scores [23], Track & Trigger systems [24, 25], and Rapid Response Teams [26]. Although few of the existing deterioration detection systems in use have been rigidly validated [27, 28], the need to identify efﬁcient means for keeping an eye on multiple patients is evident as the pressure on EDs is ever increasing. However, simply 123 J Clin Monit Comput (2017) 31:641–649 647 nurses. The percentage of patients who were identiﬁable by the monitors was highest in the early phases the data acquisition stage, and then gradually decreased. The monitor registration identiﬁcation eventually plateaued, indicating that a dedicated subset of nurses persisted in registering patients to the vital sign monitors for us. This naturally induces a permutation of selection bias that is difﬁcult to overcome in this kind of project. This selection bias is also evident as identiﬁable patients are slightly older, have longer hospital stays, are sicker, and are deemed in need of more frequent observations (Table 2). 6 Conclusion As expected, there is signiﬁcant variation concerning the how much patients are monitored during their admission to an acute admission unit, but the effect of the investigated factors varies depending on how much patients are moni- tored. We conﬁrm that patients assigned to more severe observation regimen categories, are monitored more, but also show that both distance from the wing’s nursing ofﬁce inﬂuence monitoring for most patients. Number of simul- taneously admitted patients has a small effect across all levels of monitoring. Finally, we ﬁnd a big difference between the extent to which monitoring is utilized at medical and surgical wings. 4 Discussion p g y g Cabled monitoring as found in the settings of this study has several downsides; immobilization of patients, patient induced stress due to perceived severity, and loss of data during out of bed activities [39]. Consequently, much research effort has been put into the potential of wireless monitoring, but several practical obstacles such as battery life and poor communication networks still persist [40–42]. However, given that wireless monitoring could support temporary storage of vital signs on the device, would enable a smoother transition between hospital departments and reduce loss of information in out-of-bed periods. In this scenario, all patients could achieve a monitoring load of 100 %, thereby enabling more complete representation of their states and trajectories. Another limitation is missing data, and inaccurate date and time values in the coupled registries. Issues with timestamps in data retrieved from Patient Administration Systems are well known in the scientiﬁc community. Also, the observation regimen classes originate from the triage classes assigned at arrival time, generally there is a direct mapping between triage and observation regimen for patients admitted to the acute admission unit, but not necessarily for all admissions. Finally, external validity of our ﬁndings may be challenged by the single site nature of our study. Yet, assessing the monitor load of patients may be of value to similar studies, and the design of future patient monitoring systems. Interestingly, the decreasing impact of the most inﬂu- ential coefﬁcients in our statistical analyses for patients who are continuously monitored, indicate that factors not included in our model prompt higher degrees of monitor- ing. Seeking to capture the complexity of patient moni- toring in just seven exposure variables yields a very simpliﬁed model at best, and shows that patient monitoring is a complex and subjective endeavor. In this perspective, it would be interesting to include staff speciﬁc features such as clinical experience, department seniority, team compo- sition, and clinical concern in future work. An important aspect we intentionally left out of the analysis is temporal inﬂuences. As both clinical work, and the vital signs of patients follow a circadian rhythm, these aspects may reveal valuable insight for the evaluation of existing clin- ical protocols. 4 Discussion Although, the identiﬁable admissions in our analysis only account for 27 % of the total admissions in the entire period, the linked vital signs account for 45 % of all vital signs registered in the same period. This may either be a sign of issues with linking the vital signs accurately to admissions, but is also likely a seasonal indicator as the ﬁrst 6 months had the highest inclusion rate, and took place during Q4-2013 till Q1-2014. with post-surgical situations [35, 36]. This is probably a combined effect of differences in working procedures, culture between specialties as mobilization of post-surgical patients is considered important by surgical nurses, and the fact that many pre-surgical patients are unaffected until surgery, and that many orthopedic patients are admitted with minor surgical problems. Quantifying the extent to which a patient is being monitored, may be an aid to bridge the current gap between usage of automated and manual monitoring as clinical work will continue to depend on tacit knowledge and intuition [37, 38]. Since the use of monitoring is increasing in all types of hospital departments, and as technology becomes more pervasive, the insight from this paper may provide guidance for system designers and clinicians a like. Cabled monitoring as found in the settings of this study has several downsides; immobilization of patients, patient induced stress due to perceived severity, and loss of data during out of bed activities [39]. Consequently, much research effort has been put into the potential of wireless monitoring, but several practical obstacles such as battery life and poor communication networks still persist [40–42]. However, given that wireless monitoring could support temporary storage of vital signs on the device, would enable a smoother transition between hospital departments and reduce loss of information in out-of-bed periods. In this scenario, all patients could achieve a monitoring load of 100 %, thereby enabling more complete representation of their states and trajectories. Quantifying the extent to which a patient is being monitored, may be an aid to bridge the current gap between usage of automated and manual monitoring as clinical work will continue to depend on tacit knowledge and intuition [37, 38]. 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https://openalex.org/W2736175702	https://europepmc.org/articles/pmc5646945?pdf=render	English	null	Biallelic Mutations in KDSR Disrupt Ceramide Synthesis and Result in a Spectrum of Keratinization Disorders Associated with Thrombocytopenia	The journal of investigative dermatology/Journal of investigative dermatology	2,017	cc-by	10,366	1St. John’s Institute of Dermatology, King’s College London (Guy’s Campus), London, UK; 2Department of Dermatology, Nagoya University Graduate School of Medicine, Nagoya, Japan; 3Department of Dermatology, Hospital Infantil del Nin˜o Jesu´s, Madrid, Spain; 4Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan; 5Centro Regional de Hemodonacio´n, Servicio de Hematologı´a y Oncologı´a Me´dica, Hospital Universitario Morales Meseguer, IMIB-Arrixaca, Universidad de Murcia, Centro de Investigacio´n Biome´dica en Red de Enfermedades Raras, Instituto de Salud Carlos III, Madrid, Spain; 6Viapath, St. Thomas’ Hospital, London, UK; 7Analytical Science Research Laboratories, Kao Corporation, Haga, Tochigi, Japan; 8Biological Science Research Laboratories, Kao Corporation, Haga, Tochigi, Japan; 9Department of Dermatology, Hospital Sierra de Segura, Puente de Ge´nave, Jae´n, Spain; 10Department of Dermatology, Hospital Universitario Reina Sofı´a, Co´rdoba, Spain; 11Department of Dermatology, Hiroshima City Hiroshima Citizens Hospital, Hiroshima, Japan; 12Department of Dermatology, Great Ormond Street Hospital for Children NHS Foundation Trust, London, UK; 13Centre for Cell Biology and Cutaneous Research, Blizard Institute, Barts, London, UK; 14London School of Medicine and ORIGINAL ARTICLE ORIGINAL ARTICLE Biallelic Mutations in KDSR Disrupt Ceramide Synthesis and Result in a Spectrum of Keratinization Disorders Associated with Thrombocytopenia y p Takuya Takeichi1,2, Antonio Torrelo3, John Y.W. Lee1, Yusuke Ohno4, Marı´a Luisa Lozano5, Akio Kihara4, Lu Liu6, Yuka Yasuda7, Junko Ishikawa8, Takatoshi Murase8, Ana Bele´n Rodrigo9, Pablo Ferna´ndez-Crehuet10, Yoichiro Toi11, Jemima Mellerio1,12, Jose´ Rivera5, Vicente Vicente5, David P. Kelsell13,14, Yutaka Nishimura15, Yusuke Okuno16,17, Daiei Kojima17, Yasushi Ogawa2, Kazumitsu Sugiura18, Michael A. Simpson19, W.H. Irwin McLean20, Masashi Akiyama2 and John A. McGrath1,20 Takuya Takeichi1,2, Antonio Torrelo3, John Y.W. Lee1, Yusuke Ohno4, Marı´a Luisa Lozano5, Akio Kihara4, Lu Liu6, Yuka Yasuda7, Junko Ishikawa8, Takatoshi Murase8, Ana Bele´n Rodrigo9, Pablo Ferna´ndez-Crehuet10, Yoichiro Toi11, Jemima Mellerio1,12, Jose´ Rivera5, Vicente Vicente5, David P. Kelsell13,14, Yutaka Nishimura15, Yusuke Okuno16,17, Daiei Kojima17, Yasushi Ogawa2, Kazumitsu Sugiura18, Michael A. Simpson19, W.H. Irwin McLean20, Masashi Akiyama2 and John A. McGrath1,20 Mutations in ceramide biosynthesis pathways have been implicated in a few Mendelian disorders of kerati- nization, although ceramides are known to have key roles in several biological processes in skin and other tissues. Using whole-exome sequencing in four probands with undiagnosed skin hyperkeratosis/ichthyosis, we identiﬁed compound heterozygosity for mutations in KDSR, encoding an enzyme in the de novo synthesis pathway of ceramides. Two individuals had hyperkeratosis conﬁned to palms, soles, and anogenital skin, whereas the other two had more severe, generalized harlequin ichthyosis-like skin. Thrombocytopenia was present in all patients. The mutations in KDSR were associated with reduced ceramide levels in skin and impaired platelet function. KDSR enzymatic activity was variably reduced in all patients, resulting in defective acylceramide synthesis. Mutations in KDSR have recently been reported in inherited recessive forms of pro- gressive symmetric erythrokeratoderma, but our study shows that biallelic mutations in KDSR are implicated in an extended spectrum of disorders of keratinization in which thrombocytopenia is also part of the phenotype. Mutations in KDSR cause defective ceramide biosynthesis, underscoring the importance of ceramide and sphingosine synthesis pathways in skin and platelet biology. Journal of Investigative Dermatology (2017) 137, 2344e2353; doi:10.1016/j.jid.2017.06.028 ª 2017 The Authors. Published by Elsevier, Inc. on behalf of the Society for Investigative Dermatology. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Received 20 May 2017; revised 24 June 2017; accepted 28 June 2017; accepted manuscript published online 31 July 2017; corrected proof published online 27 September 2017 Dentistry, Queen Mary University of London, Whitechapel, London, UK; 15Department of General Perinatology, Hiroshima City Hiroshima Citizens Hospital, Hiroshima, Japan; 16Center for Advanced Medicine and Clinical Research, Nagoya University Hospital, Nagoya, Japan; 17Department of Pediatrics, Nagoya University Graduate School of Medicine, Nagoya, Japan; 18Department of Dermatology, Fujita Health University School of Medicine, Toyoake, Japan; 19Department of Medical and Molecular Genetics, King’s College London, School of Medicine, Guy’s Hospital, London, UK; and 20Centre for Dermatology and Genetic Medicine, Division of Molecular Medicine, University of Dundee, Dundee, UK Correspondence: John A. McGrath, Dermatology Research Labs, Floor 9 Tower Wing, Guy’s Hospital, Great Maze Pond, London SE1 9RT, UK. E-mail: john.mcgrath@kcl.ac.uk Abbreviations: DHS, dihydrosphingosine; KDS, 3-ketodihydrosphingosine; S1P, sphingosine-1-phosphate Received 20 May 2017; revised 24 June 2017; accepted 28 June 2017; accepted manuscript published online 31 July 2017; corrected proof published online 27 September 2017 Journal of Investigative Dermatology (2017) 137, 2344e2353; doi:10.1016/j.jid.2017.06.028 Abbreviations: DHS, dihydrosphingosine; KDS, 3-ketodihydrosphingosine; S1P, sphingosine-1-phosphate INTRODUCTION indicating the likely contribution of further gene pathology (Fischer, 2009). The hereditary palmoplantar keratodermas and ichthyoses comprise a heterogeneous collection of genodermatoses caused by mutations in more than 100 genes involved in a multitude of biologic pathways and processes (Oji et al., 2010; Sakiyama and Kubo, 2016). Despite major advances in discovering the underlying molecular genetic basis of many of these disorders, several cases remain unresolved, One very recent discovery that expands the molecular pathology of ichthyosis has been the identiﬁcation of muta- tions in KDSR in four individuals with clinical phenotypes of progressive symmetric erythrokeratoderma (Boyden et al., 2017). KDSR encodes 3-ketodihydrosphingosine reductase, which catalyzes the reduction of 3-ketodihydrosphingosine Journal of Investigative Dermatology (2017), Volume 137 2344 Journal of Investigative Dermatology (2017), Volume 137 2344 T Takeichi et al. KDSR Mutations Disrupt Skin and Platelet Function T Takeichi et al. T Takeichi et al. T Takeichi et al. KDSR Mutations Disrupt Skin and Platelet Function (KDS) to dihydrosphingosine (DHS), a key step in the de novo ceramide synthesis pathway (Linn et al., 2001). Previously, mutations in a different gene in this pathway, CERS3, have also been implicated in autosomal recessive congenital ich- thyosis, emphasizing the clinical relevance of ceramide pa- thology in inherited disorders of corniﬁcation (Eckl et al., 2013; Radner et al., 2013). Ceramides also have key physi- ological roles in other organs: mutations in ELOVL4, encoding an enzyme necessary for the production of ultra- long chain ceramides in the skin, brain, and retina, lead to a recessive disorder characterized by ichthyosis, intellectual disability, and spastic quadriplegia (Aldahmesh et al., 2011). region in Spain as family 1. At age 15 months, he developed diffuse hyperkeratosis on the palms and soles, without pro- gression to the dorsae of the hands or feet (i.e., less severe than patient 1). He also developed perianal erythema and hyperkeratosis. As for patient 1, oral acitretin did not improve the hyperkeratosis. In addition, he suffered episodes of bruising with evidence of isolated thrombocytopenia. Bone marrow studies showed normal hematologic morphology. At present, he has not manifested clinically relevant signs of bleeding despite persistently low platelets (w20 109/L). g p p y p Patient 3 was the second child born to unrelated white parents from the United Kingdom (family 3, Figure 1c). His parents, older brother, and all other relatives were healthy. RESULTS Patient 4 is a 6-year-old Japanese male and is the younger of two brothers born to unrelated parents (family 4, Figure 1d). His mother and brother have atopic dermatitis, but there is no other noteworthy family history. He was delivered at 35þ3 weeks by normal spontaneous vaginal birth with a birth weight of 1.9 kg. At birth, he had thick plate-like scales with deep ﬁssuring overlying erythrodermic skin. Severe eclabium and ectropion were also observed. Skin biopsy showed marked hyperkeratosis with para- keratosis (see Supplementary Figure S1 online). These fea- tures were consistent with Harlequin ichthyosis. He was treated in the neonatal intensive care unit but did not receive systemic retinoids. Over the ﬁrst 2 months of life, the thick scales desquamated gradually, resulting in generalized erythroderma and ﬁne scaling. Platelet count was normal at birth (140e150 109/L), but since the age of 2 months this progressively decreased, and at 3 years of age he had severe thrombocytopenia (4e11 109/L). Clinical features of individuals with KDSR mutations Permission to report medical details and include clinical il- lustrations was obtained for all patients (from guardians for patients 1, 3, and 4 and from patient 2 himself). p p Patient 1 is a 15-year-old male and the only child of un- related healthy parents (family 1, Figure 1a). His parents are originally from the same geographic area in mid-southeast Spain. At the age of 12 months, he developed palmoplantar hyperkeratosis with extension to the dorsae of the hands and feet, wrists, and ankles, as well as anogenital hyperkeratosis and erythema (Figure 2aec). At age 2 years, a blood count was performed because of mucocutaneous bleeding, which showed a severe, isolated thrombocytopenia (platelet count < 30 109/L; bone marrow biopsy sample showed a normal to increased number of megakaryocytes only). A diagnosis of primary immune thrombocytopenia was made, but treatment with oral corticosteroids was suboptimal. Splenectomy at age 11 years led to a slight increase in platelets (w40 109/L), although clinically he continues to suffer recurrent nose bleeds. Light microscopy of lesional skin showed nonspeciﬁc ﬁndings of psoriasiform acanthosis, parakeratosis, and focal hypergranulosis but no epidermolytic changes (Figure 2d and e). Oral acitretin (0.5 mg/kg) prescribed for several months did not lead to any improvement in his skin. INTRODUCTION His mother’s pregnancy was uneventful until the last trimester, when oligohydramnios was noted. She had spon- taneous rupture of membranes at 33þ5 weeks and under- went elective cesarian section at 35þ2 weeks with an infant birth weight of 2.74 kg. At birth, the patient was covered in thick adherent plate-like scales, with prominent ectropion and eclabium, and pinching of all digits, collectively consistent with harlequin ichthyosis. He was treated in a humidiﬁed incubator with hourly greasy emollients and lubricating eye drops. Acitretin was started, which led to some reduction in adherent scaling, although he developed pseudomonas septicemia at age 15 days and further sepsis thereafter. At birth, platelet count was 120 109/L, but within 2 weeks this dropped to 50 109/L, and by the 3rd week to approximately 20e30 109/L and remained at this level. At day 36, he deteriorated clinically with tachypnea and hypotension associated with a profound metabolic acidosis. Despite efforts to resuscitate him, he died age 37 days. In this study, we investigated four individuals from Spain, Japan, and the United Kingdom who presented with inherited disorders of keratinization but had clinical features different from those presented by Boyden et al. (2017). Two patients displayed a milder phenotype of palmoplantar and anogen- ital hyperkeratosis, whereas the other two patients had a more severe phenotype resembling harlequin ichthyosis. An additional ﬁnding, present in all our subjects, but not featured in the Boyden et al. study, was a reduction in the number of blood platelets (thrombocytopenia). Using whole-exome sequencing, functional studies on skin and platelets, and in vitro analyses, we identiﬁed autosomal recessive mutations in KDSR in all four subjects, with only one heterozygous mutation overlapping with published ﬁndings (Boyden et al., 2017). Our ﬁndings expand the mo- lecular and clinical pathology associated with KDSR muta- tions and show that this ceramide biosynthesis pathway has important roles in both skin and platelets. T Takeichi et al. KDSR M i D (e) Higher magniﬁcation shows focal hypergranulosis and parakeratosis. Hematoxylin and eosin stain. Scale bar ¼ 10 mm. Journal of Investigative Dermatology (2017), Volume 137 2346 T Takeichi et al. KDSR M i D T Takeichi et al. KDSR Mutations Disrupt Skin and Platelet Function Patient 4 Patients 1 & 2 c.417+3A>C (Δ5 / Δ5Δ6) c.879G>A (p.Gln293Gln / Δ9) 3’ 5’ 1 2 3 5 6 7 9 10 8 4 c.413T>G (p.Phe138Cys) c.812G>A (p.Gly271Glu) c.223_224delGA (p.Glu75Asnfs2) e a b c d c.544G>A (p.Gly182Ser) Patient 3 Figure 1. Pedigrees and mutations identiﬁed in KDSR. (aed) Family pedigrees of the four patients with compound heterozygous mutations in KDSR. þ denotes the wild-type allele. (e) Schematic of KDSR to show the six compound heterozygous mutations identiﬁed in this study. d b c d d b c a Patient 4 Patients 1 & 2 c.417+3A>C (Δ5 / Δ5Δ6) c.879G>A (p.Gln293Gln / Δ9) 3’ 5’ 1 2 3 5 6 7 9 10 8 4 c.413T>G (p.Phe138Cys) c.812G>A (p.Gly271Glu) c.223_224delGA (p.Glu75Asnfs2) e c.544G>A (p.Gly182Ser) Patient 3 Figure 1. Pedigrees and mutations identiﬁed in KDSR. (aed) Family pedigrees of the four patients with compound heterozygous mutations in KDSR. þ denotes the wild-type allele. (e) Schematic of KDSR to show the six compound heterozygous mutations identiﬁed in this study. e Figure 1. Pedigrees and mutations identiﬁed in KDSR. (aed) Family pedigrees of the four patients with compound heterozygous mutations in KDSR. þ denotes the wild-type allele. (e) Schematic of KDSR to show the six compound heterozygous mutations identiﬁed in this study. (see Supplementary Figure S2 online) and segregated with disease status in family members whose DNA was available (Figures 1aed). Our study showed three missense mutations (p.Phe138Cys, p.Gly182Ser, and p.Gly271Glu), one synony- mous variant (c.879G>A, p.Gln293Gln, but affecting the last base of an exon and therefore potentially a donor splice site showed compound heterozygous mutations in KDSR, a candidate gene recently implicated in progressive symmetric erythrokeratoderma (Boyden et al., 2017). Details of the mu- tations are shown in Figure 1e and Table 1; only one of the heterozygous mutant alleles overlapped with the known KDSR mutations. The mutations were veriﬁed by Sanger sequencing heterozygous mutant alleles overlapped with the known KDSR mutations. The mutations were veriﬁed by Sanger sequencing mous variant (c.879G>A, p.Gln293Gln, but affecting the last base of an exon and therefore potentially a donor splice site Figure 2. Clinicopathologic features of patient 1. (a) Diffuse palmar keratoderma. (b) Perianal hyperkeratosis. (c) Bilateral diffuse plantar keratoderma. (d) Light microscopy of palmar skin shows psoriasiform acanthosis and hyperkeratosis. Hematoxylin and eosin stain. Scale bar ¼ 100 mm. Identiﬁcation of compound heterozygous mutations in KDSR in all affected individuals After ethics committee approval and written informed consent, whole-exome sequencing was performed using DNA from all affected probands. Candidate gene mutations were prioritized by ﬁltering for variants with a frequency of less than 0.1% in public databases such as the Exome Aggregation Consortium (ExAC), Exome Variant Server, 1000 Genomes Project, and an in-house repository. Whole-exome sequencing Patient 2 is a 21-year-old male and the older of two brothers born to healthy unrelated parents (family 2, Figure 1b). He is the only affected individual among his rel- atives. This family originates from the same geographic www.jidonline.org 2345 Journal of Investigative Dermatology (2017), Volume 137 2346 Figure 2. Clinicopathologic features of patient 1. (a) Diffuse palmar keratoderma. (b) Perianal hyperkeratosis. (c) Bilateral diffuse plantar keratoderma. (d) Light microscopy of palmar skin shows psoriasiform acanthosis and hyperkeratosis. Hematoxylin and eosin stain. Scale bar ¼ 100 mm. (e) Higher magniﬁcation shows focal hypergranulosis and parakeratosis. Hematoxylin and eosin stain. Scale bar ¼ 10 mm. Table 1. Summary of clinical and mutation details of all four affected individuals Table 1. Summary of clinical and mutation details of all four affected individuals Patient Country of Origin Dermatologic Phenotype Thrombocytopenia Mutations in KDSR and Amino Acid Change 1000 Genomes Project Frequency ExAC Frequency SIFT (Score) PolyPhen- 2 (Score) Mutation Taster 1 Spain Palmoplantar and perianal keratoderma þ c.413T>G: p.Phe138Cys 0 8.3 10e5 Damaging Probably damaging Disease- causing c.417þ3A>C 0 0 N/A N/A Disease- causing 2 Spain Palmoplantar and perianal keratoderma þ c.413T>G: p.Phe138Cys 0 8.3 10e5 Damaging Probably damaging Disease- causing c.417þ3A>C 0 0 N/A N/A Disease- causing 3 United Kingdom Harlequin ichthyosis þ c.812G>A: p.Gly271Glu 0 0 Damaging Probably damaging Disease- causing c.879G>A: p.Gln293Gln 0 3.3 10e5 N/A N/A Disease- causing 4 Japan Harlequin ichthyosis þ c.223_224delGA: p.Glu75Asnfs2 0 0 N/A N/A Disease- causing c.544G>A: p.Gly182Ser 0 8.2 10e6 Damaging Probably damaging Disease- causing Abbreviations: ExAC, Exome Aggregation Consortium; N/A, not applicable; PolyPhen-2, Polymorphism Phenotyping v2; SIFT, Sorting Tolerant From Intolerant. Patient Country of Origin Dermatologic Phenotype Thrombocytopenia Mutations in KDSR and Amino Acid Change 1000 Genomes Project Frequency ExAC Frequency SIFT (Score) PolyPhen- 2 (Score) Mutation Taster 1 Spain Palmoplantar and perianal keratoderma þ c.413T>G: p.Phe138Cys 0 8.3 10e5 Damaging Probably damaging Disease- causing c.417þ3A>C 0 0 N/A N/A Disease- causing 2 Spain Palmoplantar and perianal keratoderma þ c.413T>G: p.Phe138Cys 0 8.3 10e5 Damaging Probably damaging Disease- causing c.417þ3A>C 0 0 N/A N/A Disease- causing 3 United Kingdom Harlequin ichthyosis þ c.812G>A: p.Gly271Glu 0 0 Damaging Probably damaging Disease- causing c.879G>A: p.Gln293Gln 0 3.3 10e5 N/A N/A Disease- causing 4 Japan Harlequin ichthyosis þ c.223_224delGA: p.Glu75Asnfs2 0 0 N/A N/A Disease- causing c.544G>A: p.Gly182Ser 0 8.2 10e6 Damaging Probably damaging Disease- causing Abbreviations: ExAC, Exome Aggregation Consortium; N/A, not applicable; PolyPhen-2, Polymorphism Phenotyping v2; SIFT, Sorting Tolerant From Intolerant. yeast would not be able to grow normally if the KDSR mutants impair enzymatic activity. This assay showed that the muta- tions (illustrated for patients 1, 2, and 3; Figure 3b) diminished the ability of yeast to grow in the absence of phytosphingosine, similar to the three different mutations tested by Boyden et al. (2017). The p.Phe138Cys mutant had the mildest effect. In comparison, the D5 and D5D6 mutants (representing the c.417þ3A>C mutation) resulted in the most signiﬁcant impairment of yeast growth (Figure 3b). mutation), one other splice site mutation (c.417þ3G>A), and one out-of-frame deletion (c.223_224delGA, p.Glu75Asnfs2) (Figure 1e). Table 1. Summary of clinical and mutation details of all four affected individuals Patients 1 and 2 come from the same region of Spain, and therefore, although neither family was aware of any relatedness, the ﬁnding of identical compound heterozygotes mutations in KDSR (p.Phe138Cys and c.417þ3G>A) is likely to indicate sharing of regional founder mutations. The splicing mutation (c.417þ3A>C) was predicted to cause a reduction of 41.8% of transcripts expressing exon 5 of KDSR, based on the SPANR tool (Xiong et al., 2015), which was conﬁrmed by reverse transcription-PCR using RNA extracted from skin (pa- tients 1 and 2). Sequencing of cDNA from exon 2 to exon 7 of KDSR showed skipping of exon 5 (96 base pairs, D5) or skip- ping of exons 5 and 6 (288 base pairs, D5D6) (see Supplementary Figure S3a and b online). Both of these trun- cated transcripts restore the reading frames. By using cDNA from peripheral blood, in-frame skipping of exon 5 was shown (see Supplementary Figure S3c). The synonymous c.879G>A mutation (p.Gln293Gln) was previously identiﬁed in the study by Boyden et al. (2017). This nucleotide transition occurs within the last base of exon 9, and reverse transcription-PCR in that report showed in-frame skipping of exon 9. p y g g To assess the enzymatic activity in vitro, all mutant con- structs (including those for patient 4, performed separately under similar conditions) were introduced into HEK 293T cells (Figure 3c), and KDSR activity was measured in vitro using membrane fractions, notwithstanding an inherent limitation of this assay being that HEK 293T cells have endogenous KDSR activity, which is not abolished. Consistent with the yeast complementation assay, this showed that most of the mutants led to a signiﬁcant reduction in DHS synthesis (Figure 3d). The only exception was the p.Gly182Ser (c.544G>A) variant, which showed no signiﬁcant difference in DHS synthesis compared with the wild type (Figure 3d). The p.Gly182Ser mutation occurs within the hydrophilic domain (amino acids 22e270), similar to two mutations found by Boyden et al. (2017), and close to the canonical TyrXXXLys reductase site (amino acids 186e190). By analogy with the Boyden et al. data, p.Gly182Ser is likely to lead to a structural protein change rather than affect glycosylation or phosphorylation. KDSR mutations impair enzymatic activity and lead to defective acylceramide synthesis To assess the effect of the mutations on KDSR enzymatic ac- tivity, the new mutations identiﬁed in our patients were intro- duced into yeast and HEK 293T cells. Two mutant plasmids were designed for the c.417þ3A>C variant, one predicting skipping of exon 5 only (D5) and the other loss of both exons 5 and 6 (D5D6) (Figure 3a). Primer details for the mutant con- structs are shown in Supplementary Table S1 online. T Takeichi et al. KDSR Mutations Disrupt Skin and Platelet Function Table 1. Summary of clinical and mutation details of all four affected individuals Figure 2. Clinicopathologic features atures r fuse ws nd m. s focal osis. Scale l ( ) l of patient 1. (a) Diffuse palmar keratoderma. (b) Perianal hyperkeratosis. (c) Bilateral diffuse plantar keratoderma. (d) Light microscopy of palmar skin shows psoriasiform acanthosis and hyperkeratosis. Hematoxylin and eosin stain. Scale bar ¼ 100 mm. (e) Higher magniﬁcation shows focal hypergranulosis and parakeratosis. Hematoxylin and eosin stain. Scale bar ¼ 10 mm. bar 10 mm. Journal of Investigative Dermatology (2017), Volume 137 2346 Journal of Investigative Dermatology (2017), Volume 137 2346 Journal of Investigative Dermatology (2017), Volume 137 2346 Journal of Investigative Dermatology (2017), Volume 137 2346 T Takeichi et al. KDSR M i D (d) Total membrane fractions were incubated with 10 mmol/L KDS and 1 mmol/L NADPH at 37 C for 1 hour. Lipids were extracted and subjected to liquid chromatography- mass spectrometry/mass spectrometry analysis. DHS was detected in the MRM mode and quantiﬁed using MassLynx software. Values represent the mean standard deviations of three independent experiments. Statistically signiﬁcant differences compared with WT are indicated. P < 0.01; Tukey test. DHS, dihydrosphingosine; KDS, 3-ketodihydrosphingosine; KDSR, 3-ketodihydrosphingosine reductase; MRM, multiple reaction monitoring; NADPH, nicotinamide adenine dinucleotide phosphate; PHS, phytosphingosine; SC-URA, synthetic complete minus uracil; WT, wild type. liquid chromatography-mass spectrometry analysis (Figure 4, and Supplementary Tables S4 and S5 online for full details). In the forearms of patients 1 and 2 (uninvolved skin), there was no signiﬁcant difference in the total ceramide, ceramide components, or average carbon numbers between the affected individuals and their unaffected mothers (see Supplementary Table S1). In contrast, in the affected wrist skin, the levels of total ceramide, CER[EOS], CER[EOH], CER [NP], CER[NH], and CER[NS], were reduced in the patients’ samples. Additionally, the average carbon numbers of ceramides indicated that short chain ceramides, CER[NDS], CER[NS], and CER[AS], were relatively increased in the pa- tients’ skin. However, because of the small number of sam- ples, statistical analyses could not be performed. Likewise, in the affected palm skin samples, the level of total ceramide was decreased in patient 1 compared with his mother. In contrast, there was no difference in the levels of total or in- dividual ceramides between patient 2 and his mother. This discrepancy may be explained by the milder phenotype in patient 2 compared with patient 1. The average carbon numbers of ceramides showed that short chain ceramides, CER[NDS], CER[NS], CER[NP], CER[ADS], CER[AS], and CER[AP], were relatively increased in both patients’ palms. was performed on skin sections from patient 1, patient 2, and a control individual to examine changes in protein levels or localization. KDSR labeling was not visibly reduced in patient skin (see Supplementary Figure S5 online; antibody details in Supplementary Table S3 online). Staining with an anti-ceramide antibody showed reduced (but not absent) ceramide levels in patient skin, supporting the hypothesis that KDSR mutations lead to dysregulation of ceramide biosynthesis, although the broad reactivity of the antibody (which recognizes ceramide-2, cer- amide-3, ceramide-5, ceramide C14, ceramide C16, and dihy- droceramide C16, but not sphingosine or DHS) limits further interpretation. T Takeichi et al. KDSR M i D T Takeichi et al. KDSR Mutations Disrupt Skin and Platelet Function Figure 3. Mutations in KDSR impair enzymatic activity in vitro. (a) Total lysates prepared from KHY625 (Dtsc10) cells harboring an empty vector or the plasmid encoding WT or mutant 3xFLAG-KDSR were separated by SDS-PAGE and subjected to immunoblotting using anti-FLAG M2 antibody. (b) KHY625 cells bearing the indicated plasmid were grown serially diluted at 1:10, spotted on SC-URA plates with or without 5 mmol/L PHS, and grown at 30 C for 3 days. (c, d) HEK 293T cells were transfected with an empty vector or the plasmid encoding WT or mutant 3xFLAG-KDSR. DHS levels in the membrane fractions from the cells transfected with empty vector are due to activity of endogenous KDSR. At 24 hours after transfection, total membrane fractions were prepared. (c) Total membrane fractions (5 mg protein) were separated by SDS-PAGE and subjected to immunoblotting using anti-FLAG M2 antibody. (d) Total membrane fractions were incubated with 10 mmol/L KDS and 1 mmol/L NADPH at 37 C for 1 hour. Lipids were extracted and subjected to liquid chromatography- mass spectrometry/mass spectrometry analysis. DHS was detected in the MRM mode and quantiﬁed using MassLynx software. Values represent the mean standard deviations of three independent experiments. Statistically signiﬁcant differences compared with WT are indicated. P < 0.01; Tukey test. DHS, dihydrosphingosine; KDS, 3-ketodihydrosphingosine; KDSR, 3-ketodihydrosphingosine reductase; MRM, multiple reaction monitoring; NADPH, nicotinamide adenine dinucleotide phosphate; PHS, phytosphingosine; SC-URA, synthetic complete minus uracil; WT, wild type. Figure 3. Mutations in KDSR impair enzymatic activity in vitro. (a) Total lysates prepared from KHY625 (Dtsc10) cells harboring an empty vector or the plasmid encoding WT or mutant 3xFLAG-KDSR were separated by SDS-PAGE and subjected to immunoblotting using anti-FLAG M2 antibody. (b) KHY625 cells bearing the indicated plasmid were grown serially diluted at 1:10, spotted on SC-URA plates with or without 5 mmol/L PHS, and grown at 30 C for 3 days. (c, d) HEK 293T cells were transfected with an empty vector or the plasmid encoding WT or mutant 3xFLAG-KDSR. DHS levels in the membrane fractions from the cells transfected with empty vector are due to activity of endogenous KDSR. At 24 hours after transfection, total membrane fractions were prepared. (c) Total membrane fractions (5 mg protein) were separated by SDS-PAGE and subjected to immunoblotting using anti-FLAG M2 antibody. T Takeichi et al. KDSR M i D In keeping with the gene expression changes observed, immunoreactivity of CERS3, ﬁlaggrin, and loricrin was increased in both patients (see Supplementary Figure S5). Taken together, these alterations suggest that reduction of KDSR activity leads to diminished levels of ceramide in skin with increased or precocious expression of terminal differentiation markers such as keratin 10, involucrin, ﬁlaggrin and loricrin. liquid chromatography-mass spectrometry analysis (Figure 4, and Supplementary Tables S4 and S5 online for full details). In the forearms of patients 1 and 2 (uninvolved skin), there was no signiﬁcant difference in the total ceramide, ceramide components, or average carbon numbers between the affected individuals and their unaffected mothers (see Supplementary Table S1). In contrast, in the affected wrist skin, the levels of total ceramide, CER[EOS], CER[EOH], CER [NP], CER[NH], and CER[NS], were reduced in the patients’ samples. Additionally, the average carbon numbers of ceramides indicated that short chain ceramides, CER[NDS], CER[NS], and CER[AS], were relatively increased in the pa- tients’ skin. However, because of the small number of sam- ples, statistical analyses could not be performed. Likewise, in the affected palm skin samples, the level of total ceramide was decreased in patient 1 compared with his mother. In contrast, there was no difference in the levels of total or in- dividual ceramides between patient 2 and his mother. This discrepancy may be explained by the milder phenotype in patient 2 compared with patient 1. The average carbon numbers of ceramides showed that short chain ceramides, CER[NDS], CER[NS], CER[NP], CER[ADS], CER[AS], and CER[AP], were relatively increased in both patients’ palms. KDSR expression and ceramide immunolabeling are reduced in patient skin Quantitative PCR was performed using whole skin RNA from patient 1, patient 2, and four healthy individuals (data shown in Supplementary Figure S4 online; probe details in Supplementary Table S2 online). KDSR expression was found to be reduced in both affected individuals, but not dramatically (70e80% of control; see Supplementary Figure S4a). Expressions of FLG, CERS3, IVL, KRT10, and KRT14 were increased in both patients (seeSupplementaryFigureS4bef).Immunoﬂuorescencestaining A yeast complementation assay was performed using yeast grown on plates with or without phytosphingosine. Because sphingolipids are essential for cell viability, Dtsc10 yeast cells cannot grow normally unless the addition of phytosphingosine or DHS to the medium bypasses the requirement of de novo sphingolipid synthesis. Therefore, under these circumstances, www.jidonline.org 2347 T Takeichi et al. (d) Subanalysis of the relative percentages of esteriﬁed and non-esteriﬁed ceramides shows reduced levels of esteriﬁed ceramides for all patient samples compared with those of their respective mothers, with the exception of the palm sample from the mother of patient 2. A more detailed analysis of these data are presented in Supplementary Tables S4 and S5. b a d Figure 4. Tape stripping and liquid chromatography-mass spectrometry analysis shows reduced percentages of esteriﬁed ceramides in patient 1 and patient 2 skin. (a) In palm skin, total ceramide levels in patient 1 are reduced compared with his mother, although differences are not observed in patient 2, who had a milder clinical phenotype. (b) Total ceramide levels are also reduced in wrist skin in both patients with respect to their mothers, more so for patient 1. (c) In forearm skin (clinically normal), total ceramide levels are not reduced in patient skin. (d) Subanalysis of the relative percentages of esteriﬁed and non-esteriﬁed ceramides shows reduced levels of esteriﬁed ceramides for all patient samples compared with those of their respective mothers, with the exception of the palm sample from the mother of patient 2. A more detailed analysis of these data are presented in Supplementary Tables S4 and S5. skin. (a) In palm skin, total ceramide levels in patient 1 are reduced compared with his mother, although differences are not observed in patient 2, who had a milder clinical phenotype. (b) Total ceramide levels are also reduced in wrist skin in both patients with respect to their mothers, more so for patient 1. (c) In forearm skin (clinically normal), total ceramide levels are not reduced in patient skin. (d) Subanalysis of the relative percentages of esteriﬁed and non-esteriﬁed ceramides shows reduced levels of esteriﬁed ceramides for all patient samples compared with those of their respective mothers, with the exception of the palm sample from the mother of patient 2. A more detailed analysis of these data are presented in Supplementary Tables S4 and S5. bleeding, was decreased by 61% compared with control subjects, and the equivalent measure in patient 2 was reduced by 45% (Figure 6a). The observation that serum S1P levels compared with those of control subjects were dimin- ished in both patients by only 45% and 36%, respectively, suggests that erythrocytes contribute to most of the S1P being released in patient samples during blood clotting. T Takeichi et al. Surface- exposed ceramide in human platelets were investigated with an antibody recognizing C24:0 ceramide levels, the predominant form of ceramide present in human platelets (Chen et al., 2013). This antibody detected a signiﬁcant in- crease in ceramide levels in the plasma membrane of con- trols and carriers of the c.417þ3A>C mutation after platelet activation, whereas the intensity of immunostaining was not changed signiﬁcantly in affected patients (Figure 6b). There was a relative (but not absolute) increase of short chain ceramides in wrist and palm. Because total ceramide levels decrease, it is more likely that overall there is a decrease in longer ceramides. KDSR is one of the key enzymes involved in the de novo pathway of sphingolipid synthesis, acting between serine palmitoyl transferase and ceramide synthase. Therefore, KDSR deﬁciency may affect this cascade and lead to a reduction in the levels of synthesis of total and down- stream products. There was a relative (but not absolute) increase of short chain ceramides in wrist and palm. Because total ceramide levels decrease, it is more likely that overall there is a decrease in longer ceramides. KDSR is one of the key enzymes involved in the de novo pathway of sphingolipid synthesis, acting between serine palmitoyl transferase and ceramide synthase. Therefore, KDSR deﬁciency may affect this cascade and lead to a reduction in the levels of synthesis of total and down- stream products. KDSR mutations reduce platelet number and function Detailed analysis of platelets was performed in patients 1 and 2, their respective mothers, and healthy control subjects. No morphologic abnormalities in platelets were noted (see Supplementary Figure S6 online), although ﬂow cytometry evaluation of platelet volume was slightly increased in the patients (Table 2). The patients’ platelets expressed normal levels of adhesive surface glycoproteins, but a lower level of phosphatidylserine exposure in terms of basal annexin V binding positive percentages and also reduced thromboplastin expression in unstimulated washed platelets (Table 2). Next, we performed platelet function analysis by evaluating granule release and the conformational change of aIIbb3 integrin (CD62P and PAC-1, respectively) upon stimulation with different platelet agonists (Figure 5). The increment both in CD62Pe and PAC-1epositive platelets was lower in patients than in control subjects, speciﬁcally with agonists that are known to activate pathways that are highly dependent on Src family kinases, such as thrombin (PAR1p and PAR4p), collagen (collagen-related peptide), and adenosine diphosphate, but not as evident after arachidonic acid stimulation (Figure 5). T Takeichi et al. KDSR Mutations Disrupt Skin and Platelet Function Esterified Ceramides (%) Non-esterified Ceramides (%) Esterified Ceramides (%) Non-esterified Ceramides (%) 5.0 Each ceramide class in palm ng/μg-protein 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 4.3 4.4 7.8 4.1 95.7 95.6 92.2 95.9 Patient 1 Patient 2 Mother of Patient 2 Mother of Patient 1 a d EOP EOH EOS AP AH AS ADS NP NH 10.6 6.9 14.2 12.3 89.4 93.1 85.8 87.7 14.0 EOP EOH EOS AP AH AS ADS NP NH Each ceramide class in wrist ng/μg-protein 12.0 10.0 8.0 6.0 4.0 2.0 0.0 Patient 1 Patient 2 Mother of Patient 2 Mother of Patient 1 b 11.6 7.5 14.8 11.3 88.4 92.5 85.2 88.7 EOP EOH EOS AP AH AS ADS NP NH 16.0 Each ceramide class in forearm ng/μg-protein 12.0 14.0 10.0 8.0 6.0 4.0 2.0 0.0 Patient 1 Patient 2 Mother of Patient 2 Mother of Patient 1 c Esterified Ceramides (%) Non-esterified Ceramides (%) Esterified Ceramides (%) Non-esterified Ceramides (%) 5.0 Each ceramide class in palm ng/μg-protein 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 4.3 4.4 7.8 4.1 95.7 95.6 92.2 95.9 Patient 1 Patient 2 Mother of Patient 2 Mother of Patient 1 a d EOP EOH EOS AP AH AS ADS NP NH 10.6 6.9 14.2 12.3 89.4 93.1 85.8 87.7 14.0 EOP EOH EOS AP AH AS ADS NP NH Each ceramide class in wrist ng/μg-protein 12.0 10.0 8.0 6.0 4.0 2.0 0.0 Patient 1 Patient 2 Mother of Patient 2 Mother of Patient 1 b 11.6 7.5 14.8 11.3 88.4 92.5 85.2 88.7 EOP EOH EOS AP AH AS ADS NP NH 16.0 Each ceramide class in forearm ng/μg-protein 12.0 14.0 10.0 8.0 6.0 4.0 2.0 0.0 Patient 1 Patient 2 Mother of Patient 2 Mother of Patient 1 c Figure 4. Tape stripping and liquid chromatography-mass spectrometry analysis shows reduced percentages of esteriﬁed ceramides in patient 1 and patient 2 skin. (a) In palm skin, total ceramide levels in patient 1 are reduced compared with his mother, although differences are not observed in patient 2, who had a milder clinical phenotype. (b) Total ceramide levels are also reduced in wrist skin in both patients with respect to their mothers, more so for patient 1. (c) In forearm skin (clinically normal), total ceramide levels are not reduced in patient skin. KDSR mutations lead to variable alterations in skin ceramides The levels of 11 major ceramide species in the skin of the forearm, wrist, and palm were assessed by tape stripping and Journal of Investigative Dermatology (2017), Volume 137 2348 Journal of Investigative Dermatology (2017), Volume 137 2348 2348 T Takeichi et al. KDSR M t ti D Values presented are the mean of MFI standard error of mean in duplicate samples from the two compound heterozygote patients (black bars), their mothers (white bars), and two parallel controls (gray bars). Signiﬁcant differences (P < 0.05, Mann-Whitney test) compared with control samples. M, mol/L; MFI, median ﬂuorescence intensity. Figure 5. Functional differences are present in platelets from patients 1 and 2 compared with control samples. Platelets from compound heterozygotes of the mutations c.413T>G and c.417þ3A>C in KDSR, their heterozygous mothers (carrying the c.417þ3A>C mutation), and healthy unrelated control subjects (combined data from two subjects) were stimulated under static conditions (30 minutes at room temperature) with agonist (1.6 mmol/L arachidonic acid [AA], 10 mmol/L adenosine diphosphate [ADP], 25 mmol/L PAR1 peptide [PAR1p], 100 mmol/L PAR4 peptide [PAR4p], 2 mg/ml convulxin [CVX], and 2 mg/ml collagen-related peptide [CRP]) in the presence of both PAC-1-FITC and anti-CD62P-PE monoclonal antibodies. The samples were evaluated by ﬂow cytometry, and the MFIs for (a) alpha granule release (anti- CD62P-PE) and (b) aIIbb3 integrin activation (anti PAC-1-FITC) are shown. Values presented are the mean of MFI standard error of mean in duplicate samples from the two compound heterozygote patients (black bars), their mothers (white bars), and two parallel controls (gray bars). Signiﬁcant differences (P < 0.05, Mann-Whitney test) compared with control samples. M, mol/L; MFI, median ﬂuorescence intensity. formation is the salvage pathway, which delivers 50e90% of the ceramide and uses hydrolysis of sphingomyelin by sphingomyelinase (Linn et al., 2001). Ceramide can be also be synthesized de novo in the endoplasmic reticulum (Linn et al., 2001). The ﬁrst step in the de novo pathway of cer- amide synthesis is catalyzed by serine palmitoyl transferase, condensing L-serine and a fatty acid to generate 3-KDS. Subsequently, reduction of 3-KDS by KDSR produces DHS. DHS is the substrate of ceramide synthases, a group of six enzymes, which bind fatty acids of varying lengths to the amide group of DHS, thus giving rise to a variety of dihy- droceramides (Levy and Futerman, 2010). Finally, dihy- droceramide desaturase creates a double bond between positions 4 and 5, generating ceramide. The mutations we identiﬁed in KDSR are predominantly loss of function, leading to reduced ceramide synthesis with a relative reduction of esteriﬁed ceramides evident in our tape stripping and liquid chromatography-mass spectrometry an- alyses. T Takeichi et al. KDSR M t ti D T Takeichi et al. KDSR M i D T Takeichi et al. KDSR Mutations Disrupt Skin and Platelet Function T Takeichi et al. KDSR Mutations Disrupt Skin and Platelet Function Table 2. Blood parameters, platelet size, glycoprotein expression, and annexin V and tissue factor binding in patients 1 and 2, their mothers, and control subjects P1 P2 Mother of P1 Mother of P2 Control 1 Control 2 WBC (109/L) 16.1 8.2 5.2 8.6 7.2 7.8 Hb (g/dl) 13.9 12.7 12.4 13.5 12.8 14.3 Ht (%) 40.2 37.9 36.6 39.9 37.7 42.6 Platelets (109/ L) 24 7 213 226 207 206 FSC (MFI) 32.9 32.9 26.8 27.9 25.7 25.6 CD42b (MFI) 127.2 122.8 152.7 186.2 161.3 199.5 CD42a (MFI) 187.2 189.2 188.1 207.7 180.6 212.8 CD61 (MFI) 209.8 215.3 194.5 237.8 216.7 226.4 CD49b (MFI) 29.5 28.5 33.6 41.1 39.5 31.6 Annexin V (% positive) 2.5 1.7 5.9 4.2 4.7 3.6 Tissue factor (% positive) 4.2 4.4 7.8 5.9 7.5 6.2 Abbreviations: FSC, forward side scatter; Hb, hemoglobin; Ht, hematocrit; MFI, median ﬂuorescence intensity; P1, patient 1; P2, patient 2; WBC, white blood cells. Figure 5. Functional differences are present in platelets from patients 1 and Figure 5. Functional differences are present in platelets from patients 1 and 2 compared with control samples. Platelets from compound heterozygotes of the mutations c.413T>G and c.417þ3A>C in KDSR, their heterozygous mothers (carrying the c.417þ3A>C mutation), and healthy unrelated control subjects (combined data from two subjects) were stimulated under static conditions (30 minutes at room temperature) with agonist (1.6 mmol/L arachidonic acid [AA], 10 mmol/L adenosine diphosphate [ADP], 25 mmol/L PAR1 peptide [PAR1p], 100 mmol/L PAR4 peptide [PAR4p], 2 mg/ml convulxin [CVX], and 2 mg/ml collagen-related peptide [CRP]) in the presence of both PAC-1-FITC and anti-CD62P-PE monoclonal antibodies. The samples were evaluated by ﬂow cytometry, and the MFIs for (a) alpha granule release (anti- CD62P-PE) and (b) aIIbb3 integrin activation (anti PAC-1-FITC) are shown. Values presented are the mean of MFI standard error of mean in duplicate samples from the two compound heterozygote patients (black bars), their mothers (white bars), and two parallel controls (gray bars). Signiﬁcant differences (P < 0.05, Mann-Whitney test) compared with control samples. M, mol/L; MFI, median ﬂuorescence intensity. Table 2. T Takeichi et al. KDSR M t ti D Blood parameters, platelet size, glycoprotein expression, and annexin V and tissue factor binding in patients 1 and 2, their mothers, and control subjects Abbreviations: FSC, forward side scatter; Hb, hemoglobin; Ht, hematocrit; MFI, median ﬂuorescence intensity; P1, patient 1; P2, patient 2; WBC, white blood cells. present, there does not appear to be a basis for clear genotype-phenotype correlation. Before the discovery of human mutations in KDSR, data linking this gene to disease have been very limited, aside from a missense variant in the bovine ortholog of KDSR that was proposed to cause spinal muscular atrophy (Krebs et al., 2007). Intriguingly, however, a de novo deletion of human chromosome arm 18q has been reported previously in an infant with lethal harlequin ichthyosis (Stewart et al., 2001): this child’s karyotype was 46,XY,del(18)(q21.3). The authors hypothesized that the causative gene may be located at or distal to 18q21.3 and that this deletion may have unveiled this autosomal recessive disorder. Indeed, KDSR is located at 18q21.33, and thus we speculate that loss of KDSR may have been responsible for this individual’s phenotype. The vast majority of cases of harlequin ichthyosis have biallelic mu- tations in the lipid transporter gene ABCA12 (Akiyama, 2014), but this previous report (Stewart et al., 2001) and our current ﬁndings in two further subjects with harlequin ichthyosis identify KDSR as a possible additional candidate gene for non-ABCA12 harlequin ichthyosis. Figure 5. Functional differences are present in platelets from patients 1 and 2 compared with control samples. Platelets from compound heterozygotes of Figure 5. Functional differences are present in platelets from patients 1 and 2 compared with control samples. Platelets from compound heterozygotes of the mutations c.413T>G and c.417þ3A>C in KDSR, their heterozygous mothers (carrying the c.417þ3A>C mutation), and healthy unrelated control subjects (combined data from two subjects) were stimulated under static conditions (30 minutes at room temperature) with agonist (1.6 mmol/L arachidonic acid [AA], 10 mmol/L adenosine diphosphate [ADP], 25 mmol/L PAR1 peptide [PAR1p], 100 mmol/L PAR4 peptide [PAR4p], 2 mg/ml convulxin [CVX], and 2 mg/ml collagen-related peptide [CRP]) in the presence of both PAC-1-FITC and anti-CD62P-PE monoclonal antibodies. The samples were evaluated by ﬂow cytometry, and the MFIs for (a) alpha granule release (anti- CD62P-PE) and (b) aIIbb3 integrin activation (anti PAC-1-FITC) are shown. DISCUSSION In this study, we identiﬁed biallelic mutations in KDSR in patients with defective keratinization and thrombocytopenia, implicating KDSR in the pathobiology of hereditary palmo- plantar keratodermas and ichthyosis, as recently shown by Boyden et al. (2017), but also showing that KDSR has an important additional role in platelet biology. Our data suggest that mutations in KDSR impair ceramide biosynthesis path- ways and function in skin and platelets. Clinically, the skin phenotypes in our patients were diverse: two patients had localized hyperkeratosis/keratoderma, and the other two had generalized harlequin-like ichthyosis. Neither of these forms of abnormal corniﬁcation resembled the progressive symmetric erythrokeratoderma reported in association with KDSR mutations by Boyden et al. (2017). Collectively, most of the mutations are loss-of-function but, at The plasma sphingosine-1-phosphate (S1P) concentration in patient 1, who presented with more severe clinical www.jidonline.org 2349 T Takeichi et al. KDSR M t ti D T Takeichi et al. KDSR M t ti D Ceramides are a class of sphingolipids, a family of lipids present in eukaryotes, which are involved in a variety of key physiologic functions in the skin, brain, immune sys- tem, and blood vessels (Wegner et al., 2016). Ceramides are vital not only for membrane structure integrity but are also essential for critical signaling processes such as cell cycle arrest, migration, chemotaxis, adhesion, and differentiation (Wegner et al., 2016). Additionally, ceramides are relevant to proliferation, inﬂammation, apoptosis, and autophagy in the context of stress (Uchida, 2014). There are more than 1,000 ceramide species, most of which are present in skin stratum corneum (Kihara, 2016). The major route of ceramide Patients with mutations in KDSR also exhibit progressive thrombocytopenia and a moderate functional platelet defect that develops early in life. The most likely explanation for the reduction in platelet count is diminished S1P synthesis. With Journal of Investigative Dermatology (2017), Volume 137 50 2350 T Takeichi et al. KDSR Mutations Disrupt Skin and Platelet Function T Takeichi et al. T Takeichi et al. KDSR Mutations Disrupt Skin and Platelet Function * 400 300 a 200 Plasma S1P (ng/L) 100 0 * * Serum 10 8 b 6 4 Ceramide expression (MFI) 2 0 Figure 6. KDSR mutations reduce sphingosine 1 phosphate (S1P) and ceramide expression in plasma, serum, and activated platelets. (a) S1P concentration in blood plasma and serum samples obtained from patients 1 and 2 and healthy control subjects. Red bars represent the affected individuals, and violet bars represent two parallel control subjects. (b) Ceramide expression in stimulated platelets in response to 250 mmol/L PAR1p. Bars colored in lighter shades of red, orange, and violet represent unstimulated cells, and the bars in darker shades of each respective color indicate activated platelets. The values shown are the means of duplicate samples of (a) S1P concentration and (b) MFI. The error bars indicate the standard error of the mean. Signiﬁcant differences (P < 0.05, Mann-Whitney test) (a) in plasma S1P levels between patients and control subjects and (b) in ceramide expression between unstimulated and activated platelets. MFI, median ﬂuorescence intensity; PAR1p, PAR1 peptide. 400 300 a 200 Plasma S1P (ng/L) 100 0 Serum receptor-targeting drugs that act as agonists upon initial binding to the S1P receptor. MATERIALS AND METHODS The full description of all materials and methods used in this study for venous blood sampling for DNA, platelet, plasma, and serum studies, as well as methodology for whole-exome sequencing (including reads and coverage), cell culture and transfection, immunoﬂuorescence microscopy, quantitative PCR, and platelet microscopy and ﬂow cytometry are provided in the Supplementary Materials online. relevance to onset of platelet loss, transplacental sphingosine stored in erythrocytes during fetal life may account for thrombocytopenia not being evident at birth. Instead, this phenomenon may manifest once the release from these cells, with a life span of 4 months, is compromised. In thrombo- poiesis, both extracellular and intracellular normal levels of this lipid mediator are essential in pro-platelet shedding from megakaryocytes in genetically deﬁcient mice (Zhang et al., 2012, 2013). Therefore, defects in platelet formation and release in the ﬁnal stage of thrombopoiesis may contribute to the pathogenesis of thrombocytopenia in KDSR patients. Moreover, the functional defects associated with mutations in KDSR could be related to the reduced synthesis of not only S1P but also ceramide. Previous studies in knockout mice have shown that platelets defective in S1P or ceramide fail to activate normally and that exogenous ceramide or S1P is able to rescue the phenotype of defective platelet secretion and aggregation (Munzer et al., 2014; Urtz et al., 2015). relevance to onset of platelet loss, transplacental sphingosine stored in erythrocytes during fetal life may account for thrombocytopenia not being evident at birth. Instead, this phenomenon may manifest once the release from these cells, with a life span of 4 months, is compromised. In thrombo- poiesis, both extracellular and intracellular normal levels of this lipid mediator are essential in pro-platelet shedding from megakaryocytes in genetically deﬁcient mice (Zhang et al., 2012, 2013). Therefore, defects in platelet formation and release in the ﬁnal stage of thrombopoiesis may contribute to the pathogenesis of thrombocytopenia in KDSR patients. Moreover, the functional defects associated with mutations in KDSR could be related to the reduced synthesis of not only S1P but also ceramide. Previous studies in knockout mice have shown that platelets defective in S1P or ceramide fail to activate normally and that exogenous ceramide or S1P is able to rescue the phenotype of defective platelet secretion and aggregation (Munzer et al., 2014; Urtz et al., 2015). T Takeichi et al. KDSR M t ti D Signiﬁcant differences (P < 0.05, Mann-Whitney test) (a) in plasma S1P levels between patients and control subjects and (b) in ceramide expression between unstimulated and activated platelets. MFI, median ﬂuorescence intensity; PAR1p, PAR1 peptide. ceramide expression in plasma, serum, and activated platelets. (a) S1P concentration in blood plasma and serum samples obtained from patients 1 and 2 and healthy control subjects. Red bars represent the affected individuals, and violet bars represent two parallel control subjects. (b) Ceramide expression in stimulated platelets in response to 250 mmol/L PAR1p. Bars colored in lighter shades of red, orange, and violet represent unstimulated cells, and the bars in darker shades of each respective color indicate activated platelets. The values shown are the means of duplicate samples of (a) S1P concentration and (b) MFI. The error bars indicate the standard error of the mean. Signiﬁcant differences (P < 0.05, Mann-Whitney test) (a) in plasma S1P levels between patients and control subjects and (b) in ceramide expression between unstimulated and activated platelets. MFI, median ﬂuorescence intensity; PAR1p, PAR1 peptide. Yeast strain and medium The yeast Saccharomyces cerevisiae strain KHY625 (MATa ura3 his3 trp1 leu2 Dtsc10::LEU2; Kihara and Igarashi, 2004) harboring a URA3 marker-containing plasmid was grown on synthetic complete minus uracil (0.67% yeast nitrogen base, 2% D-glucose, 0.5% casamino acids, 20 mg/L adenine, and 20 mg/L tryptophan) plates with or without 5 mmol/L phytosphingosine and 0.0015% Nonidet P- 400 (dispersant) at 30 C. T Takeichi et al. KDSR M t ti D Fingolimod administration cau- ses a rapid increase in platelet numbers in mice (Zhang et al., 2012), suggesting acute agonistic action of the drug on megakaryocyte S1P receptor-induced platelet release. Thus, it could be possible, in patients with reduced but not absent KDSR enzymatic activity, to therapeutically regulate platelet deﬁciencies by targeting the S1P receptor. Regarding treat- ment of the skin, we saw no or limited response to systemic retinoid (acitretin) in three subjects (patients 1, 2, and 3). In contrast, use of isotretinoin in individuals with KDSR muta- tions and a progressive symmetric erythrokeratoderma phenotype was reported to be very effective (Boyden et al., 2017). Between our study (patient 3) and that of Boyden et al. (2017), there were two patients who died in early in- fancy. Although there are currently no data speciﬁcally implicating KDSR mutations as being any more likely to lead to increased infant mortality over other forms of congenital ichthyosis, this potentially poor outcome will need to be reviewed as more cases of KDSR mutations are documented. a * * 10 8 b 6 4 Ceramide expression (MFI) 2 0 b In conclusion, our data add to recent ﬁndings by Boyden et al. (2017) in showing that defective ceramide biosyn- thesis due to mutations in KDSR is responsible for some forms of local hyperkeratosis and generalized ichthyosis. Moreover, we show that the KDSR mutations we identiﬁed are also associated with accompanying thrombocytopenia. Our work therefore extends knowledge about ceramides in skin disease and provides original insights into ceramides and platelet biology, with collective implications for patient diagnostics, prognostics, and therapeutics. Figure 6. KDSR mutations reduce sphingosine 1 phosphate (S1P) and ceramide expression in plasma, serum, and activated platelets. (a) S1P Figure 6. KDSR mutations reduce sphingosine 1 phosphate (S1P) and ceramide expression in plasma, serum, and activated platelets. (a) S1P concentration in blood plasma and serum samples obtained from patients 1 and 2 and healthy control subjects. Red bars represent the affected individuals, and violet bars represent two parallel control subjects. (b) Ceramide expression in stimulated platelets in response to 250 mmol/L PAR1p. Bars colored in lighter shades of red, orange, and violet represent unstimulated cells, and the bars in darker shades of each respective color indicate activated platelets. The values shown are the means of duplicate samples of (a) S1P concentration and (b) MFI. The error bars indicate the standard error of the mean. ACKNOWLEDGMENTS The Centre for Dermatology and Genetic Medicine is supported by a Well- come Trust Strategic Award (reference 098439/Z/12/Z). The work was sup- ported by the UK Biotechnology and Biological Sciences Research Council (BBSRC), the Medical Research Council (MRC), and the National Institute for Health Research (NIHR) comprehensive Biomedical Research Centre (BRC) award to Guy’s and St. Thomas’ NHS Foundation Trust, in partnership with the King’s College London and King’s College Hospital NHS Foundation Trust. This work was also supported by funding from Advanced Research and Development Programs for Medical Innovation (AMED-CREST) from the Japan Agency for Medical Research and Development (AMED). This work was also supported by a Grant-in-Aid for Scientiﬁc Research (B) 15H04887, and by a Grant-in-Aid for Young Scientists (B) 16K19717 from the Japan So- ciety for the Promotion of Science (JSPS). REFERENCES Akiyama M. The roles of ABCA12 in epidermal lipid barrier formation and keratinocyte differentiation. Biochim Biophys Acta 2014;1841:435e40. Aldahmesh MA, Mohamed JY, Alkuraya HS, Verma IC, Puri RD, Alaiya AA, et al. Recessive mutations in ELOVL4 cause ichthyosis, intellectual disability, and spastic quadriplegia. Am J Hum Genet 2011;89:745e50. Boyden LM, Vincent NG, Zhou J, Hu R, Craiglow BG, Bayliss SJ, et al. Mu- tations in KDSR cause recessive progressive symmetric erythroker- atoderma. Am J Hum Genet 2017;100:978e84. SUPPLEMENTARY MATERIAL Supplementary material is linked to the online version of the paper at www. jidonline.org, and at http://dx.doi.org/10.1016/j.jid.2017.06.028. Plasmid generation Human FVT-1/KDSR cDNA was digested from the pAK591 plasmid (Kihara and Igarashi, 2004) and cloned into pCE-puro 3xFLAG-1, the mammalian expression vector designed for N-terminal 3xFLAG-tag- ged protein production. Four of the identiﬁed mutations (F138C, D5, D5D6, and Q271E) were created using the QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA), and the primers listed in Supplementary Table S1. The E75Nfs2 mutant was produced by amplifying the mutated KDSR gene using the primers KDSR-F and KDSR E75Nfs2, respectively (see Supplementary Table S1), followed by cloning into the pCE-puro 3xFLAG-1 vector. For expression in yeast, wild-type and mutant KDSR plasmids were The platelet abnormalities in patients proved difﬁcult to treat with conventional approaches, but an alternative strat- egy might be to use drugs such as ﬁngolimod and related S1P www.jidonline.org 2351 In vitro 3-KDS reductase assay y Cells were suspended in buffer A (50 mmol/L Tris-HCl [pH 7.5], 10% glycerol, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 protease inhibitor mixture [Complete EDTA free; Roche Diagnostics, Basel, Switzerland], 1 mmol/L phenylmethylsulfonyl ﬂuoride, and 1 mmol/L dithiothreitol]) and lysed by sonication. After ultracentrifugation (100,000g, 30 minutes, 4 C), the pellet was suspended in buffer A and was used as the total membrane fraction. Protein amounts were quantiﬁed using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientiﬁc, Waltham, MA). In vitro KDS reductase assay was performed by incubating the total membrane fraction (1 mg) with 1 mmol/L nicotinamide adenine dinucleotide phosphate and 10 mmol/L KDS (C18, Matreya, State College, PA) at 37 C for 1 hour. Lipids were extracted by mixing with successive additions of 3.75 volume of chloroform/methanol/HCl (100:200:1, volume/volume/volume), 1.25 volume of chloroform, and 1.25 volume of water. Phases were separated by centrifugation (20,000g, room temperature, 3 minutes). The resulting organic (lower) phase was recovered, dried, and dis- solved in methanol. The reaction product DHS was detected by ultra performance liquid chromatography coupled with electrospray ioni- zation tandem triple quadrupole MS (Xevo TQ-S; Waters, Milford, MA). The ultra performance liquid chromatography solvent systems and electrospray ionization condition were described previously (Yamamoto et al., 2016). DHS was detected by multiple reaction monitoring byselecting the m/z value of 302.2 at Q1 and the m/z value of 266.0 at Q3 with the collision energy setting at 20 V in positive ion mode (see Supplementary Table S5). DHS levels were quantiﬁed using a standard curve plotted from serial dilutions of DHS (Avanti Polar Lipids, Alabaster, AL) standard. Data were analyzed using MassLynx software (Waters). KDSR Mutations Disrupt Skin and Platelet Function and an L-column octadecylsilyl (2.1 mm inside diameter 150 mm; Chemicals Evaluation and Research Institute). Chromatographic separation of the lipids was achieved at a ﬂow rate of 0.2 ml/minute using a mobile phase of binary gradient solvent system. Each ceramide species was detected by selected ion monitoring of m/z [MþCH3COO]. Soluble proteins were extracted from the other half-strip with a 0.1-mol/L NaOH of 1% sodium dodecyl sulfate aqueous solution at 60 C for 150 minutes. The extract solutions were then neutralized with an HCl aqueous solution. After that, soluble proteins were measured using a BCA protein assay kit (Thermo Fisher Scientiﬁc, Waltham, MA). Samples were taken from two unaffected mothers (families 1 and 2) as a control. transferred into pAKNF316 (CEN, URA3 marker), the yeast expression vector designed to produce N-terminally 3xFLAG-tagged protein under the control of a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter. Immunoblotting Immunoblotting was performed as described previously (Kitamura et al., 2015) using anti-FLAG M2 antibody (1.85 mg/ml; Sigma, St. Louis, MO) as the primary antibody and an horseradish peroxidase- conjugated anti-mouse IgG F(ab0)2 fragment (diluted 1:7,500; GE Healthcare Life Sciences, Piscataway, NJ) as the secondary antibody. ORCIDs ORCIDs David P Kelsell: http://orcid.org/0000-0002-9910-7144 John A. McGrath: http://orcid.org/0000-0002-3708-9964 WH Irwin McLean: http://orcid.org/0000-0001-5539-5757 David P Kelsell: http://orcid.org/0000-0002-9910-7144 John A. McGrath: http://orcid.org/0000-0002-3708-9964 WH Irwin McLean: http://orcid.org/0000-0001-5539-5757 T Takeichi et al. KDSR M t ti D KDSR Mutations Disrupt Skin and Platelet Function KDSR Mutations Disrupt Skin and Platelet Function KDSR Mutations Disrupt Skin and Platelet Function KDSR Mutations Disrupt Skin and Platelet Function Kitamura T, Takagi S, Naganuma T, Kihara A. Mouse aldehyde dehy- drogenase ALDH3B2 is localized to lipid droplets via two C-terminal tryptophan residues and lipid modiﬁcation. Biochem J 2015;465: 79e87. Stewart H, Smith PT, Gaunt L, Moore L, Tarpey P, Andrew S, et al. De novo deletion of chromosome 18q in a baby with harlequin ichthyosis. Am J Med Genet 2001;102:342e5. Stewart H, Smith PT, Gaunt L, Moore L, Tarpey P, Andrew S, et al. De novo deletion of chromosome 18q in a baby with harlequin ichthyosis. Am J Med Genet 2001;102:342e5. Uchida Y. Ceramide signaling in mammalian epidermis. Biochim Biophys Acta 2014;1841(3):453e62. Krebs S, Medugorac I, Rother S, Strasser K, Forster M. A missense mutation in the 3-ketodihydrosphingosine reductase FVT1 as candidate causal muta- tion for bovine spinal muscular atrophy. Proc Natl Acad Sci USA 2007;104: 6746e51. Urtz N, Gaertner F, von Bruehl ML, Chandraratne S, Rahimi F, Zhang L, et al. Sphingosine 1-phosphate produced by sphingosine kinase 2 intrinsically controls platelet aggregation in vitro and in vivo. Circ Res 2015;117:376e87. Levy M, Futerman AH. Mammalian ceramide synthases. IUBMB Life 2010;62:347e56. Wegner MS, Schiffmann S, Parnham MJ, Geisslinger G, Grosch S. The enigma of ceramide synthase regulation in mammalian cells. Prog Lipid Res 2016;63:93e119. Linn SC, Kim HS, Keane EM, Andras LM, Wang E, Merrill AH Jr. Regulation of de novo sphingolipid biosynthesis and the toxic consequences of its disruption. Biochem Soc Trans 2001;29:831e5. Xiong HY, Alipanahi B, Lee LJ, Bretschneider H, Merico D, Yuen RK, et al. RNA splicing. The human splicing code reveals new insights into the ge- netic determinants of disease. Science 2015;347(6218):1254806. Munzer P, Borst O, Walker B, Schmid E, Feijge MA, Cosemans JM, et al. Acid sphingomyelinase regulates platelet cell membrane scrambling, secretion, and thrombus formation. Arterioscler Thromb Vasc Biol 2014;34:61e71. Yamamoto S, Yako Y, Fujioka Y, Kajita M, Kameyama T, Kon S, et al. A role of the sphingosine-1-phosphate (S1P)-S1P receptor 2 pathway in epithelial defense against cancer (EDAC). Mol Biol Cell 2016;27:491e9. Ohno Y, Nakamichi S, Ohkuni A, Kamiyama N, Naoe A, Tsujimura H, et al. 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The samples were then subjected to liquid chromatography-mass spectrometry analysis to assess the levels of 11 major ceramide species (Ishikawa et al., 2013; Ohno et al., 2015). The strips were cut into two half-strips, one for lipid analysis and the other for protein analysis. The lipids within the ﬁrst half-strip were dissolved in 2 ml of chloroform/methanol/ 2-propanol (10:45:45, volume/volume/volume). N-heptadecanoyl- D-erythro-sphingosine (d18:1/17:0) (Avanti Polar Lipids) was added as an internal control, and its ﬁnal concentration was 50 nmol/L. This lipid solution was subjected to reversed-phase liquid chroma- tography/mass spectrometry. The system was an Agilent 1100 Series LC/MSD SL system equipped with a multi-ion source, ChemStation software, a 1,100-well plate auto-sampler (Agilent Technologies) Eckl KM, Tidhar R, Thiele H, Oji V, Hausser I, Brodesser S, Preil MK, et al. 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https://openalex.org/W2904369629	http://sajie.journals.ac.za/pub/article/download/1948/895	English	null	THE FACTORS THAT AFFECT PUBLIC PARTICIPATION FOR EFFECTIVE MUNICIPAL SERVICE DELIVERY: A CASE OF WARD COMMITTEES	South African journal of industrial engineering	2,018	cc-by	7,948	South African Journal of Industrial Engineering December 2018 Vol 29(4), pp 82-93 South African Journal of Industrial Engineering December 2018 Vol 29(4), pp 82-93 OPSOMMING Hierdie studie het gepoog om die faktore wat die publiek se deelname in munisipale dienslewering bepaal deur onderhoude met raadslede in die Ekurhuleni Metropool Stadsraad in Suid-Afrika te voer. Data is versamel deur ŉ gedeeltelik gestruktureerde meningspeiling. Daarmee saam is faktoranalise en strukturele vergelykingsmodellering uitgevoer. Sewe faktore is saam gegroepeer as (1) diensleweringfasiliteringsfaktore (die publiek se deelname, aanspreeklikheid en deursigtigheid, mens- gesentreerdheid, kommunikasie, geslag verteenwoordiging en gesonde verhoudings) en (2) faktore wat dienslewering belemmer (magstryde). Gebied, ras en kennis van die Batho Pele-beginsels het die persepsie van dienslewering bepaal. Die fasiliteringfaktore van die publiek se deelname vir doeltreffende dienslewering moet aangemoedig word, terwyl magstryde geminimeer moet word vir effektiewe dienslewering in Suid-Afrika. Hierdie studie het gepoog om die faktore wat die publiek se deelname in munisipale dienslewering bepaal deur onderhoude met raadslede in die Ekurhuleni Metropool Stadsraad in Suid-Afrika te voer. Data is versamel deur ŉ gedeeltelik gestruktureerde meningspeiling. Daarmee saam is faktoranalise en strukturele vergelykingsmodellering uitgevoer. Sewe faktore is saam gegroepeer as (1) diensleweringfasiliteringsfaktore (die publiek se deelname, aanspreeklikheid en deursigtigheid, mens- gesentreerdheid, kommunikasie, geslag verteenwoordiging en gesonde verhoudings) en (2) faktore wat dienslewering belemmer (magstryde). Gebied, ras en kennis van die Batho Pele-beginsels het die persepsie van dienslewering bepaal. Die fasiliteringfaktore van die publiek se deelname vir doeltreffende dienslewering moet aangemoedig word, terwyl magstryde geminimeer moet word vir effektiewe dienslewering in Suid-Afrika. THE FACTORS THAT AFFECT PUBLIC PARTICIPATION FOR EFFECTIVE MUNICIPAL SERVICE DELIVERY: A CASE OF WARD COMMITTEES C. Naidoo1* & R.R. Ramphal2 C. Naidoo1* & R.R. Ramphal2 ARTICLE INFO Article details Submitted by authors 19 Mar 2018 Accepted for publication 8 Oct 2018 Available online 10 Dec 2018 Contact details * Corresponding author calvin.naidoo@ekurhuleni.gov.za Author affiliations 1 Ekurhuleni Metropolitan Municipality, South Africa 2 Graduate School of Business Leadership, University of South Africa, South Africa DOI http://dx.doi.org/10.7166/29-4-1948 ABSTRACT ARTICLE INFO This study sought to determine the factors that affect public participation (PP) for effective municipal service delivery by interviewing ward committee members in the Ekurhuleni Metropolitan Municipality in South Africa. Data were collected using a semi-structured questionnaire, and factor analysis and structural equation modelling were done. Seven factors were grouped as (1) facilitation of service delivery (SD) factors (i.e., public participation; accountability and transparency; people centeredness; communication; gender representation; and healthy relationships) and (2) factors that impede service delivery (i.e., power struggles). Region, race, and knowledge of the Batho Pele principles determined the perception of SD. The facilitation factors of PP for effective SD should be encouraged, while power struggles must be minimised for effective SD in South Africa. 1.1 Context Globally, local governments (LGs) are facing challenges in meeting their mandate of providing basic services that conform to the expectations of communities [1]; [2]. This makes LG the ‘coalface’ of any government’s developmental strategy. In South Africa (SA), the national, provincial, and local governments work together, based on a strong principle of intergovernmental relations in the execution of their mandates, powers, and functions. This is because national and provincial governments are primarily responsible for initiating and formulating policy, while LGs operationalise such policies and transform them into tangible service delivery (SD). However, the implementation of policy is often characterised by a lack of both effective feedback mechanisms and effective management in respect of monitoring and evaluation systems [3] — a state of affairs that tends to result in an implementation gap between the design and implementation processes. 82 In order to strengthen its service delivery plans, the SA government introduced the Batho Pele principles to serve as the acceptable policy and legislative framework for SD in the public service. Batho Pele is a ‘tool’ that is nested in strategies that are intended to enhance community or pubic participation (PP) and SD [4]. PP can be defined as a process by which potential interested and affected parties are given an opportunity to comment on, or raise issues relevant to, an application. SA is experiencing massive backlogs, a poor skills base, and persistent SD protests, often characterised by the violent destruction of public property. One major reason for such protests appears to be the exclusion or inadequate participation of the society to be served by the integrated development plan (IDP) process. This results in dissatisfaction with services, such as running water and sanitation, electricity, roads, housing, and schools [5]; [6]; [7]; [8]; [9] but, above all, the ineffective PP process [10], as promised by municipalities. Participation [3] often works best for all concerned when each of the key interest groups is satisfied with the level of participation at which they are involved, and see value in their participation. This study therefore sought to identify the factors of public participation for improving municipal service delivery in SA. .2 The existing PP framework In SA, the national government prescribes regulations for provincial and local governments to implement. LG in SA consists of municipalities that are governed by municipal councils that are elected every five years. LG is mandated by provincial government to carry out the prescribed pieces of legislation. LG has to implement the respective regulations and laws pertaining to local governance within the allocated timeframes for the effective governance of its citizens, and PP is one of the requirements that must be implemented. There are various sub–committees within the LG PP process that enhance the overall participation process. These include voter engagement, the budgeting process, the IDP, PP, and petitions. A local mayor may communicate directly with the public, or the public may directly contact the mayor about problems that concern them. Another important stakeholder are ward committees, which are made up of a ward councillor and a maximum of 10 people who are elected from the ward — an administrative division of a city — and who serve voluntarily on the ward committee. The municipal council determines the rules for the election of ward committee members. The purpose of a ward committee is to assist the democratically elected representative of a ward. The ward councillor spreads important information to the community, encourages participation in the community, and is supposed to enhance effective public participation. Eikenberry (2009) asserted that effective public participation can assist municipalities to eliminate major challenges that hinder service delivery to the citizens, including corrupt practices, power struggles, and gender misrepresentation. The ward committee must make the municipal council aware of the needs and concerns of the community, and keep the community informed of developments in the municipal council. The ward committee can make recommendations to the municipal council, but it does not have decision-making powers. Ward committee members are people within a community who know sectors of the community well, and who are thus able to assist the ward councillor with certain issues of governance. It is important to note that ward committee meetings do not replace public meetings of the ward, where all stakeholders are represented. The ward committee works with the ward councillor to ensure that the public participation process in a particular ward represents the full diversity of interests of that ward. Ward committees are meant to encourage participation by the community. 2.1 The PP process According to DWAF [15], the PP process must involve the activities shown in Figure 1: Figure 1: Activities essential to the PP process: The International Association for PP [10] n the figure, Inform means that the community should be provided with balanced and objecti nformation to enable people to understand the problem, the alternatives, and the possible solution Consult involves obtaining feedback on analysis, alternatives, and decisions. Involve means worki directly with the community throughout the process to ensure that community issues and concerns a nderstood and considered at an early stage. The objective of Collaborate is to involve the commun s equal partners in each aspect of decision-making, including the development of alternatives and t dentification of the preferred solutions. Empower aims to place the final decision in the hands of t Inform Consult Involve Collaborate Empower Figure 1: Activities essential to the PP process: The International Association for PP [10] Inform Consult Involve Collaborate Empower Inform Empower Collaborate Consult Involve Figure 1: Activities essential to the PP process: The International Association for PP [10] gure 1: Activities essential to the PP process: The International Association for PP [10] In the figure, Inform means that the community should be provided with balanced and objective information to enable people to understand the problem, the alternatives, and the possible solutions. Consult involves obtaining feedback on analysis, alternatives, and decisions. Involve means working directly with the community throughout the process to ensure that community issues and concerns are understood and considered at an early stage. The objective of Collaborate is to involve the community as equal partners in each aspect of decision-making, including the development of alternatives and the identification of the preferred solutions. Empower aims to place the final decision in the hands of the community in order to ensure that it easily accepts the decisions taken by the municipal council. According to The World Bank [10], there are three types of community participation: passive participation, which involves only the dissemination of information to stakeholders; consultative participation, which occurs when stakeholders are consulted before a municipality makes a decision, and interactive participation, which occurs when stakeholders are involved in collaborative analysis and decision-making. 83 2.2 The existing PP framework .2 The existing PP framework As already alluded to, their job is to make the municipal council aware of the needs and concerns of residents, and to keep people informed of the activities of the municipal council. The committees spread information about what the community wants from the municipal council and what the council is doing. Figure 2 shows the interdependency between the social actors. 84 Figure 2 shows the interdependency between the social actors. Figure 2: Interdependency within the municipal SD process (Source: Author) At the macro level, the national government formulates policies and allocates funds for certain basic requirements and basic services. At the micro level, the delivery of public services to the members of the local community can take place through the state/province and municipality or, on behalf of the state, by a voluntary community organisation or private sector company. This bottom-up approach means that individuals or members of the local community, through PP and the ward committees that represent them, can influence the policy decision-making and the resources that affect them. Figure 2: Interdependency within the municipal SD process (Source: Author) At the macro level, the national government formulates policies and allocates funds for certain basic requirements and basic services. At the micro level, the delivery of public services to the members of the local community can take place through the state/province and municipality or, on behalf of the state, by a voluntary community organisation or private sector company. This bottom-up approach means that individuals or members of the local community, through PP and the ward committees that represent them, can influence the policy decision-making and the resources that affect them. 84 2.3 The integrated development plan Figure 3: Intended process of IDP [12] According to the figure, consultation is supposed to occur at every step, as the participation process is envisaged to address internal and external circumstances that impact on priority issues, objectives, strategies, projects, and programmes of integrated planning. This might therefore imply that SD protests mimic some sort of non-adherence to, or a lack of alignment with, the intended process. 2.4 The IDP implementation framework 2.3 The integrated development plan Municipalities have recently adopted the integrated development plan (IDP) as a vital tool for planning and development, and to ensure that available resources are optimally used to promote sustainable economic and social development. The value of the IDP is embedded in the formulation of focused plans that are based on developmental priorities. This approach assists with curbing wasteful expenditure and breaking perpetual past spending patterns. The legislative mandate is such that the success of the IDP process hinges on public (and other stakeholder) participation, as enshrined in Chapter 2 of the Constitution, in the Bill of Rights. This mandate is visually represented in Figure 3. Constitution, in the Bill of Rights. This mandate is visually represented in Figure 3. Figure 3: Intended process of IDP [12] According to the figure, consultation is supposed to occur at every step, as the participation process is envisaged to address internal and external circumstances that impact on priority issues, objectives, strategies, projects, and programmes of integrated planning. This might therefore imply that SD protests mimic some sort of non-adherence to, or a lack of alignment with, the intended process. 2.4 The IDP implementation framework In order to manage the IDP process effectively municipalities institutionalise implementation strategies Figure 3: Intended process of IDP [12] 2.4 The IDP implementation framework In order to manage the IDP process effectively, municipalities institutionalise implementation strategies and structures for internal organisational arrangements that are meant to give effect to an inclusive participatory approach. In terms of section 74(a) of the Municipal Structures Act 58 of 1999 [79], municipalities can establish ward communities whose primary function is to act as a communication channel between the community and the municipality. Municipalities have to establish IDP representative forums, including representatives from business, labour, community, and ward committees, as well as traditional leaders or their representatives and other organisations. Public involvement in the process of public services leads to services being perceived as better [13]. The emphasis on the importance of participation is intended to strengthen a self-development process for inclusion in the policy and decision-making process, thus creating citizen satisfaction. According to DPLG [14], the gap between the social and political spheres can be closed if they are linked, as shown in figure 4. 85 Figure 4: Linking approaches to participation [14] The overlapping in the figure represents the links between social participation, citizenship, political participation, and participatory methods. If these are linked properly, then there is effective participation in local authorities. Barriers to participation can be overcome using workshops to facilitate the relationship between citizens and municipal officials. Blair [15] discusses a formula (represented in figure 5) that would enrich participation for both citizens and state institutions. In the figure, symbolises ‘an increase in’: Figure 4: Linking approaches to participation [14] Figure 4: Linking approaches to participation [14] Figure 4: Linking approaches to participation [14] The overlapping in the figure represents the links between social participation, citizenship, political participation, and participatory methods. If these are linked properly, then there is effective participation in local authorities. Barriers to participation can be overcome using workshops to facilitate the relationship between citizens and municipal officials. Blair [15] discusses a formula (represented in figure 5) that would enrich participation for both citizens and state institutions. In the figure, symbolises ‘an increase in’: Figure 5: Formula on participation [15] 2.5 Theoretical framework This study was underpinned by six theories: (1) the SD theory of a configuration of technology [66], stakeholder engagement theory [67], PP budgetary theory [68], project integration theory [69], inter- organisational ICT-supported reforms of SD theory [70], and systems theory. Of these, the main theories are systems theory and stakeholder theory. Systems theory allows one to view an organisation (e.g., EMM) as a social system composed of sub-systems that interact with one another in a holistic way. Viewed in terms of inputs, throughputs, and outcomes, the system requires knowledge of the basic resources and needs of the community that the local authority is providing (i.e., services such as water, electricity, sanitation, infrastructure, land, and housing). It also requires the input of various community groups working via elected officials, and hence it is political in nature. This system does not operate in a vacuum, but takes place in an environment where various stakeholder groups are involved: central government, citizens, businesses, policing authorities, health authorities, non-governmental organisations (NGOs), pressure groups, employees, councillors, and the media. There are also social norms and various role expectations that are associated with formal organisations, such as local authorities, that have been established for the specific purpose of service delivery [71]. Stakeholder engagement is “a process in which the organisations involve the citizens or those who are affected by the decisions which it makes” [67]. The main idea is to develop an assertive understanding of the stakeholders of an organisation, what the stakeholders care about, and in what way they relate to the goal you are urging them to reach. The organisation should get to know about their stakeholders, about their complexity and scope. The enterprise-wide programmes in the organisation need a comprehensive scan to identify the stakeholders with their needs and interests. And the next step is to engage them as early as possible, for nobody likes a sudden change in his or her entity [72]. Enterprise-wide management supplies programmes that are planned and developed among themselves, and the final plan is delivered to stakeholders. Stakeholders should be involved in the programmes, and should be encouraged to participate throughout the lifecycle of every programme. [73] states that the organisation must use the right mechanisms to keep the stakeholders happy. They will not participate if not given proper guidance and ways to improve the participatory process. One must be open-minded so that the stakeholders will continue participating. 2.5 Theoretical framework Communication with stakeholders is important if the organisation is to have a good relationship with them. In order to obtain stakeholder participation in a collaborative way, the researcher suggests that the throughput process should address the following factors: accountability and transparency, social background, health relationships, putting people first by using a people centeredness approach, open communication with all stakeholders, relevant knowledge of the social background in which the local authority operates, knowledge of the presence of power struggles, and equal gender representation in the important decision-making processes. Figure 5: Formula on participation [15] Figure 5: Formula on participation [15] The horizontal arrows represent pressure and force for good governance [13]. Participation of democratic LGs ensures a certain degree of participation so that citizens can vote for their own local council members, thus involving them in the political process. There is a need for more representation on councils [15], which would lead to empowerment. Representation translates into benefits being fairly distributed (see [15]) and poverty deduction [16]; [17]; [18]. PP is seen as a vehicle to promote and instil a culture of good governance at the LG level [19]; [9]; [8]; [19]; [16]; [17]. Projects have to be transparent so that all stakeholders have a better understanding of the method or process employed, especially when many people are involved [20]; [18]; [8]; [9]. With collective participation, LGs must cater for participants from villages and rural areas or districts; but the problem occurs when ward committees work in silos, isolated from each other [21]; [5]. Table 1: The factors of PP for effective SD include: Table 1: The factors of PP for effective SD include: Factors Literature Accountability and transparency Accountability [6]; [22]; [23]; [7]; [24]; [25] [26]; [27]; [28]; [9] Transparency [29]; [30]; [31]; [8]; [32]; [17]; [1]; [33]; [34]; 35]; [36]; [37]; [38]. People-centredness [27]; [18] to make the public more involved in municipal affairs [39]; Communication [40]; [14]; [41]; [42]. Power struggles [43]; [44]; [14]; [41]; [46]; [47]; [48]; [64]; [49]. Gender representation [50]; [51]; [52]. Other factors: knowledge and social background, representativeness gap, and health relationships. Workforce turnovers, resource gap (cost) and trust gap [53]; [54], [55]; [56]; [57], [58], [59]; [60]; [61]; [62]; [63]; [35]. [64]; [65]; [51]. 86 3.1 Data In this study, the Ekurhuleni Metropolitan Municipality (EMM) was used as a case, and the quantitative approach and conclusive survey research design were adopted. Data were intended to be collected from a random sample of the 400 ward committee members, using anonymous online semi-structured questionnaires, but only 108 responded positively, yielding a response rate of 27 per cent. The questionnaire was validated by addressing the different types of validity: face, constructive, and content. It was also piloted using at least 40 (10% of 400) members of the target population, who were not used again in the main survey [74]. The sample size was chosen in accordance with the suggestion of [75] that exploratory factor analysis (EFA) requires a minimum number of cases if reliable results are to be at least five times the number of questions or variables. The pilot study helped the researcher to evaluate how the questionnaire was understood, and was therefore adjusted accordingly. All the constructs were measured on a scale of agreement ranging from 1= disagree to 7 = strongly agree. To analyse the data, the scores for the concepts making up a construct were added, and the sum divided by the number of scores added to obtain the mean scores. These mean scores of the constructs were used in the analyses. 87 Figure 6 shows the model that was analysed. Factors affecting public participation Latent factors Figure 6: A model for optimising public participation in the service delivery processes  Accountability & transparency,  Effective communication  Gender representation  Power struggles  People centeredness  Social background  Healthy relationships  Facilitating and impeding factors Effective municipal service delivery Figure 6: A model for optimising public participation in the service delivery processes The figure indicates that public participation is affected by several factors: accountability and transparency, effective communication, gender representation, power struggles, people centredness, social background, and healthy relationships. The latent factors are the underlying factors (i.e., facilitating and impeding) that determine the level of municipal service delivery, which is the dependent variable. The figure indicates that public participation is affected by several factors: accountability and transparency, effective communication, gender representation, power struggles, people centredness, social background, and healthy relationships. The latent factors are the underlying factors (i.e., facilitating and impeding) that determine the level of municipal service delivery, which is the dependent variable. 3.2 Data analysis Descriptive data analysis involved data summaries and descriptive statistics to identify patterns in the data — including frequencies and cross-tabulations, percentages, means, mode and standard deviation, and graphical representation of the data. Factor analysis and structural equation modelling (SEM) were done. SPSS AMOS 23.0 was used for this. 3.1 Data Transparency is about being easy to understand, and being open, frank, and honest in all communications, transactions, and operations [6]. Accountability is about being responsible to citizens for actions taken, and about being able to explain, clarify, and justify actions. It implies that citizens have a right to know and to hold the local government to account, and that the local government has a duty to explain and account for its actions. Accountability and transparency go hand-in-hand, and involve being aware of to whom local governments are accountable, what the important pieces of information are, and how they can be communicated most effectively to the public [8]; [32]; [6]; [7]. Power-struggles are avenues for gaps in public participation and service delivery in local governments [48]; [26]; and with power-struggles, the authors assert that collective participation and transparency and accountability can hardly be realised. [35] suggested the development of a participatory culture and capacity among public officials through communities of practice and more effective communication, while [40] linked lack of fulfilment of the goals of developmental local government to lack of effective communication between the local government and community members. This was noted as a gap that needs to be bridged if effective public participation and service delivery are to be attained [40]; [76]. Facilitating inclusivity, including gender representation, is important for effective SD [50]. [27] noted that the size of a municipal territory has a direct impact on public participation in municipal service delivery: smaller municipalities foster people-centred practices in public participation [18]; [39]. There should also be healthy relationships among all the stakeholders for effective participation and SD to take place. 4.2.1 Common factors Referring to Figure 6, the PP factor explained 80.58 per cent of the variation in the perception of SD (with a Cronbach’s alpha coefficient of 0.85 and a mean score of 5.4). Accountability and transparency explained 79.2 per cent (with a Cronbach’s alpha coefficient of 0.89 and a mean score of 6.0). The communication factor explained 52.7 per cent of the variation (with a Cronbach’s alpha coefficient of 0.76 and a mean score of 4.7). Knowledge and social background explained 50.62 per cent of the variance (with a Cronbach’s alpha coefficient of 0.485 and a mean score of 5.39). Power struggles explained 51.9 per cent of the variance (with a Cronbach’s alpha coefficient of 0.91 and a mean score of 5.4). Health relationships explained 51.9 per cent of the variation (with a Cronbach’s alpha coefficient of .85 and a mean score of 5.3), and gender representation explained 78.13 per cent of the variance (with a Cronbach’s alpha coefficient of 0.829 and a mean score of 5.93). In general, the respondents agreed with the factors of PP, accountability and transparency, people centredness, healthy relationships, and gender representation; but they only partially agreed with the factors of communication, knowledge and social background, and power struggles. Two main factors were extracted, and all the constructs loaded on the first main factor, the PP facilitating factor. An exception was power struggles, which loaded on the second factor, the impeding factor. All the resulting factors had reliability coefficients that would be considered acceptable (rule of thumb >0.70) [77]. The impeding factor contained only power struggles (with a loading of 0.958 and a mean score of 5.01). Public participation in effective service delivery (for ward committee members) is affected by two main factors: one related to aspects that facilitate service delivery, and the other based on aspects that impede service delivery. Facilitation of service delivery is composed of six first-order factors (public participation, accountability and transparency, people centredness, communication, gender representation, and healthy relationships), and the impeding factor is formed by one first-order factor — power struggles — for effective service delivery. The impeding factor consists of items that hinder or hamper public participation in effective service delivery. 4.1 Descriptive analysis 88 In the sample, 64 (59.3%) out of 108 were males. The age group 19 to 35 years had 24 (22.2%) respondents; the majority, 75 (77.8%), were between 36 and 65 years old. Most of the respondents were black, with 85 (91.7%) respondents, while there were 2 (2.1%) Indian, and 17 (15.8%) white. Most of the ward committee members, 84 (77.8%), had a matric or lower educational qualification; some had a diploma or degree (21.3%). One (.9%) omitted this question. Most respondents (64%) indicated good or very good knowledge of Batho Pele principles, and 52.7 per cent were happy with communication on SD from the EMM offices. (The reader is cautioned that Indian committee members’ perception of the presence of public participation, accountability, and transparency in their wards, and of SD in general, is based on only a very small number of observations.) degree (21.3%). One (.9%) omitted this question. Most respondents (64%) indicated good or very good knowledge of Batho Pele principles, and 52.7 per cent were happy with communication on SD from the EMM offices. (The reader is cautioned that Indian committee members’ perception of the presence of public participation, accountability, and transparency in their wards, and of SD in general, is based on only a very small number of observations.) 4.2.1 Common factors The mean score of 5.32 of the facilitating factor indicated a partial agreement with the first-order factors forming it, whereas the overall mean score of 5.01 of the second main factor, power struggles, indicated that the ward committee members partially agreed that party politics impeded service delivery in the EMM. 4.3.1 Conclusions Seven factors were found to be involved in obtaining the largest possible public participation in service delivery processes. These were grouped as (1) factors that facilitate service delivery, and (2) factors that impede service delivery. The facilitating factors were public participation, accountability and transparency, people centredness, communication, gender representation, and healthy relationships. The impeding factor was power struggles. For ‘region of residence’, significant differences were present only with respect to the facilitating factor. Ward committee members from previously disadvantaged regions agreed significantly more strongly with the facilitating factor than did ward committee members from previously advantaged regions. The factors concerned were public participation, accountability and transparency, healthy relationships, and gender representation. Black ward committee members agreed statistically significantly more strongly with the facilitating factor, including public participation and accountability and transparency, than did white ward committee members. Very good knowledge of the Batho Pele principles — which was mostly associated with black respondents — was associated with stronger agreement that power struggles influenced public participation and service delivery issues. 4.3.2 Recommendations  There should be a co-development by the community and the municipality of a comprehensive strategy to enhance the first-order factors associated with the facilitating factor. As the facilitation of effective service delivery is caused by public participation, accountability and transparency, effective communication, and gender representation, a comprehensive strategy needs to be designed to enhance the factors associated with the facilitation factor and to suppress power struggles — the factor associated with the impeding factor. Such a strategy must be the result of collaboration between the municipality and the various community groups involved with service delivery issues. The drawing up of the annual municipal budget could serve as a good example of collaborative planning.  There should be a co-development by the community and the municipality of a comprehensive strategy to enhance the first-order factors associated with the facilitating factor. As the facilitation of effective service delivery is caused by public participation, accountability and transparency, effective communication, and gender representation, a comprehensive strategy needs to be designed to enhance the factors associated with the facilitation factor and to suppress power struggles — the factor associated with the impeding factor. Such a strategy must be the result of collaboration between the municipality and the various community groups involved with service delivery issues. The drawing up of the annual municipal budget could serve as a good example of collaborative planning. p g  In the light of the point above, the annual budget should be a collaborative planning issue between the municipality and the various stakeholder public groups, through which it is subjected to open communication, and where all involved can give their honest opinion about the money allocated to the various services p g  In the light of the point above, the annual budget should be a collaborative planning issue between the municipality and the various stakeholder public groups, through which it is subjected to open communication, and where all involved can give their honest opinion about the money allocated to the various services 4.2.2 Associations with independent variables The non-parametric equivalent of the independent t-test, the Mann-Whitney U-test, was used, as the constructs were not normally distributed. It was also found that gender, the number of years resident in a particular area, and the education of a ward committee member did not affect their perceptions about public participation in service delivery processes. ‘Region where one lives’ was found to be associated with their perceptions, but only for the facilitation factors. The only factors that differed significantly according to ‘region where one lives’ were public participation, accountability and transparency, healthy relationships, and gender representation. Ward committee members in the disadvantaged areas agreed more strongly with public participation, accountability and transparency, healthy relationships, and gender representation than did the ward committee members from previously advantaged areas. Race affected the perceptions of ward committee members about public participation in service delivery processes too, as far as blacks and whites and the facilitation factors were concerned. Facilitation factors involved were public participation, and accountability and transparency. Seemingly, for both factors, 89 black ward committee members agreed more strongly with the view that there is public participation, and accountability and transparency in their wards than did the white ward committee members. black ward committee members agreed more strongly with the view that there is public participation, and accountability and transparency in their wards than did the white ward committee members. Knowledge of the Batho Pele principles is another important variable affecting the impeding factor. The ward committee members who indicated that they had very good knowledge of the Batho Pele principles were associated with a stronger agreement that power struggles influence public participation and service delivery issues than were the others. 5 FUTURE RESEARCH Oxford: page 1-10,Oxford University Press. [22] I tit t f F d i i d D fi i t bilit d t & k i i l A il bl t White, G. 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Klientų dalyvavimas viešosiose paslaugose: bendrasis p Viešoji politika ir administravimas, 27, pp. 54-62. j p , , pp [24] Meng, B. 2008. Pathways towards citizen participation: Insights on community planning in Cambodian cities. Journal of Contemporary Easter Asia, 7(2), pp. 49-55. f p y ( ), pp [25] United Nations. 2011. Good governance practices for the protection of human rights. Office of the United Nations High Commissioner for Human Rights. [26] United Nations. 2012. Good governance practices for the protection of human rights. Office of the United Nations High Commissioner for Human Rights. [27] Glaser, M.A., Yeager, S.J. and Parker, L.E. 2008. Involving citizens in the decisions of government and community: Neighborhood-based vs. government-based citizen engagement. Public Administration Review, Summer, pp. 177-217. [28] Siebert, H. 2008. Economics of the environment: Theory and policy. Berlin: Sprin [29] Transparency International. 2008. Corruption perception index. Available at [28] Siebert, H. 2008. Economics of the environment: Theory and policy. Berlin: Spring [29] Transparency International. 2008. Corruption perception index. Available at [ ] , f y p y p g g [29] Transparency International. 2008. Corruption perception index. Available at http://www.transparency.org/policy_research/surveys_indices/cpi/2008. Accessed 28 November 2014. 5 FUTURE RESEARCH c research suggestions that emerged from this empirical investigation include the followin Specific research suggestions that emerged from this empirical investigation include the  More items relating to knowledge and social background need to be included.  The actual reasons why respondents differed statistically significantly from one another in some of the latent factors involved needs to be further investigated. 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https://openalex.org/W2299681553	https://ejournal.undip.ac.id/index.php/ijms/article/download/10054/pdf	Indonesian	null	Diversity and Abundance of Sand Crabs on the South Coast of Central Java (Diversitas dan Kelimpahan Kepiting Pasir di Pantai Selatan Jawa Tengah)	Deleted Journal	2,014	cc-by-sa	3,703	Ali Mashar1, Yusli Wardiatno2, Mennofatria Boer2, Nurlisa A. Butet2, dan Achmad Farajallah3 ar1, Yusli Wardiatno2, Mennofatria Boer2, Nurlisa A. Butet2, dan Achmad Farajallah3 Ali Mashar1, Yusli Wardiatno2, Mennofatria Boer2, Nurlisa A. Butet2, dan Ac 1Program Studi Pengelolaan Sumberdaya Perairan, Sekolah Pascasarjana IPB 2Departemen Manajemen Sumberdaya Perairan, Fakultas Perikanan dan Ilmu Kelautan IPB 3Departemen Biologi, Fakulitas Matematika dan Ilmu Pengetahuan Alam IPB Jl. Agatis, Kampus Institut Pertanian Bogor, Dramaga, Indonesia 16680 Email: alimashar75@gmail.com Abstrak Kepiting pasir atau undur-undur laut merupakan biota bentik yang hidup di pantai berpasir yang mempunyai nilai ekologi dan nilai ekonomi cukup penting. Adanya tekanan penangkapan mengharuskan adanya pengelolaan yang bijak yang disesuaikan dengan karakteristik populasi kepiting pasir. Informasi tentang jenis dan kelimpahan kepiting pasir penting untuk diketahui terlebih dahulu sebagai langkah awal upaya pengelolaan lestari kepiting pasir. Penelitian ini bertujuan untuk mengetahui komposisi jenis dan kelimpahan setiap jenis kepiting pasir yang terdapat di dua lokasi penelitian, yaitu pantai Bocor, Kabupaten Kebumen, dan pantai Bunton, Kabupaten Cilacap. Hasil penelitian menunjukkan bahwa di kedua lokasi penelitian ditemukan tiga jenis kepiting pasir, yaitu Emerita emeritus, Hippa adactyla, dan Albunea symmysta. Kepiting pasir E. emerita ditemukan dengan persentase komposisi paling besar, 70,5%-75,3%; disusul H. adactyla 22,5%-24,7%; dan A. symmysta 2,2%- 4,8%. Kepiting pasir betina dijumpai dengan persentase komposisi paling tinggi, yaitu 78,2% hingga 92,8%. Kepiting pasir E. emeritus juga ditemukan dengan kelimpahan rata-rata paling tinggi di kedua lokasi penelitian, yaitu 5 dan 34 ekor/100m2. Kepiting pasir E. emeritus dan H. adactyla yang ditemukan di pantai Bunton, Cilacap mempunyai kelimpahan lebih tinggi dari yang ditemukan di pantai Bocor, Kebumen, adapun kelimpahan rata-rata Albunea symmysta relatif sama di kedua lokasi penelitian, yaitu sekitar 1 ekor/100m2. Implikasi dari hasil penelitian ini adalah diversitas kepiting pasir di pesisir selatan Jawa Tengah relatif tinggi dengan kelimpahan yang berbeda-beda yang dipengaruhi oleh intensitas aktivitas manusia di wilayah pantai berpasir. Kata kunci: Albunea symmysta, Emerita emeritus, Hippa adactyla, kelimpahan, kepiting pasir, komposisi jenis ILMU KELAUTAN Desember 2014 Vol 19(4):226-232 ILMU KELAUTAN Desember 2014 Vol 19(4):226-232 ISSN 0853-7291 ) Corresponding author © Ilmu Kelautan, UNDIP Diversity and Abundance of Sand Crabs on the South Coast of Central Java Sand crabs or mole crabs are benthic fauna that live in the sandy beaches. They have ecological and economic value. Because of fishing pressure to this organism, it requires wise management based on the characteristics of the sand crab population. Information on the type and abundance of sand crabs is important as an initial step in sustainable management of sand crabs. This study aims to determine the species composition and abundance of each species of sand crabs. Sand crab specimens were collected from two study sites, namely Bocor beach, Kebumen, and Bunton beach, Cilacap. The results showed that in both sites found three species of sand crabs, namely Emerita emeritus, Hippa adactyla, and Albunea symmysta. E. emerita found in greatest composition percentage, 70.5% -75.3%; followed by H. adactyla 22.5% -24.7%; and A. symmysta 2.2% -4.8%. Females sand crab found in highest composition percentage, which is 78.2% to 92.8%. E. emeritus were also found in highest abundance average in both sites, 5 and 34 ind.100m-2. E. emeritus and H. adactyla were found on Bunton beach, Cilacap have abundance higher than that found on Bocor beach, Kebumen, while average abundance ofAlbunea symmysta relatively similar in both study sites, which is about 1 ind.100m-2. Implication of this research is high diversity of sand crabs relatively on the southern coast of Central Java with varying abundance that affected by intensity of human activities in the sandy beach area. Keywords: abundance, Albunea symmysta, Emerita emeritus, Hippa adactyla, sand crab, species co Diterima/Received : 26-10-2014 Disetujui/Accepted : 20-11-2014 ijms.undip.ac.id ILMU KELAUTAN Desember 2014 Vol 19(4):226-232 Pendahuluan makin meningkat, seiring dengan makin dikenalnya kepiting pasir, terutama karena dapat dikonsumsi. Hal tersebut ditandai dengan semakin banyak jumlah nelayan penangkap kepiting pasir. Sebagai gambaran, berdasarkan hasil wawancara dengan nelayan kepiting pasir, di pesisir Cilacap bagian timur, jumlah nelayan kepiting pasir meningkat dari 5 (lima) orang pada tahun 2007 menjadi sekitar 50 orang pada tahun 2013. Kondisi tersebut jika tidak dikendalikan dapat memberikan tekanan makin tinggi pada populasi dan habitat kepiting pasir. Oleh karena itu, diperlukan pengelolaan sumber daya kepiting pasir agar tetap lestari, baik secara ekologi maupu ekonomi. Kepiting pasir atau undur-undur laut merupakan biota bentik yang hidup di di pantai berpasir (Efford 1976; Haley 1982). Pantai berpasir Indonesia merupakan salah satu daerah sebaran kepiting tersebut, diantaranya pesisir barat Sumatera, pantai selatan Jawa, dan Maluku (Boyko dan Harvey, 1999; Haye et al., 2002; Boyko, 2002). Di pantai selatan Jawa, kepiting pasir banyak ditemukan di pantai berpasir selatan Jawa Tengah, diantaranya di pantai Kebumen dan Cilacap (Osawa et al., 2010; Mashar dan Wardiatno, 2013ab; Wardiatno et al., 2014). Kepiting pasir memiliki beberapa peran ekologis cukup penting di daerah intertidal, diantaranya sebagai makanan bagi hewan pantai dan sebagai bioindikator pencemaran pestisida atau DDT, tumpahan merkuri, dan indikasi kandungan asam domoik (Siegel dan Wenner, 1984, Wenner, 1988; Pérez, 1999; Dugan et al., 2005; Lafferty et al., 2013). Kepiting pasir juga mengandung protein dan omega-3 serta omega-6 cukup tinggi, yaitu kandungan protein 32,32% (Hartono et al., 2011), omega-3 12,49% (Hartono et al., 2011), dan omega-6 11,80% - 12,94% (Mursyidin, 2007) sehingga cukup baik untuk dikonsumsi, terutama untuk anak-anak dalam masa pertumbuhan. Sebagai langkah awal perlu diketahui jenis kepiting pasir yang terdapat di pantai berpasir Kebumen dan Cilacap, komposisi per jenis, dan kondisi kelimpahannya. Diversitas dan Kelimpahan Kepiting Pasir (A. Mashar, et al) Materi dan Metode Penelitian ini dilakukan di pantai berpasir wilayah Kabupaten Kebumen dan Kabupaten Cilacap, tepatnya di pantai Bocor, Kecamatan Buluspesantren, Kabupaten Kebumen, dan pantai Bunton, Kecamatan Adipala, Kabupaten Cilacap (Gambar 1.). Kepiting pasir ditemukan pada daerah atau zona gelombang pecah di pantai (swash zone) yang lebarnya dapat mencapai 8–30 meter tergantung kondisi gelombang (Mann, 2000; Wardiatno et al., 2013; 2014). Pantai berpasir Kebumen dan Cilacap merupakan dua wilayah di selatan Jawa yang banyak ditemukan kepiting pasir. Di kedua wilayah tersebut, kegiatan eksploitasi kepiting pasir juga Gambar 1. Lokasi penelitian. Kotak hitam menunjukkan lokasi pengambilan sampel kepiting pasir Sumber peta: www.d-maps.com/asia/indonesia Gambar 1. Lokasi penelitian. Kotak hitam menunjukkan lokasi pengambilan sampel kepiting pasir Sumber peta: www.d-maps.com/asia/indonesia Diversitas dan Kelimpahan Kepiting Pasir (A. Mashar, et al) 227 ILMU KELAUTAN Desember 2014 Vol 19(4):226-232 Analisis data Analisis data yang dilakukan adalah analisis komposisi setiap jenis kepiting pasir secara deskriptif. Selain itu dilakukan analisis kelimpahan setiap jenis kepiting pasir dengan membandingkan jumlah kepiting pasir yang tertangkap dengan luas daerah sapuan sorok, yang secara sederhana dapat ditulis dengan notasi matematika sebagai berikut: dimana: K = kelimpahan kepiting pasir (ekor.m-2) N = jumlah kepiting pasir yang tertangkap (ekor) A = luas area sapuan sorok (m2). Berdasarkan Tabel 1 diketahui bahwa dari sisi jumlah, kepiting pasir jenis Emerita emeritus paling banyak terkumpul di kedua lokasi penelitian, kemudian disusul Hippa adactyla, dan yang paling sedikit sampel kepiting pasir yang terkumpul dari jenis Albunea symmysta. Secara persentase, berdasarkan Tabel 2, komposisi hasil pengumpulan sampel kepiting pasir di kedua lokasi adalah E. emeritus 70,5%-75,3%; H. adactyla 22,5%-24,7%; dan A. symmysta 2,2%-4,8%. Kepiting pasir E. emeritus selalu didapatkan dalam jumlah yang paling besar di setiap pengampilan sampel kepiting pasir, dan A. symmysta selalu dijumpai dalam jumlah yang paling sedikit, bahkan di beberapa waktu pengambilan sampel tidak didapatkan kepiting pasir A. symmysta. Pengumpulan sampel dan genetik. Ketiga jenis kepiting pasir tersebut telah mendapatkan konfirmasi dari taksonom kepiting pasir, yaitu Dr. Christopher B. Boyko dari Division of Invertebrate Zoology, American Museum of Natural History, New York, USA (Komunikasi pribadi 2013). dan genetik. Ketiga jenis kepiting pasir tersebut telah mendapatkan konfirmasi dari taksonom kepiting pasir, yaitu Dr. Christopher B. Boyko dari Division of Invertebrate Zoology, American Museum of Natural History, New York, USA (Komunikasi pribadi 2013). Pengumpulan kepiting pasir dilakukan pada waktu yang berbeda. Pengumpulan sampel kepiting pasir di pantai Bocor, Kebumen, dilakukan selama 12 bulan dari Maret 2012 hingga Februari 2013. Pengumpulan sampel kepiting pasir di pantai Bunton, Cilacap, dilakukan selama 12 bulan dari Juni 2013 hingga Mei 2014. Penangkapan sampel kepiting pasir di kedua wilayah tersebut dilakukan dengan alat tangkap tradisional nelayan setempat yang dinamakan sorok. Alat tangkap sorok bentuknya seperti alat sorok padi pada saat dijemur, terbuat dari sebatang bambu sebagai pegangan dengan panjang antara sekitar 1,8 meter, ujungnya dipasang papan kecil dengan ukuran 20x60 cm yang fungsinya untuk menyorok. Prinsip kerja alat ini cukup sederhana, yaitu dengan menyorok atau menyisir permukaan pantai berpasir di daerah swash zone, ketika pada pasir basah yang terlewati alat tersebut tampak ada gundukan kecil yang berdenyut-denyut, maka segera gali gundukan tersebut karena kemungkinan di dalamnya terdapat kepiting pasir. Jumlah keseluruhan kepiting pasir yang tertangkap dan terkumpul selama penelitian adalah 1.513 ekor di pantai Bocor, Kebumen, dan 6.559 ekor di pantai Bunton, Cilacap. Ditemukannya kepiting pasir famili Hippidae dan Albuneidae dalam satu lokasi juga merupakan kenyataan yang jarang terjadi dan tidak terjadi di semua lokasi atau habitat kepiting pasir. Hal tersebut dikarenakan habitat kedua famili kepiting pasir tersebut memang berbeda, yaitu famili Hippidae hidup di daerah intertidal, sedangkan famili Albuneidae umumnya hidup di daerah subtidal. Maka apabila di suatu pantai berpasir ditemukan kepiting pasir famili Hippidae, jarang sekali ditemukan kepiting pasir famili Albuneidae, begitu juga sebaliknya. Kepiting pasir famili Albuneidae genus Albunea masih dapat ditemukan pada kedalaman sekitar 50-150 meter (Boyko dan Harvey, 1999; Corsini-Foka dan Kalogirou, 2013). Kejadian ini sama seperti ditemukannya kepiting pasir famili Hippidae dan Albuneidae di pantai Phuket Thailand pada penelitian tahun 1971-1973 (Boonruang dan Phasuk, 1975). Kenyataan ini menunjukkan bahwa perairan Indonesia mempunyai keanekaragaman sumber daya hayati yang tinggi, termasuk keanekaragaman kepiting pasir tersebut. Komposisi kepiting pasir Berdasarkan hasil pengumpulan sampel kepiting pasir selama penelitian, secara keseluruhan didapatkan 8.072 ekor kepiting pasir, dimana 1.513 ekor berhasil dikumpulkan dari pantai Bocor, Kebumen, dan 6.559 ekor dikumpulkan dari pantai Bunton, Cilacap. Secara lengkap, komposisi hasil tangkapan kepiting pasir untuk setiap jenis dan setiap lokasi penelitian disajikan pada Tabel 1 dan Tabel 2. K = N/A Jenis kepiting pasir Didapatkan 3 (tiga) jenis kepiting pasir, baik di pantai Bocor, Kebumen, maupun di pantai Bunton, Cilacap, yaitu 2 (dua) jenis dari famili Hippidae (Emerita emeritus dan Hippa adactyla), dan 1 (satu) jenis dari famili Albuneidae, yaitu Albunea symmysta. Ditemukannya tiga jenis kepiting pasir tersebut semakin memperkuat hasil penelitian Wardiatno et al. (2013; 2014) yang mengidentifikasi ketiga jenis kepiting pasir tersebut secara morfologi 228 Diversitas dan Kelimpahan Kepiting Pasir (A. Mashar, et al) ILMU KELAUTAN Desember 2014 Vol 19(4):226-232 Tabel 1. Komposisi hasil tangkapan per jenis kepiting pasir di kedua lokasi penelitian Lokasi Penelitian Jenis Kepiting Pasir Hasil Tangkapan (ekor) Jantan Betina Total Pantai Bocor, Kebumen Emerita emeritus 76 991 1.067 Hippa adactyla 171 202 373 Albunea symmysta 32 41 73 279 1.234 1.513 Pantai Bunton, Cilacap Emerita emeritus 1.077 3.860 4.973 Hippa adactyla 311 1.162 1.473 Albunea symmysta 47 102 149 1.435 5.124 6.559 8.072 Tabel 2. Persentase komposisi hasil tangkapan per jenis kepiting pasir di kedua lokasi penelitian Lokasi Penelitian Jenis Kepiting Pasir Proporsi Hasil Tangkapan (%) Persentase Total (%) Jantan Betina Pantai Bocor, Kebumen Emerita emeritus 7,1% 92,9% 70,5% Hippa adactyla 45,8% 54,2% 24,7% Albunea symmysta 43,8% 56,2% 4,8% 18,4% 81,6% Pantai Bunton, Cilacap Emerita emeritus 21,8% 78,2% 75,3% Hippa adactyla 21,1% 78,9% 22,5% Albunea symmysta 31,5% 68,5% 2,2% 21,9% 78,1% Kenyataan bahwa kepiting pasir E. emeritus Sedangkan daerah pengambilan sampel kepiting Tabel 1. Komposisi hasil tangkapan per jenis kepiting pasir di kedua lokasi penelitian Lokasi Penelitian Jenis Kepiting Pasir Hasil Tangkapan (ekor) Jantan Betina Total Pantai Bocor, Kebumen Emerita emeritus 76 991 1.067 Hippa adactyla 171 202 373 Albunea symmysta 32 41 73 279 1.234 1.513 Pantai Bunton, Cilacap Emerita emeritus 1.077 3.860 4.973 Hippa adactyla 311 1.162 1.473 Albunea symmysta 47 102 149 1.435 5.124 6.559 8.072 Tabel 1. Komposisi hasil tangkapan per jenis kepiting pasir di kedua lokasi penelitian Tabel 2. Persentase komposisi hasil tangkapan per jenis kepiting pasir di kedua lokasi penelitian Lokasi Penelitian Jenis Kepiting Pasir Proporsi Hasil Tangkapan (%) Persentase Total (%) Jantan Betina Pantai Bocor, Kebumen Emerita emeritus 7,1% 92,9% 70,5% Hippa adactyla 45,8% 54,2% 24,7% Albunea symmysta 43,8% 56,2% 4,8% 18,4% 81,6% Pantai Bunton, Cilacap Emerita emeritus 21,8% 78,2% 75,3% Hippa adactyla 21,1% 78,9% 22,5% Albunea symmysta 31,5% 68,5% 2,2% 21,9% 78,1% Tabel 2. Persentase komposisi hasil tangkapan per jenis kepiting pasir di kedua lokasi penelitian Kenyataan bahwa kepiting pasir E. Jenis kepiting pasir emeritus selalu didapatkan jauh lebih banyak dari jenis kepiting pasir lainnya menunjukkan adanya dominasi populasi kepiting pasir E. emeritus. Kondisi ini semakin memperkuat informasi dari Boyko dan McLaughlin (2010) dan Haye et al. (2002) bahwa kepiting pasir atau undur-undur laut genus Emerita, terutama jenis E. emeritus banyak tersebar di pantai berpasir wilayah Asia Tenggara. Adapun kepiting pasir atau undur-undur laut genus Hippa secara umum banyak dijumpai di perairan Australia (Poore, 2004). Jadi diduga, keberadaan kepiting pasir jenis H. adactyla yang ditemukan di lokasi penelitian yang berada di bagian utara Samudra Hindia ada kaitannya dengan kepiting pasir genus Hippa yang tersebar di perairan Australia berada di bagian timur Samudra Hindia. Sedangkan daerah pengambilan sampel kepiting pasir pada penelitian ini adalah daerah intertidal bagian atas, sehingga wajar jika kepiting pasir A. symmysta ditemukan dalam jumlah sedikit. Kepiting pasir A. symmysta yang didapatkan di daerah intertidal diduga sedang mencari makanan, diantaranya berupa bangkai-bangkai kepiting pasir famili Hippidae. Berdasarkan jenis kelamin, kepiting pasir betina selalu lebih banyak, baik di kedua lokasi penelitian maupun untuk setiap jenis kepiting pasir tersebut. Bahkan, untuk jenis E. emerita, betina ditemukan jauh lebih banyak dari jantannya, dengan komposisi betina mencapai 78,2%-92,9%. Hasil yang sama juga dijumpai pada penelitian Boonruang dan Phasuk (1975) bahwa kepiting pasir betina selalu dijumpai dengan komposisi lebih tinggi dari jantan, bahkan untuk jenis E. emeritus komposisi betina bisa mencapai hingga sekitar 95%. Adapun kepiting pasir A. symmysta ditemukan dalam jumlah yang paling sedikit, bahkan terkadang tidak didapatkan pada pengambilan sampel, merupakan kondisi yang wajar. Hal tersebut dikarenakan habitat kepiting pasir A. symmysta sebenarnya ada di daerah intertidal bagian bawah hingga daerah tidal, bahkan pernah ditemukan pada kedalaman sekitar 50 dan 150 m (Boyko dan Harvey, 1999; Corsini-Foka dan Kalogirou, 2013). Komposisi kepiting pasir betina yang jauh lebih besar dibanding jantan diduga sebagai bentuk adaptasi alami dari kepiting pasir tersebut. Sebagaimana diketahui bahwa dalam siklus reproduksi kepiting pasir, setelah telur-telur menetas, mereka mengalami fase planktonik yang Diversitas dan Kelimpahan Kepiting Pasir (A. Mashar, et al) 229 ILMU KELAUTAN Desember 2014 Vol 19(4):226-232 Oleh karena itu, untuk mempertahankan eksistensinya di dalam, kepiting pasir didominasi oleh betina sehingga diharapkan semakin banyak telur yang dihasilkan untuk mengimbangi kehilangan larva yang tinggi pada fase planktonik. cukup lama, antara 3-4 bulan (Ricketts et al., 1992). cukup lama, antara 3-4 bulan (Ricketts et al., 1992). Selama fase planktonik tersebut, peluang kelangsunganhidupnya relatif kecil, terutama karena adanya peluang predasi yang tinggi terhadap larva- larva kepiting pasir tersebut di daerah intertidal. Tabel 3. Kelimpahan rata-rata per jenis kepiting pasir di kedua lokasi penelitian Lokasi Penelitian Jenis Kepiting Pasir Kelimpahan Rata-rata (ekor.100 m-2) Pantai Bocor, Kebumen Emerita emeritus 5 Hippa adactyla 2 Albunea symmysta 1 Pantai Bunton, Cilacap Emerita emeritus 34 Hippa adactyla 10 Albunea symmysta 1 Gambar 2. Fluktuasi kelimpahan bulanan kepiting pasir di setiap lokasi penelitian Tabel 3. Kelimpahan rata-rata per jenis kepiting pasir di kedua lokasi penelitian Lokasi Penelitian Jenis Kepiting Pasir Kelimpahan Rata-rata (ekor.100 m-2) Pantai Bocor, Kebumen Emerita emeritus 5 Hippa adactyla 2 Albunea symmysta 1 Pantai Bunton, Cilacap Emerita emeritus 34 Hippa adactyla 10 Albunea symmysta 1 Gambar 2. Fluktuasi kelimpahan bulanan kepiting pasir di setiap lokasi penelitian Gambar 2. Fluktuasi kelimpahan bulanan kepiting pasir di setiap lokasi penelitian Diversitas dan Kelimpahan Kepiting Pasir (A. Mashar, et al) 230 ILMU KELAUTAN Desember 2014 Vol 19(4):226-232 Ucapan Terima Kasih Ucapan terima kasih kepada DIKTI melalui program beasiswa BPPS (BPPDN) dan hibah penelitian BOPTN yang telah membantu pembiayaan penelitian ini. Ucapan terima kasih disampaikan pula kepada Bapak Sugeng (nelayan kepiting pasir di Cilacap), Agus Alim Hakim, Surya Gentha Akmal, Wahyu Muzammil, Eni Megawati, dan Rani Nuraisah yang telah membantu dalam pengumpulan sampel kepiting pasir. Rendahnya kelimpahan kepiting pasir di pantai Bocor tersebut, terutama untuk famili Hippidae, disamping karena jumlah tangkapan kepiting pasir di pantai Bocor lebih rendah dan daerah sapuan soroknya lebih luas, juga dikarenakan lokasi penangkapan kepiting pasir di pantai Bocor berdekatan (satu hamparan pantai) dengan lokasi wisata pantai di pantai Bocor, sedangkan di pantai Bunton, Cilacap, kondisi pantainya masih relatif alami dan belum ada aktivitas wisata pantai, yang ada hanya kegiatan pemancingan dari pinggir pantai dan itupun jumlahnya sedikit. Adapun pantai Bocor, sebagian pantainya adalah pantai wisata yang banyak dikunjungi ketika hari libur, dan daerah penangkapan kepiting pasir tersebut juga terkadang menjadi bagian dari kegiatan wisata pantai. Kondisi ini mengakibatkan populasi kepiting pasir di daerah tersebut tidak berkembang optimal dan untuk menjaga eksistensinya, diduga kepiting pasir tersebut berpindah habitatnya mencari lokasi atau sisi pantai berpasir lainnya yang benar-benar aman atau masih sedikit atau bahkan belum ada aktivitas manusia, terutama wisata pantai. Kesimpulan Berdasarkan Tabel 3 dan Gambar 2 terlihat bahwa dari sisi jenis, kepiting pasir E. emeritus mempunyai kelimpahan paling tinggi dibanding dua jenis kepiting pasir lainnya, hampir 3 kali lipat dari kelimpahan kepiting pasir H. adactyla, baik di pantai Bocor, Kebumen maupun di pantai Bunton, Cilacap. Berdasarkan pengamatan bulanan, terlihat bahwa kelimpahan kepiting pasir tertinggi ditemukan pada pengamatan bulan Oktober di pantai Bocor dan bulan Juli dan Oktober di pantai Bunton. Jika dilihat per lokasi penelitian, kelimpahan kepiting pasir famili Hippidae yang dijumpai di pantai Bunton, Cilacap jauh lebih tinggi dibanding yang ditemukan di pantai Bocor Kebumen. Dikarenakan belum ada kajian tentang kelimpahan ketiga jenis kepiting pasir tersebut di lokasi lain, maka nilai kelimpahan untuk setiap kepiting pasir pada penelitian ini hanya bisa dibandingkan antar lokasi penelitian pada penelitian ini. Keragaman kepiting pasir di pesisir selatan Jawa Tengah relatif tinggi dengan ditemukannya tiga jenis kepiting pasir di lokasi penelitian, yaitu Emerita emeritus, Hippa adactyla, dan Albunea symmysta, dengan kepiting pasir betina umumnya didapatkan selalu lebih banyak dibanding jantan. Kelimpahan kepiting pasir E. emeritus selalu dijumpai paling tinggi dari kepiting pasir jenis lainnya. Namun, kelimpahan kepiting pasir pada setiap wilayah pantai berpasir dapat berbeda-beda, tergantung kepada intensitas aktivitas manusia di daerah pantai berpasir tersebut. Daftar Pustaka Boonruang, P. & B. Phasuk. 1975. Species composition and abundance distribution of anomuran sand crabs, and population bionomics of Emerita emeritus (L) along the Indian ocean coast of Thailand (Decapoda: Hippidae). Research Bulletin 8. Published by the Center. Thailand, pp. 1-19. Boyko, C.B. 2002. A worldwide revision of the recent and fossil sand crabs of the Albuneidae Stimpson and Blepharipodidae, new family (crustacea: decapoda: anomura: hippoidea). Bull. American Mus. Nat. History. 272–396. doi:10.1206/0003-0090(2002)272<0001:AW ROTR>2.0.CO;2. Boyko, C.B. & A.W. Harvey. 1999. Crustacea Decapoda: Albuneidae and Hippidae of the tropical Indo-West Pacific region, in Crosnier A. (ed.), Résultats des Campagnes MUSORSTOM. Volume 20. Mémoires du Muséum national d’Histoire naturelle. 180: 379-406. Satu hal yang menarik dari kelimpahan kepiting pasir tersebut adalah kelimpahan A. symmysta. Kelimpahan A. symmysta di kedua lokasi penelitian tersebut relatif sama, yaitu sekitar 1 ekor.100m-2, sangat rendah dibandingkan dengan dua jenis kepiting pasir lainnya. Kenyataan ini semakin memperkuat pernyataan sebelumnya bahwa daerah intertidal memang bukan habitat sebenarnya dari A. symmysta. Mereka berenang ke daerah intertidal, terutama hanya untuk mencari makan. Boyko, C.B. & P.A. Mclaughlin. 2010. Annotated checklist of anomuran decapod crustaceans of the world (exclusive of the kiwaoidea and families chirostylidae and galatheidae of the Diversitas dan Kelimpahan Kepiting Pasir (A. Mashar, et al) 231 ILMU KELAUTAN Desember 2014 Vol 19(4):226-232 dari pantai berpasir Kabupaten Kebumen. J. Biol. Tropis. 13(2):119-127. galatheoidea). The Raffles Bull. Zool. 23:139– 151. Mursyidin, D.H. 2007. Kandungan asam lemak omega 6 pada ketam pasir (Emerita spp.) di pantai selatan Yogyakarta. Bioscientiae. 4 (2):79-84 Corsini-Foka, M. & S. Kaligirou. 2013. First record of Albunea carabus (Linnaeus, 1758) (Decapoda: Anomura: Hippoidea) in the Aegean Sea. Cah. Biol. Mar. 54:297-299. Osawa, M., C.B. Boyko & T.Y. Chan. 2010. [Part I. Hippoidea (mole crabs)]. In: [Crustacean fauna of Taiwan: crab-like anomurans (Hippoidea, Lithodoidea, Porcellanidae)]. Chan TY (ed), Institute of Marine Biology, Taiwan, pp. 1-40 Dugan, J.E., G. Ichikawa, M. Stephenson, D.B. Crane, J. McCall, & K. Regalado. 2005. Monitoring of coastal contaminants using sand crabs.Central Coast Regional Water Quality Control Board. 37p. Pérez, D. 1999. Mercury levels in mole crabs Hippa cubensis, Emerita brasiliensis, E. portoricensis, and Lepidopa richmondi (Crustacea: Decapoda: Hippidae) from a sandy beach at Venezuela. Bull. Environ. Contam. Toxicol. 63:320-326. doi: 10.1007/s001289900983 Efford, I.E. 1976. Distribution of the sand crabs in the genus Emerita (Decapoda, Hippidae). Crustaceana. 30:169-183. doi:10.1163/1568 54076X00558. Haley, S.R. 1982. Diversitas dan Kelimpahan Kepiting Pasir (A. Mashar, et al) Daftar Pustaka Zonation by size of the Pacific mole crab, Hippa pacifica Dana (Crustacea: Anomura: Hippidae), in Hawaii. J. Exp. Mar. Biol. Ecol. 58:221-231. doi:10.1016/0022-0981(8 2)90 131-9. Poore, G.C.B. 2004. Marine decapods crustacea of southern Australia: a guide to identification. CSIRO Publishing Ricketts, E.F., J. Calvin, D.W. Phillips & J.W. Hedgpeth. 1992. Open-coast sandy beaches. Between Pacific Tides (5thed.) Stanford University Press. pp. 249–265 Hartono, E., E.S. Rejeki & A.A. Puspitasari. 2011. Pengaruh asupan makanan undur-undur laut terhadap kandungan omega 3 pada telur itik. J. Biomedika. 4:60-65. Siegel, P.R. & A.M. Wenner. 1984. Field and laboratory studies of sand crab growth. In: Wenner AM (ed.) Crustacean Growth, Crustacean Issues, Vol II. Balkema Press, Rotterdam. Haye, P.A., Y.K. Tam & I. Kornfield. 2002. Molecular phylogenetics of mole crabs (Hippidae:Emerita). J. Crust. Biol. 22(4):903-915. doi:10.1651/02 78-0372(2002)022[0903:MPOMHE]2.0.CO;2. Lafferty, K.D., J.P. McLaughlin & J.E. Dugan. 2013. Novel foraging in the swash zone on pacific sand crabs (Emerita analoga, Hippidae) by Mallards. The Wilson J. Ornithology. 125(2): 423-426. doi:http://dx.doi.org/10.1676/12-14 1.1. Wardiatno, Y., I.W. Nurjaya & A. Mashar. 2013. Eksplorasi informasi biologi undur-undur laut Emerita sp. dan Hippa sp. (Crustacea: Hippidae). Tahun Pertama. Laporan Akhir: Penelitian Unggulan Perguruan Tinggi. Tahun Pertama. Lembaga Penelitian dan Pengabdian kepada Masyarakat. Institut Pertanian Bogor. Bogor. Mann, K.H. 2000. Sandy beaches. Ecology of Coastal Waters with Implications for Management. Volume 8 of Studies in Ecology (2nded.). Wiley-Blackwell. pp. 218-236. Wardiatno, Y., I.W. Nurjaya & A. Mashar. 2014. Karkateristik habitat undur-undur laut (Famili Hippidae) di pantai berpasir, Kabupaten Cilacap. J. Biol. Tropis. 14(1):1-8. Mashar, A. & Y. Wardiatno. 2013a. Aspek pertumbuhan undur-undur laut, Emerita emeritus dari pantai berpasir Kabupaten Kebumen. J. Biol. Tropis. 13(1):29-38. Wenner, A.M. 1988. Crustaceans and other invertebrates as indicators of beach pollution. In: Soule DF, Kleppel GS (Eds.) Marine Organisms as Indicators, pp. 199-229. Springer-Verlag, New York. Mashar, A. & Y. Wardiatno. 2013b. Aspek pertumbuhan undur-undur laut, Hippa adactyla Diversitas dan Kelimpahan Kepiting Pasir (A. Mashar, et al) 232
https://openalex.org/W2066071775	https://europepmc.org/articles/pmc3546622?pdf=render	English	null	Space Flight Calcium: Implications for Astronaut Health, Spacecraft Operations, and Earth	Nutrients	2,012	cc-by	11,135	Nutrients 2012, 4, 2047-2068; doi:10.3390/nu4122047 nutrients ISSN 2072-6643 www.mdpi.com/journal/nutrients Review Space Flight Calcium: Implications for Astronaut Health, Spacecraft Operations, and Earth Scott M. Smith 1,, Torin McCoy 1, Daniel Gazda 2, Jennifer L. L. Morgan 3, Martina Heer 4,5 and Sara R. Zwart 6 1 Human Health and Performance Directorate, NASA Lyndon B. Johnson Space Center, Houston, TX 77058, USA; E-Mail: torin.mccoy-1@nasa.gov 2 Wyle Science, Technology & Engineering Group, Houston, TX 77058, USA; E-Mail: daniel.b.gazda@nasa.gov 3 Oak Ridge Associated Universities/NASA Post-Doctoral Fellow, NASA Lyndon B. Johnson Space Center, Houston, TX 77058, USA; E-Mail: jennifer.morgan@nasa.gov 4 Profil Institut für Stoffwechselforschung GmbH, Hellersbergstrasse 9, 41460 Neuss, Germany; E-Mail: martina.heer@profil.com 5 University of Bonn, 53115 Bonn, Germany 6 Division of Space Life Sciences, Universities Space Research Association, Houston, TX 77058, USA; E-Mail: sara.zwart-1@nasa.gov Author to whom correspondence should be addressed; E-Mail: scott.m.smith@nasa.gov; Tel.: +1-(281)-483-7204; Fax: +1-(281)-483-2888. Received: 11 October 2012; in revised form: 13 November 2012 / Accepted: 10 December 2012 / Published: 18 December 2012 Abstract: The space flight environment is known to induce bone loss and, subsequently, calcium loss. The longer the mission, generally the more bone and calcium are lost. This i id hi t f b d l i t di l t d t fli ht d hi hli ht OPEN ACCESS Nutrients 2012, 4, 2047-2068; doi:10.3390/nu4122047 nutrients ISSN 2072-6643 www.mdpi.com/journal/nutrients Review Space Flight Calcium: Implications for Astronaut Health, Spacecraft Operations, and Earth Scott M. Smith 1,, Torin McCoy 1, Daniel Gazda 2, Jennifer L. L. Morgan 3, Martina Heer 4,5 and Sara R. Zwart 6 1 Human Health and Performance Directorate, NASA Lyndon B. Johnson Space Center, Houston, TX 77058, USA; E-Mail: torin.mccoy-1@nasa.gov 2 Wyle Science, Technology & Engineering Group, Houston, TX 77058, USA; E-Mail: daniel.b.gazda@nasa.gov 3 Oak Ridge Associated Universities/NASA Post-Doctoral Fellow, NASA Lyndon B. Johnson Space Center, Houston, TX 77058, USA; E-Mail: jennifer.morgan@nasa.gov 4 Profil Institut für Stoffwechselforschung GmbH, Hellersbergstrasse 9, 41460 Neuss, Germany; E-Mail: martina.heer@profil.com 5 University of Bonn, 53115 Bonn, Germany 6 Division of Space Life Sciences, Universities Space Research Association, Houston, TX 77058, USA; E-Mail: sara.zwart-1@nasa.gov Author to whom correspondence should be addressed; E-Mail: scott.m.smith@nasa.gov; Tel.: +1-(281)-483-7204; Fax: +1-(281)-483-2888. Received: 11 October 2012; in revised form: 13 November 2012 / Accepted: 10 December 2012 / Published: 18 December 2012 Abstract: The space flight environment is known to induce bone loss and, subsequently, calcium loss. The longer the mission, generally the more bone and calcium are lost. This review provides a history of bone and calcium studies related to space flight and highlights issues related to calcium excretion that the space program must consider so that urine can be recycled. It also discusses a novel technique using natural stable isotopes of calcium that OPEN ACCESS nutrients ISSN 2072-6643 www.mdpi.com/journal/nutrients OPEN ACCESS Space Flight Calcium: Implications for Astronaut Health, Spacecraft Operations, and Earth Scott M. Smith 1,, Torin McCoy 1, Daniel Gazda 2, Jennifer L. L. Morgan 3, Martina Heer 4,5 and Sara R. Zwart 6 Nutrients 2012, 4, 2047-2068; doi:10.3390/nu4122047 Nutrients 2012, 4, 2047-2068; doi:10.3390/nu4122047 2. Bone Loss Space flight-induced bone and calcium loss have been documented for decades, and have been the subject of many reviews [1–5]. There have also been several evaluations of ground-based analogs, including the most common, bed rest [6,7]. Bed rest is a viable model of space flight-induced bone loss, producing metabolic changes and bone loss that are qualitatively similar to those brought on by space flight but have a smaller magnitude. Bone loss in bed rest is about half of that observed in space flight [8,9]. Documentation of negative calcium balance and increased calcium excretion during space flight first came from Gemini and Apollo missions of the 1960s and early 1970s [10,11], but bed rest studies documenting negative calcium balance go back even further, with initial documentation in the 1940s [12]. Although urinary and fecal calcium excretion were shown clearly to increase on short-duration space flights (1–2 weeks of flight), not until the longer Skylab missions were flown (1973–1974) were actual changes in bone observed using densitometry [13,14]. In the 1990s the Russian space station Mir provided a platform for long-duration studies on changes in bone during space flight. Dual-energy X-ray absorptiometry (DXA) scans on astronauts and cosmonauts began to better characterize and quantify bone loss during space flight [2,15,16]. Although considerable site-to-site variability exists, along with crewmember-to-crewmember variability, in general, a 1.0%–1.5% loss of bone mineral density occurred per month of space flight [2]. Bone biochemistry studies in the 1990s also expanded significantly, largely fueled by the identification of collagen crosslinks as markers that could be used to assess bone resorption, and development of immunoassays to measure these crosslinks along with markers of bone formation. Novel techniques are being developed to assess overall bone balance using stable isotopes of calcium. These techniques will be beneficial because they are noninvasive (only a urine sample is needed), might be possible to do during flight, and will give a picture of overall bone balance instead of resorption or formation alone. These techniques will be discussed in detail below. Early flight experiments with animals pointed to a decrease in bone formation as the metabolic key to bone loss, whereas human studies (both space flight and bed rest) clearly pointed to an increase in bone resorption, with bone formation being either unchanged or slightly decreased [17–22]. Scott M. Smith 1,, Torin McCoy 1, Daniel Gazda 2, Jennifer L. L. Morgan 3, Martina Heer 4,5 and Sara R. Zwart 6 1 Human Health and Performance Directorate, NASA Lyndon B. Johnson Space Center, Houston, TX 77058, USA; E-Mail: torin.mccoy-1@nasa.gov 2 Wyle Science, Technology & Engineering Group, Houston, TX 77058, USA; E-Mail: daniel.b.gazda@nasa.gov 3 Oak Ridge Associated Universities/NASA Post-Doctoral Fellow, NASA Lyndon B. Johnson Spac Center, Houston, TX 77058, USA; E-Mail: jennifer.morgan@nasa.gov 4 Profil Institut für Stoffwechselforschung GmbH, Hellersbergstrasse 9, 41460 Neuss, Germany; E-Mail: martina.heer@profil.com 5 University of Bonn, 53115 Bonn, Germany 6 Division of Space Life Sciences, Universities Space Research Association, Houston, TX 77058, USA; E-Mail: sara.zwart-1@nasa.gov * Author to whom correspondence should be addressed; E-Mail: scott.m.smith@nasa.gov; Tel.: +1-(281)-483-7204; Fax: +1-(281)-483-2888. Received: 11 October 2012; in revised form: 13 November 2012 / Accepted: 10 December 2012 / Published: 18 December 2012 Abstract: The space flight environment is known to induce bone loss and, subsequently, calcium loss. The longer the mission, generally the more bone and calcium are lost. This review provides a history of bone and calcium studies related to space flight and highlights issues related to calcium excretion that the space program must consider so that urine can be recycled. It also discusses a novel technique using natural stable isotopes of calcium that will be helpful in the future to determine calcium and bone balance during space flight. Keywords: bed rest; bone; calcium; collagen crosslinks; dual-energy X-ray absorptiometry; space flight Nutrients 2012, 4 Nutrients 2012, 4 Nutrients 2012, 4 2048 1. Introduction Bone and calcium metabolism have been a concern for space travelers, literally since before human space flight was a reality. A little more than a half century after the first human space flight, we have improved our understanding of the effects of space flight on calcium and bone, but we still have much to learn. We review here the current state of knowledge and describe ongoing studies, including application of the findings to space exploration and implications for the general population. 3. Calcium Isotopes and Relevance for Bone Turnover Analytical techniques to assess bone health, bone loss, and bone metabolism continue to evolve with technology. Although densitometry techniques (such as DXA and quantitative computerized tomography) provide valuable assessment of specific bones, these techniques detect only relatively large changes in bone, and it takes several months for changes of this magnitude to occur. Studying calcium requires either intensive balance studies or tracer kinetic studies. Bone biochemical markers can provide more rapid assessments of changes in bone formation or resorption, but assessing the relative association of these two factors has not been possible to date, and thus it is difficult to assess net changes in bone calcium content. A new technique to rapidly detect and quantitatively predict changes in bone mineral balance (the ratio of bone formation to resorption) has recently been validated in a bed rest model [27]. Changes in bone mineral balance as a result of bed rest can be detected by measuring the ratios of stable calcium isotopes in urine from individuals who have not received any stable isotope tracers [28]. This calcium isotope biomarker is based on natural, biologically induced variations. These variations are a result of the 6 naturally occurring calcium isotopes (40Ca, 42Ca, 43Ca, 44Ca, 46Ca, and 48Ca) reacting at different rates depending on their mass [29]. In general, isotopic selectivity can occur in all elements with multiple stable isotopes, and has been measured in light elements such as hydrogen, oxygen, carbon, and nitrogen for many decades. These variations arise because the vibrational frequencies of any chemical bonds are a function of the masses of the constituent nuclides. As a result, more energy is required to break bonds formed from heavy isotopes than for the same bond with a lighter isotope. In chemical reactions that do attain equilibrium (which are rare in biology), heavier isotopes are preferentially concentrated in the strongest chemical bonding environments. In chemical reactions that do not attain equilibrium (e.g., kinetic reactions), the bonds incorporating lighter isotopes usually react more quickly than those incorporating heavier isotopes. Kinetic processes like diffusion, evaporation, and precipitation, preferentially select lighter isotopes because they move faster than heavy isotopes [30,31]. In soft tissue (e.g., blood, urine), variations in the calcium isotope composition exist because bone formation depletes soft tissue of lighter calcium isotopes most likely as a result of isotope selectivity during osteoblast-induced calcium precipitation. Bone resorption releases that isotopically light calcium back into soft tissue. Nutrients 2012, 4 Nutrients 2012, 4 when interpreting results from space flight studies. Young animals are commonly used in research, and provide an added benefit for space flight studies in that more animals may be flown in the limited space available for animal research on these missions. Although any model has limitations, and these need to be carefully accounted for, animal studies do provide the ability to do more extensive research on mechanisms of bone loss. In 2000, the first crews took up residence on the International Space Station (ISS), marking the beginning of what many hope will be a permanent human presence off the planet. One of the novel aspects of the ISS was the inclusion of resistance exercise equipment. Details of these efforts will be described below, but although initial evaluations were disappointing [24], possibly because of altered kinematics [25], recent evidence illustrates the potential of proper nutrition and exercise regimens to prevent whole-body and regional loss of bone mineral density [26] during extended space flight. 2. Bone Loss Further research documented that the discrepancy between the animal and human data was not caused by a problem with the animal model itself, but rather by an issue with the age of the animals used in those early flight studies. In young rats, bone formation is suppressed during space flight. In mature rats, bone resorption predominates and little change is seen in bone formation [23], as evidenced in hindlimb-suspension studies. These differences in mechanism of bone metabolism must be considered 2049 3. Calcium Isotopes and Relevance for Bone Turnover Therefore, when bone is being resorbed, as 2050 Nutrients 2012, 4 Nutrients 2012, 4 As the relationship between calcium isotopes and bone mineral balance is well established based on the isotope selectivity principles described above and preliminary work measuring the offset between soft tissue and bone [32,33], this relationship can be used to quantitatively translate the changes in the calcium isotope ratio in urine to changes in bone mineral density using a simple model (Figure 2) [27]. Using this model it was estimated that subjects lost 0.25% ± 0.07% (1 SD) of their bone mass from day 7 to day 30 of bed rest [27]. This rate of loss extrapolates to a loss of 1.36% ± 0.38% of skeletal mass over 119 days, which is equivalent, within error, to bone loss rates determined by DXA scans in long-term (119-day) bed rest studies [34]. Figure 2. Schematic of model pools and fluxes used to quantify bone loss. Fdiet is the flux of calcium absorbed from diet, Fbone and Fresorp are the bone formation and resorption fluxes, and Furine and Fbile are the net excretion fluxes of calcium through urine and bile respectively. 44/42Casoft and δ44/42Cabone are the Ca isotopic compositions of the soft tissue and bone pools, δ44/42Cadiet and δ44/42Caurine are the isotopic compositions of the fluxes of Ca absorbed from diet and excreted in urine, and ε44/42Cabone, ε44/42Caurine, and ε44/42Cabile are the isotopic variations associated with forming bone, urine, and bile, respectively, from the soft tissue pool. Note: This figure is adapted with permission from [27], Copyright © 2012 National Academy of Sciences. A preliminary study examined the calcium isotope shift in bed rest subjects undergoing three different treatments (untreated bed rest, the bisphosphonate alendronate, and intense resistance exercise) [35,36]. In this study there were significant differences in isotope response: the control group’s calcium isotope composition shifted in a direction consistent with bone resorption, while the alendronate and resistance exercise groups’ calcium isotope composition shifted in a manner suggesting maintained and slightly increased bone formation, respectively [28]. A preliminary study examined the calcium isotope shift in bed rest subjects undergoing three different treatments (untreated bed rest, the bisphosphonate alendronate, and intense resistance exercise) [35,36]. In this study there were significant differences in isotope response: the control group’s calcium isotope composition shifted in a direction consistent with bone resorption, while the alendronate and resistance exercise groups’ calcium isotope composition shifted in a manner suggesting maintained and slightly increased bone formation, respectively [28]. Nutrients 2012, 4 is the case during bed rest, the urinary calcium isotope abundance shifts toward lighter values (i.e., −δ44/42Ca; more 42Ca and less 44Ca relative to baseline). Applying this technique to a bed rest study, it was shown that the calcium isotope ratio shifted in a direction consistent with bone loss after just 7 days of bed rest, long before detectable changes in bone density occur. Consistent with this interpretation, the calcium isotope variation accompanied changes observed in N-telopeptide, while bone-specific alkaline phosphatase, a bone-formation biomarker, was unchanged (Figure 1) [27]. Figure 1. Variations in bone biochemical markers N-telopeptide (NTX, top panel) and bone-specific alkaline phosphatase (BSAP, middle panel), along with calcium isotopes (bottom panel), during and after bed rest (days 0 to 40). Percent changes were calculated as the difference between the measured value at each time point and the average of the pre-bed rest values (baseline, days to left of day 0) for that individual. All values are mean ± SD. The calcium isotopes shift in a direction consistent with bone loss after just 7 days of bed rest and track the signal observed in NTX while BSAP remains unchanged. Note: This figure is adapted with permission from [27], Copyright © 2012 National Academy of Sciences. Data are from 12 subjects. 2051 Nutrients 2012, 4 Nutrients 2012, 4 in bone formation and resorption are not only being altered by space flight itself but are being manipulated by various countermeasures. in bone formation and resorption are not only being altered by space flight itself but are being manipulated by various countermeasures. 4. Dietary Calcium When bone loss is considered, calcium intake is an obvious initial concern. Skylab crews consumed an average of 894 ± 142 (SD) mg calcium/day [8] while participating in carefully planned and executed metabolic balance studies over the entire course of their missions (28, 59, and 84 days) [14,37]. Based on reviews of available information, a decision was made requiring that the ISS food system shall provide 1000–1200 mg calcium per day [8], similar to Earth-based recommendations. Proximate analysis of the foods provided in the ―standard‖ menu revealed that calcium content of the ISS menu averages 1020 ± 109 mg calcium/day [8]. Although the crews self-select meals while they are on board the ISS, dietary intake estimates using a food frequency questionnaire designed for space flight documented actual intakes of calcium during flight that were in the same range as in the ―standard‖ menu, 1068 ± 384 mg calcium/day in one report [38], and 912 ± 229 and 1025 ± 309 mg calcium/day in another [26]. Thus, intakes typically are close to or meeting planned calcium intake during flight, which is similar to the Earth-based recommended intake. Nutrients 2012, 4 Given the rapid signal observed using calcium isotope measurements and the potential to quantitatively assess bone loss, this technique is ideally suited for space flight studies in which changes 2052 6. Bone Loss Countermeasures Along with the identification of space flight-induced bone loss, came a significant effort to find a means to counteract this loss. Many countermeasures have been proposed and studied, including physical, nutritional, and pharmacological measures. As with virtually all space flight research, ground testing is required to document proof of concept before a countermeasure is implemented on a space mission. Thus, numerous bed rest and related analog studies have been done in which bone countermeasures were evaluated. Physical countermeasures tested in bed rest studies have included exercise, vibration, and centrifugation. Resistance exercise has shown the most promise in combating bone loss that results from skeletal unloading. Heavy resistance exercise (such as weight-lifting) results in increased bone formation but has little or no effect on resorption, while maintaining bone mineral density [36]. When heavy resistance exercise was combined with an aerobic type of exercise countermeasure (treadmill exercise within a lower-body negative pressure device), the effect was similar, although a striking dose-response relationship of the effect was observed [47]. That is, when subjects performed resistance exercise only every other day (3–4 days/week), the increase in bone formation was half of what it was in subjects who performed resistance exercise 6 days per week. Low-level vibration seemed promising as a bone loss countermeasure judging by a pilot study with human subjects and animal model data. However, although in bed rest vibration had some beneficial effects on bone marrow and vertebral disks [50–52], it did not protect bone mineral density as was initially hoped [53]. Although vibration of higher intensity or a combination of vibration and resistance exercise produced more encouraging results with bone and muscle [48,54–58], some concerns have been raised about potential risks of repetitive-motion injuries. Space flight-induced bone loss is clearly related to a lack of the gravity found on Earth. Preliminary studies have been conducted to evaluate the counteraction of bone loss by artificial gravity, produced either by centrifugation [59,60] or by simply standing or walking for specified time periods during the bed rest phase of bed rest studies. Although standing or walking for 2–4 h daily during bed rest reduced hypercalciuria [61], an hour of centrifugation daily failed to have a positive effect on bone health [44,62]. Many have proposed that subjects exercise during centrifugation, and although this has proven beneficial for the muscle and cardiovascular systems [63], clear effects on bone have yet to be documented. Nutrients 2012, 4 Nutrients 2012, 4 limits) [46,47]. Not all studies have shown decreases in circulating parathyroid hormone concentrations during bed rest; some have shown trends that were not statistically significant [47,49], others have shown clearly unchanged concentrations [42,43]. Many factors are likely to lead to these discrepancies, including study design and controls; subject age, gender, and pre-study fitness levels; laboratory analytical methods; and inherent lab-to-lab variability. Despite these discrepancies, bed rest remains the best analog of space flight with regard to bone and calcium metabolism. 5. Calcium Absorption, Metabolism, and Excretion In space flight and bed rest models, changes in bone biochemistry occur rapidly after humans enter the altered environment. This is hypothesized to be related to a loss of bone in response to unloading, and the release of calcium from bone, which suppresses parathyroid hormone. This suppression of parathyroid hormone in turn is associated with a drop in circulating 1,25-dihydroxyvitamin D and a decrease in calcium absorption. In space flight studies, serum total and ionized calcium are tightly regulated, and do not manifest as consistent measurable changes during flight [21,26,39,40]. Parathyroid hormone decreases during flight [26], although because of the small numbers of subjects, this change is not always statistically significant [18,22]. Circulating concentrations of the active form of vitamin D—1,25-dihydroxyvitamin D—are decreased during flight (vitamin D stores will be addressed below). Calcium absorption has been shown by calcium tracer kinetic studies to be decreased during flight [21,22,41]. This decreased absorption has also been indirectly evidenced through increased fecal calcium during flight [10,14,37]. Urinary calcium excretion is also increased during flight (this will be addressed in greater detail below). In bed rest, findings generally similar to those from space flight have been documented: unchanged total circulating calcium [42–45], unchanged [44,45] or slightly increased ionized calcium [42], decreased parathyroid hormone [44–48] and 1,25-dihydroxyvitamin D concentrations [42,45,46], decreased calcium absorption [42], and increased urinary [42,43,45–49] and fecal calcium [42]. As mentioned earlier, although the biochemical and physiological changes in bed rest are similar to those observed in space flight, the magnitudes of the changes are less in the ground-based analog. As with any area of research, particularly research with human subjects, not all studies reveal the same effects on all analytes. For example, some studies have shown increased serum calcium levels (within normal 2053 Nutrients 2012, 4 Decreasing the intake of sulfur-containing amino acids and decreasing the intake of sodium chloride have been documented to mitigate bone loss in bed rest studies, and both of these nutritional countermeasures are being tested on the ISS. In one study the impact of altering the ratio of animal protein to potassium in the diet (not from supplements) is being examined, using animal protein as a proxy for sulfur-containing amino acids and potassium as an estimate for organic base precursor salts (carbonate, etc.) in the diet [64–67]. The literature contains many conflicting studies of the effects of protein on bone, but bed rest (and space flight) provides a unique situation, in which generally healthy individuals are placed in an environment that induces bone loss. The negative effect of protein on bone seems to occur during bed rest (but not during an ambulatory control phase), and is more pronounced with greater duration of bed rest [64–67]. Negative effects of protein on bone have also been found in studies of individuals with inadequate intake of calcium, vitamin D, protein, or other nutrients, or individuals with mobility challenges. However, in many studies of healthy, ambulatory, well-nourished individuals, no effect (or at least no negative effect) of protein on bone was found [68]. This is an area where space flight studies might help to bridge gaps in the literature, in documenting that neither case is ―right‖ or ―wrong,‖ but rather that the conditions of the experiments in essence provide different models for the effect of protein on bone metabolism. Most of the offered space food up to now is rather high in sodium, mainly sodium chloride, content. This results in a high dietary sodium intake for residents on the ISS, whose average intake is more than 5000 mg of sodium per day, with individual intakes exceeding 12,000 mg/day [8,9,38,69,70]. In the general population, high sodium chloride intake induces increased urinary calcium excretion [71–77] and renal stone risk [78]. Controlled studies in humans have shown that high sodium chloride intake may lead to low-grade metabolic acidosis [79,80]. As the resorbing cells, osteoclasts, need an acidic environment to be activated [81–83], the acidosis that results from high sodium chloride intake might cause increased bone resorption as shown by bone resorption markers [80]. This effect is exacerbated when bone-resorbing mechanisms are already activated because of the immobilization in bed rest [84]. 6. Bone Loss Countermeasures Artificial gravity should clearly, at some point, have an effect on bone health, but the ability to provide this on spacecraft will be an engineering challenge. Nutritional countermeasures also have the potential to mitigate bone loss. One potential nutritional countermeasure is mitigation of low-level metabolic acidosis that can result from metabolism of dietary components, including excess amounts of sulfur-containing amino acids and sodium chloride. 2054 Nutrients 2012, 4 Nutrients 2012, 4 Nutrients 2012, 4 during flight if supplemental intake of this vitamin is inadequate [22,38]. Ground analog studies have been conducted in Antarctica [91,92], where crews who stay during the winter also get little or no ultraviolet light exposure. Recent studies have documented that supplementation with 800 IU vitamin D/day will maintain vitamin D stores in astronauts on 6-month space missions [26]. Although vitamin D is likely not a countermeasure for space flight-induced bone loss, vitamin D deficiency will surely exacerbate the problem. Studies with other nutrients have also been attempted, with the aim of providing higher intake of calcium [43] or vitamin K [93,94], but to date these have not provided enough convincing ground-based data to warrant further investigation of calcium or vitamin K intake [95]. Pharmacologic studies focusing on bisphosphonates have been conducted in bed rest (and related human and animal models) with several forms of these antiresorptive drugs [35,96–99]. Initial in-flight testing with alendronate was conducted recently [100]. Initial results seem promising, but concerns over side effects arise with any pharmaceutical agent. Exogenous testosterone is also often proposed as a potential countermeasure for bone and muscle loss of space flight. This idea is based on data from a 1993 Space Shuttle flight, with 4 subjects and one data point for each subject [101]. It is noteworthy that these 4 subjects were also consuming about half of their energy requirement in the days before data collection [69]. Detailed analysis of data from 15 crewmembers on long-duration flights to the ISS found no change in circulating testosterone during flight in well-nourished crewmembers [102]. Similar findings were also reported in bed rest subjects [102]. Combinations of countermeasures, including heavy resistance exercise and good nutritional intakes, have recently been associated with maintenance of bone mineral density during space flight. ISS crewmembers consuming an adequate diet (maintaining intake of energy and other nutrients, including maintaining optimal vitamin D status) and exercising with an enhanced suite of exercise equipment, including treadmill, cycle, and a high-load resistance exercise device were able to maintain their whole-body and regional bone mineral density at preflight levels [26]. This regimen led to an increase in bone formation without suppressing the space flight-induced increase in bone resorption, and thus questions and concerns remain about bone strength. Nutrients 2012, 4 Nonetheless, after more than a half century of space travel and many proposed countermeasures that were unable to mitigate bone mineral density loss, this is a significant step forward. Nutrients 2012, 4 Bone formation, on the other hand, does not seem to be affected by acidosis, as shown by bone formation markers [84]. How much high sodium chloride intake affects bone turnover during space flight is currently being examined on the ISS in controlled studies, by comparing bone turnover in phases of low and high sodium intake. To what extent the increased amount of dietary sodium chloride is responsible for the loss of bone mass in space flight is not known. However, taking into account the extremely high sodium intake in the daily diet of astronauts [8,9,38,69], it is very likely that bone loss can be decreased by lowering daily sodium chloride intake. As a consequence of that, and further undesirable effects of high sodium chloride intake in space flight, NASA has made a tremendous effort to decrease the salt content in the American space food products. Omega-3 fatty acid intake has been shown to mitigate bone resorption in bed rest [85]. This was supported by cell culture studies in which eicosapentaenoic acid suppressed NFκB activation in osteoclasts [85]. Controlled studies in humans and animals also support a protective role of fish and omega-3 fatty acids in bone health [86–90]. Controlled studies during space flight have not yet been possible, but a somewhat crude negative association between fish intake and bone loss has been established [85]. Vitamin D is a concern for space travelers, in part because their dietary sources of vitamin D are insufficient, and in part because they lack ultraviolet light exposure [8]. Vitamin D stores decline 2055 8. Urinary Calcium and Spacecraft Water Reclamation Renal stone risk and bone loss are well known to be medical concerns for space flight, and recently urinary calcium precipitation became a concern for operation of the ISS Program for an entirely different reason. In this section, we will walk through the process undertaken by NASA’s Nutritional Biochemistry Laboratory and Water and Food Analytical Laboratory to make recommendations to the ISS Program related to urinary calcium concentration and fluid intake, and we will provide the in-flight urine data that were used to make these decisions. In late 2008 NASA deployed the Urine Processor Assembly (UPA), a system designed to reclaim water from urine. Reclaiming water from urine is a significant step toward closing the life-support loop on the ISS and significantly reduces the resupply requirement for this critical resource. The UPA works on the principle of reduced-pressure distillation. Preserved urine is collected in a metal bellows tank that feeds a fixed-volume recirculation loop. As water is removed from the preserved urine, additional urine flows from the bellows tank to maintain the volume in the recirculation loop. The loop also contains a brine tank that is emptied at the end of each concentration cycle. The amount of water recovered during each concentration cycle is calculated as a percentage of the total amount of urine added to the recirculation loop. The initial goal for the UPA was to recover 85% of the water in urine. This corresponds to 232 L of water for a 41-L brine tank and 119 L of water for a 21-L brine tank. The brine that is collected during each concentration cycle is discarded in the non-reusable resupply vehicles that deliver cargo to the ISS. After initial deployment and checkout of the UPA, and several months of normal operations, a hard failure occurred in the system. This failure was later determined to be the result of precipitation in the distillation assembly and recirculation loop. Several brine samples and hardware components were returned as part of the failure investigation, and after extensive ground analyses the precipitate was identified as calcium sulfate. The primary source of the sulfate was sulfuric acid, which is one component of the preservative added to urine when it is collected in the waste hygiene compartment (toilet). The preservative is added to inhibit biological activity and prevent ammonia formation while the urine is stored before it is processed. Nutrients 2012, 4 2056 However, in some cases urine chemistry changes may indicate that additional in-flight countermeasures are required [113]. One such potential countermeasure is potassium citrate, which has an alkalinizing effect on urine and thus will reduce stone-forming potential [114]. 7. Urinary Calcium and Renal Stone Risk One of the immediate concerns about increased urinary calcium during space flight is the increased risk of renal stone formation. This increased risk has been documented during both short- and long-duration space missions [103–105], as well as in bed rest [106–110]. Given that the occurrence of a kidney stone on orbit would likely result in the medical evacuation of the affected crewmember (and likely end the mission for all crewmembers, given the nature of spacecraft), the likelihood of a kidney stone occurring has warranted a fair amount of attention. Many factors can increase or decrease renal stone risk, but increased fluid intake is clearly the easiest, and a proven effective, means to reduce this risk [111]. Space-flight studies suggest that increased fluid intake is a viable countermeasure after flight, when stone risk remains increased relative to preflight levels [112]. 8. Urinary Calcium and Spacecraft Water Reclamation The sulfate combined with the calcium in urine to form the precipitate, which coated the surface of the distillation assembly (Figure 3). An extensive review of urinary calcium excretion during space flight was conducted after the failure occurred, because preflight testing of the UPA had shown no evidence of precipitation. Evaluation of existing in-flight urinary calcium data shed new light on the cause of the UPA failure and, by extension, the risk of renal stone formation during space flight. Data were initially evaluated in 2010 and then the updated data set was reanalyzed in 2012. By then, the data set had expanded to include samples from 10 additional subjects (2–5 in-flight samples for each subject). The intent of revisiting the data was to determine if the findings of the initial assessment were still valid. We report both data sets here and elaborate on the impact of the assessments on water reclamation on the ISS and on evaluation of renal stone risk. 2057 Nutrients 2012, 4 After the initial data review and analysis in 2010, a decision was made to reduce the water recovery rate to 65%, which significantly reduced the amount of water recovered during each concentration cycle. Eventually, the recovery rate was increased to 70%, but that was still much lower than the initially targeted 85%. When the data were reevaluated in 2012, the expanded data set indicated that the average of all combined urinary calcium concentrations had dropped by 8% relative to the average of data available in 2010. This was largely due to the fact that the average urinary calcium concentrations for the 10 additional subjects were 18% lower than those for the first 13 subjects (Figure 4). When daily urinary calcium excretion and total urine volumes were examined, this difference in calcium concentration was found to be almost entirely the result of increased urine volumes (Figure 4). Data from in-flight food frequency questionnaires (FFQs) allowed us to compare fluid intake over time; the average fluid intake increased from 1904 ± 414 mL/day in crews that launched in 2006–2007 to 2284 ± 184 mL/day in crews that launched in 2010. This increased fluid intake was consistent with the observed higher urine volumes and may be attributed to a number of factors, including education of the crews on the importance of fluid intake. The need for adequate fluid intake was emphasized in preflight briefings with crews after the initial precipitation event. The UPA served as a perfect analog for the human kidney to illustrate the potential for formation of precipitates (renal stones). Another contributing factor could be launching and installation of the U.S. Potable Water Dispenser in early 2009. This provided a second dispensing point for potable water on the ISS and simplified access to water. Importantly, the chance that the more recent crews simply happened to habitually drink more water cannot be ignored. Regardless of the cause, in evaluating these data, it was determined that consumption of fluid at >32 mL/kg body weight was associated with urinary calcium concentrations that would allow recovery of water at 75% with minimal risk of precipitation (Figure 5) and also reduce the risk of renal stone formation. Figure 5. In-flight fluid intake and associated in-flight urinary calcium concentration of crewmembers on the ISS. Nutrients 2012, 4 Nutrients 2012, 4 Figure 3. Photograph of calcium sulfate precipitates formed inside the Urine Processor Assembly (UPA) distillation assembly on the International Space Station (ISS), and scanning electron micrograph (inset) of precipitates formed during UPA ground tests. Photo and micrograph courtesy of the NASA Marshall Space Flight Center Environmental Control and Life Support (ECLS) Project. Water recovery in the UPA is limited by the concentration of calcium in the urine, and not the total amount of calcium excreted per day. For this reason, total urine volume was a key variable in the assessments. Figure 4 shows urinary calcium excretion, urine volume, and urinary calcium concentration data before and during flight, separated by year of collection. Independent of time, urinary calcium excretion is increased during flight (as noted above), and urine volume is decreased. This combination of effects results in higher urinary calcium concentrations during space flight. The red lines in the right panel reflect the calculated values above which precipitation would be expected when recovering 85% of the water. As shown in Figure 4, given the number of data points above the lines in 2009, the initial precipitation event that occurred on orbit should have been expected. Figure 4. Urinary calcium excretion (left), urine volume (middle), and urinary calcium concentration (right) before and during flight, by year of collection on the ISS. Each symbol represents a 24-h urine collection; 23 crewmembers may have each provided up to five 24-h collections during flight, and at least 4 before flight. Within data sets, the horizontal lines represent the mean ± 1 SD. The green dashed line in the right panel represents a calcium concentration of 28.3 mg/dL, the expected calcium precipitation point for UPA water recovery at 70%, and the red dashed line represents calcium concentrations of 14.2 mg/dL, the expected calcium precipitation point for UPA water recovery at 85%. A subset of these data, in a different form, have been published, along with details of urine collection procedures [26]. 2058 Nutrients 2012, 4 For the 23 crewmembers included in the data set, 32 mL/kg body weight equals an average of 2.5 L/day of fluid, the amount of water that the water recovery system on the ISS is designed to provide for crewmembers. Thus, this level of water consumption would not be considered extraordinary or even atypical. The fluid intake requirement for ISS crewmembers is 1.0–1.5 mL/kcal, but not less than 2000 mL/day [8]. From an ISS Program point of view, it would be ideal if one could predict in-flight urinary calcium concentrations on the basis of preflight data. This would allow personalized consultations with individual crewmembers to minimize their health risks during flight and tailor water recovery to reduce precipitation risks for a given crew complement. To this end, the available data were evaluated from two perspectives: using 24-h pools (Figure 6, left panel), and using single-void urinary calcium concentration data (Figure 6, right panel) to determine whether a predictable relationship existed between preflight and in-flight urinary calcium concentrations. These data suggest that although variability is substantial, there does seem to be a relationship. Figure 6. Relationships between preflight and in-flight urinary calcium concentrations. Left panel: Each symbol represents a 24-h urine collection, and on the x axis individual crewmember preflight data were averaged, with individual in-flight data points shown on the graph. Right panel: Each point reflects the average of all individual urine voids, and the error bars reflect one standard deviation of the data before flight (horizontal) or during flight (vertical). The green dashed lines represent calcium concentrations of 28.3 mg/dL, the expected calcium precipitation point for UPA water recovery at 70%, and the red dashed lines represent calcium concentrations of 14.2 mg/dL, the expected calcium precipitation point for UPA water recovery at 85%. In the attempt to understand the nature of urinary calcium and operational aspects of this system, many facets were examined. Data were evaluated to determine if urinary calcium concentrations In the attempt to understand the nature of urinary calcium and operational aspects of this system, many facets were examined. Data were evaluated to determine if urinary calcium concentrations changed over the course of a mission, but no significant relationship was observed. That is, urinary calcium concentration was essentially the same earlier and later in the 6-month mission. Nutrients 2012, 4 Each symbol represents a 24-h urine collection; individual crewmembers may have provided up to five 24-h collections during flight, and at least 4 before flight. Dashed horizontal lines reflect urinary calcium concentrations above which calcium precipitation would be expected for UPA water recoveries at 70% and 75%. 2059 Nutrients 2012, 4 70 mg of alendronate was administered once weekly during flight, and also was true for those not taking alendronate (Figure 7). Figure 7. Creatinine (left panel) and calcium (right panel) concentration in single-void (SV) urine samples, by time of day when samples were collected. Some ISS crewmembers were participating in an experiment on bisphosphonate (alendronate) use as a bone loss countermeasure. These data are shown with filled symbols, but did not have a significant effect on calcium concentration. Results from the expanded data set and documented efforts to educate crews on the importance of adequate fluid intake led the ISS Program in 2012 to make the decision to increase water recovery by the UPA from 70% to 74%–75%. This decision required the periodic reevaluation of urinary calcium concentrations to assess whether or not the trend for increased fluid intake and urine output is being maintained. Increasing the water recovery percentage in the UPA will reclaim an additional 60–80 L of water per year. When one considers the cost of launching 60–80 kg of water, the 4%–5% increase in water recovery yields a significant cost savings. An additional benefit of increasing the water recovery rate in the UPA is the reduction in crew time associated with changing the brine tank. Crew time is arguably the most valuable resource on the ISS, and running the UPA at 74%–75% recovery is estimated to save 6–8 h of crew time each year. Results from the expanded data set and documented efforts to educate crews on the importance of adequate fluid intake led the ISS Program in 2012 to make the decision to increase water recovery by the UPA from 70% to 74%–75%. This decision required the periodic reevaluation of urinary calcium concentrations to assess whether or not the trend for increased fluid intake and urine output is being maintained. Increasing the water recovery percentage in the UPA will reclaim an additional 60–80 L of water per year. When one considers the cost of launching 60–80 kg of water, the 4%–5% increase in water recovery yields a significant cost savings. An additional benefit of increasing the water recovery rate in the UPA is the reduction in crew time associated with changing the brine tank. Crew time is arguably the most valuable resource on the ISS, and running the UPA at 74%–75% recovery is estimated to save 6–8 h of crew time each year. Nutrients 2012, 4 Single-void data were also evaluated to assess whether excluding the first morning void from water recovery would eliminate higher average calcium concentrations in urine, but again, somewhat surprisingly, this was found not to be the case (Figure 7). This was true for subjects participating in a study in which In the attempt to understand the nature of urinary calcium and operational aspects of this system, many facets were examined. Data were evaluated to determine if urinary calcium concentrations changed over the course of a mission, but no significant relationship was observed. That is, urinary calcium concentration was essentially the same earlier and later in the 6-month mission. Single-void data were also evaluated to assess whether excluding the first morning void from water recovery would eliminate higher average calcium concentrations in urine, but again, somewhat surprisingly, this was found not to be the case (Figure 7). This was true for subjects participating in a study in which 2060 Nutrients 2012, 4 Nutrients 2012, 4 References 1. Sibonga, J.D.; Cavanagh, P.R.; Lang, T.F.; LeBlanc, A.D.; Schneider, V.S.; Shackelford, L.C.; Smith, S.M.; Vico, L. Adaptation of the skeletal system during long-duration spaceflight. Clin. Rev. Bone Miner. Metab. 2008, 5, 249–261. 1. Sibonga, J.D.; Cavanagh, P.R.; Lang, T.F.; LeBlanc, A.D.; Schneider, V.S.; Shackelford, L.C.; Smith, S.M.; Vico, L. Adaptation of the skeletal system during long-duration spaceflight. Clin. Rev. Bone Miner. Metab. 2008, 5, 249–261. 2. 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https://openalex.org/W3197345385	https://www.frontiersin.org/articles/10.3389/fphar.2021.700696/pdf	English	null	Integrated Proteomics and Metabolomics Link Acne to the Action Mechanisms of Cryptotanshinone Intervention	Frontiers in pharmacology	2,021	cc-by	11,159	Integrated Proteomics and Metabolomics Link Acne to the Action Mechanisms of Cryptotanshinone Intervention Zhaoming Zhu 1, Tingting Chen 1, Zhuxian Wang 1, Yaqi Xue 1, Wenfeng Wu 1, Yuan Wang 1, Qunqun Du 1, Yufan Wu 1, Quanfu Zeng 1, Cuiping Jiang 1, Chunyan Shen 1, Li Liu 1, Hongxia Zhu 2* and Qiang Liu 1* 1School of Traditional Chinese Medicine, Southern Medical University, Guangzhou, China, 2Integrated Hospital of Traditional Chinese Medicine, Southern Medical University, Guangzhou, China The label-free methods of proteomic combined with metabolomics were applied to explore the mechanisms of Cryptotanshinone (CPT) intervention in rats with acne. The model group consisted of rats given oleic acid (MC), then treated with CPT, while control groups did not receive treatment. The skin samples were signiﬁcantly different between control, model and CPT-treated groups in hierarchical clustering dendrogram. Obvious separations of the skin metabolic proﬁles from the three groups were found through PCA scoring. In total, 231 and 189 differentially expressed proteins (DEPs) were identiﬁed in MC and CPT groups, respectively. By the KEGG analysis, ﬁve protein and metabolite pathways were found to be signiﬁcantly altered. These played important roles in response to oleic acid-induced acne and drug treatment. CPT could negatively regulate glycolysis/ gluconeogenesis and histidine metabolisms to decrease keratinocyte differentiation and improve excessive keratinization and cellular barrier function. CPT could down-regulate the IL-17 signaling pathway and regulate the acne-driven immune response of sebum cells. The biosynthesis of unsaturated fatty acids metabolism, glycerophospholipid metabolism and linoleic acid pathways could signiﬁcantly alter sebum production and control sebaceous gland secretion after CPT treatment. The gap junction was up-regulated after CPT treatment and the skin barrier turned back to normal. Krt 14, Krt 16 and Krt 17 were signiﬁcantly down-regulated, decreasing keratinization, while inﬂammatory cell inﬁltration was improved by down-regulation of Msn, up-regulation of linoleic acid and estrogen pathways after CPT treatment. These results propose action mechanisms for the use of CPT in acne, as a safe and potential new drug. Citation: Zhu Z, Chen T, Wang Z, Xue Y, Wu W, Wang Y, Du Q, Wu Y, Zeng Q, Jiang C, Shen C, Liu L, Zhu H and Liu Q (2021) Integrated Proteomics and Metabolomics Link Acne to the Action Mechanisms of Cryptotanshinone Intervention. Front. Pharmacol. 12:700696. doi: 10.3389/fphar.2021.700696 Edited by: Gokhan Zengin, Selcuk University, Turkey Edited by: Gokhan Zengin, Selcuk University, Turkey Reviewed by: Chao-Zhan Lin, Guangzhou University of Chinese Medicine, China Sakina Mohamed Yagi, University of Khartoum, Sudan Correspondence: Hongxia Zhu gzzhx2012@163.com Qiang Liu liuqiang@smu.edu.cn Reviewed by: Chao-Zhan Lin, Guangzhou University of Chinese Medicine, China Sakina Mohamed Yagi, University of Khartoum, Sudan Reviewed by: Chao-Zhan Lin, Guangzhou University of Chinese Medicine, China Sakina Mohamed Yagi, University of Khartoum, Sudan Correspondence: Hongxia Zhu gzzhx2012@163.com Qiang Liu liuqiang@smu.edu.cn Correspondence: Hongxia Zhu gzzhx2012@163.com Qiang Liu liuqiang@smu.edu.cn Specialty section: This article was submitted to Ethnopharmacology, a section of the journal Frontiers in Pharmacology Specialty section: This article was submitted to Ethnopharmacology, a section of the journal Frontiers in Pharmacology Received: 26 April 2021 Accepted: 20 August 2021 Published: 01 September 2021 Keywords: Cryptotanshinone1, Acne2, label-free Proteomic3, metabolomics4, action mechanisms5 ORIGINAL RESEARCH published: 01 September 2021 doi: 10.3389/fphar.2021.700696 Keywords: Cryptotanshinone1, Acne2, label-free Proteomic3, metabolomics4, action mechanisms5 INTRODUCTION Acne vulgaris has become one of the most common skin diseases (Oules et al., 2020) since more than 85% of teenagers and young adults have been affected worldwide (Kang et al., 2015). Acne vulgaris is considered a chronic skin inﬂammation caused by pilosebaceous (Saurat, 2015), and sebaceous glands (SG) abnormally increased in the hair follicles (HF) (Li Z. et al., 2021). Although the pathogenesis of acne remains unclear, four major factors are involved, namely: excessive sebum September 2021 \| Volume 12 \| Article 700696 Frontiers in Pharmacology \| www.frontiersin.org Mechanisms of Cryptotanshinone Intervene Acne Zhu et al. growth, excessive hair follicle keratinization, skin bacteria colonization and skin inﬂammation (O’Neill and Gallo, 2018; Harper, 2020). determines biological functions and regulatory mechanisms, while metabolites are the main source of substances and the basis of phenotypes. Proteomics studies can only explain the function and mechanism, but lack a direct description of phenotype (Monti et al., 2019). In this study, we compared the differential protein expression between rats with acne and rats with acne treated with CPT, and used the KEGG pathway enrichment analysis to assess the differential metabolic pathways between the two groups. Finally, we tried to explain the differential pathways with metabolomics analysis to illustrate the mechanism for CPT intervention in acne. p Although oral isotretinoin is an effective therapy, its use is limited by adverse effects (Markovics et al., 2019). Thus, more research is needed to ﬁnd new, safe and effective therapeutic drugs with few side effects (Williams et al., 2012). Cryptotanshinone (CPT), a major lipophilic compound extracted from Salvia miltiorrhiza demonstrated various pharmacological effects, including anti-tumor (Li H. et al., 2021), anti-inﬂammatory (Tang et al., 2018), anti-bacterial, anti-proliferation (Ashraﬁzadeh et al., 2021), anti-androgen (Xu et al., 2012), pulmonary ﬁbrosis protection, cardio protection, anti-metabolic disorders, anti-angiogenic (Maione et al., 2018) and liver protection (Nagappan et al., 2020). In addition, it is often used to treat multiple chronic diseases, including angiocardiopathy, hyperlipidemia, acne vulgaris and chronic renal failure, with few side effects (Rahman et al., 2016; Zhang et al., 2019). Previous research has improved CPT dosage forms to strengthen its anti-acne activity (Yu et al., 2016; Zuo et al., 2016; Wang et al., 2020), leading to the development of CPT cerasomes, CPT ethosomes and 3D-Printed CPT niosomal hydrogel. Our group has made contributions to these studies (Ruan et al., 2020). INTRODUCTION However, the underlying mechanisms of the anti-acne effects of CPT have not been studied yet. Materials Cryptotanshinone (Lot number 17071601, purity ≥98%, high- performance liquid chromatography; HPLC; Baoji Herbest Bio- Tech Co., Ltd., Baoji, China), carbomer 940 (Macklin Biochemical Co., Ltd., Shanghai, China), glycerin (Aladdin Chemical Reagent Co., Ltd., Shanghai, China), pentobarbital (Sigma, United States), oleic acid (Macklin Biochemical Co., Ltd., Shanghai, China), other chemical substances were analytically pure (AR). Acne Model and CPT Treatment Acne Model and CPT Treatment Adult male SD (Sprague-Dawley) rats (weight: 200 ± 20 g) were acquired from the Experimental Animal Center of the Southern Medical University (SMU). After a 1-week adaptation, rats were randomly separated into three groups: Blank control group (BC), Model group (MC) and CPT treatment group (CPT) with eight rats per group. Three percent sodium pentobarbital was used to anesthetize the rats. The back hair of the rats was removed for further study. Then, 0.5 ml of 80% oleic acid was evenly smeared on the back skin of each rat, for 14 days once per day, except for the BC group (Zuo et al., 2016). CPT gels were then applied to the back skin of CPT rats once per day for 1 week. The procedures of this research were in accordance to the Guide for the Care and Use of Laboratory Animals (Worlein et al., 2011) (eighth versions, revised in 2011), which was approved by the Laboratory Animal Ethics Committee of SMU. Our study was designed to ﬁnd underlying molecular mechanisms of CPT treatment in a rat acne model through the use of label-free quantitative proteomics and metabolomics. Indeed, metabolomic can complement the proteomic analysis and represent disease phenotype (Gertsman and Barshop, 2018). Metabolomics can therefore be used effectively in diagnosing and identifying therapeutic targets of diseases and investigating the mechanisms of biological processes (Patti et al., 2012). Metabolites can be regulated by proteins, while, protein expression may also be inﬂuenced by metabolites (Ma et al., 2020). The functional interpretation of proteomics by metabolomics facilitates the comprehension of the investigated biological phenomenon (Gui et al., 2018). Proteomics mainly Preparation of CPT Gel y As a high-throughput technology, omics technique has been widely used in drug discovery (Tricarico et al., 2019; Worheide et al., 2021). Indeed, high-throughput omics techniques are being used to screen and identify speciﬁc molecular biomarkers for CPT in acne. Proteomics can discover biomarkers to illuminate the underlying mechanisms and reveal novel diagnostic and therapeutic targets by investigating the proﬁle of protein alterations (Nesvizhskii, 2014). We have previously used proteomics to elucidated the potential mechanisms of Licorice ﬂavonoid in acne (Ruan et al., 2020). Quanico J et al. have studied the response pathways and pathophysiological differences for microcysts and papule lesions of acne using proteomic and transcriptomic techniques (Quanico et al., 2017). These results have contributed to identifying targeted therapy for acne vulgaris. However, the pathogenesis of acne is very complex and, in addition to inﬂammation, there should be other mechanisms such as bacteria, sebum and androgens. Nevertheless these issues have not been elucidated yet, while the development of anti-acne drugs based on proteomics is still a rare practice. The CPT was dissolved with anhydrous ethanol (sonication for 1 min) and was ﬁltered with 0.22 μm ﬁlters. Then, we took 3 g of carbomer 940, dissolved in 100 ml pure water for 24 h. Next, 3 g glycerol and CPT were dissolved in ethanol. Followed by addition of triethanolamine. The CPT gel was an orange transparent semi- solid preparation with a ﬁnal CPT concentration of 2.2 mg/g. Frontiers in Pharmacology \| www.frontiersin.org Identiﬁcation and Quantitation of Proteins Identiﬁcation and Quantitation of Proteins The raw data for each sample were combined and searched using the Max Quant 1.5.3.17 software. The parameters were set as follows: enzyme choosing trypsin; max missed cleavages setting two; ﬁxed and variable modiﬁcations respectively being carbamidomethyl (C) and oxidation (M); main and ﬁrst search respectively being 6 and 20 ppm; MS/MS Tolerance: 20 ppm; database pattern: Reverse; Include contaminants: True; protein and peptide FDR both being ≤0.01; peptides used for protein quantiﬁcation using a razor and unique peptides; time window being set at 2 min; minimum ratio count of 1. The p-value was obtained with the t-test. Proteins were signiﬁcantly changed if fold changes >2.0, or <0.5, and p < 0 0.05, which were considered as DEPs. In the same way, the SDEPs were set as fold changes >2.0, or <0.5, and p < 0.01 (O’Connell et al., 2018). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral. proteomexchange.org) via the iProX partner repository with the dataset identiﬁer: PXD027219. Quantitative Proteomic Analysis Skin samples were analyzed by LC-MS/MS on a Q Exactive mass spectrometer (Thermo Scientiﬁc). The peptides were loaded onto a nano Viper C18-reversed phase trap column (Thermo Scientiﬁc Acclaim PepMap100, 100 μm × 2 cm), then they were connected to a C18-reversed phase analytical column (Thermo Scientiﬁc Easy Column, 75 μm × 10 cm, 3 μm resin). Next, 0.1%-Formic acid (A phase) and B phase (84%-acetonitrile: 0.1%-Formic acid) Histopathological Examination p g Rat dorsal skin tissues were ﬁxed and stored in a 10% formaldehyde solution. Then the skin tissues were dehydrated in a gradient of 80–100% alcohol. Then the tissues were embedded with parafﬁn. The parafﬁn blocks were cut into 3–5 μm thickness sections. Next Hematoxylin-eosin (HE, Sigma) was used for the section dyeing. Histopathological September 2021 \| Volume 12 \| Article 700696 Frontiers in Pharmacology \| www.frontiersin.org 2 Mechanisms of Cryptotanshinone Intervene Acne Zhu et al. were used for gradient elution. The ﬂow rate was set at 300 nL/ min. Samples were analyzed with the positive ion mode. MS data were acquired from the 300 m/z to 1800 m/z dynamic survey scan mode to choose the most abundant precursor ions for higher energy collisional dissociation fragmentation. Automatic gain control target was set to 3×106, and maximum injection time was 10 ms. Dynamic exclusion duration was 40.0 s. Survey scans were acquired at a resolution of 70,000 atm/z 200. The resolution for higher energy collisional spectra was set to 17,500 atm/z 200. The isolation width was set at 2 m/z. Normalized collision energy was 30 eV. The under ﬁll ratio was 0.1%. The samples were analyzed in peptide recognition mode. sections were analyzed by an optical microscope (magniﬁcations ×200 and ×100: type BX53, Olympus; magniﬁcations ×40: type Eclipse E100, Nikon). The pathophysiology of acne was judged based on a previous reference (Gollnick and Dreno, 2015). Proteomic Bioinformatics Analysis A total of 200 μg of proteins for each skin tissue were mixed with 30 μL of 4% SDS, 100 mM DTT, 150 mM Tris-HCl, pH 8.0. The eluent was removed. Next, DTT and small molecule components were ﬁltered out with a UA of 8 M Urea in 150 mM Tris-HCl, pH 8.0. Then, 100 μL of iodoacetamide in UA with 100 mM IAA was added, and the skin tissues were incubated in the dark for 30 min. Filters were washed three times with 100 μL of UA, and 100 μL of 25 mM NH4HCO3. The buffer was used to digest 4 μg of trypsin in 40 μL of 25 mM NH4HCO3 for 18 h, at 37°C, then the dissolved peptides were collected. The digested peptides were subjected to desalination processing with a C18 solid phase extraction column (Empore™SPE, 7 mm × 3 ml, Sigma). Then, the concentration under vacuum centrifugation and reconstitution was performed with 40 µL of 0.1% formic acid. The peptides were detected with a UV light spectral (280 nm). y Cluster analysis of phosphorylated peptides was performed by Cluster 3.0 and Java Treeview softwares. Z-score (label-free or metabolism) mode was set as the standard method. The distance type was set as euclidean, and the clustering algorithm was set as average. Then, GO annotation of the differentially expressed proteins (DEPs) was performed with the software program Blast 2 GO, and the top 20 terms of GO enrichment results were drawn in a bar graph. The top 20 Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathway (p-value < 0.05) terms were searched in the KEGG database. Parameters were set as follows: graphic style: bar graph; data type: proteome; screening data: standardized processing; p-value/FDR: p; p-value threshold: 0.05; legend style: standardized processing. Proteins that met the fold change (FC) > 2.0, or FC < 0.5, and p < 0.05 were considered DEPs. Both FC > 2.0 and p < 0.01 values were considered to be SDEPs (Chen et al., 2020). Proteomics Analysis Protein Extraction The skin tissues were lysed and proteins were extracted with SDT buffer (4% SDS, 1 mM DTT, 100 mM Tris-HCl, pH 7.6). The protein content was quantitatively analyzed by a protein- detection kit (Bio-Rad, United States). Protein digestion with trypsin was performed through the ﬁlter-aided sample preparation (FASP) procedure from Matthias Mann (Wisniewski et al., 2009). The digested peptides of every skin were desalination processing with a C18 solid phase extraction column (Empore™SPE, 7 mm × 3 ml, Sigma). Then, the concentration under vacuum centrifugation and reconstitution was performed with 40 µL of 0.1% formic acid. Lesions Analysis The stratum corneum (SC) thickness and the long diameter and short diameter of sebaceous glands (SG) of the rat skin were measured with Olympus cellSens software in histopathological sections magniﬁed 200 times. (Li et al., 2018). Cytokine Levels The serum samples from rats were tested by the ELISA kits of rat of IL-6, IL-8, TNF-α and IL-1β. According to the instructions of these kits, the OD values of IL-6, IL-8, TNF-α and IL-1β were detected at speciﬁc wavelengths, and their contents were calculated by graphpad 8.02 software (Han et al., 2018). CPT Improved Acne Compared with the BC group, in the model group, the SC thickness of rat skin was signiﬁcantly increased. Compared with the MC group, after CPT treatment, the SC thickness of rat skin was signiﬁcantly reduced. The diameter of SG in the model group was signiﬁcantly increased, while that in the CPT group was signiﬁcantly decreased (Figure 2A). The serum contents of IL-6, IL-8, TNF-α and IL-1β were signiﬁcantly higher in the MC group (p < 0.05), whereas the contents were signiﬁcantly lower in the CPT group (p < 0.05). The results showed that the highly expressed cytokine levels in model group rats could be reduced by CPT, thus inhibiting the inﬂammation of the acne rats (Figure 2B). Skin Samples Preparation for Metabolomic Skin Samples Preparation for Metabolomic performed by comparing the accuracy m/z value (<10 ppm), and MS/MS spectra with an in-house database established with available authentic standards. Principle component analysis (PCA) was performed with SIMCA (Version 14.1) using quantitative data. Skin Samples Preparation for Metabolomic The skin tissues were immediately frozen in liquid nitrogen. Then the samples were cut on 80 mg of dry ice into a 2 ml tube. The skin tissues with ﬁve ceramic beads were homogenized. 1 ml methanol-acetonitrile aqueous solution (2:2:1, V/V) was added to the homogenized solution for metabolite extraction. This mixture was centrifuged for 30 min (14,000 × g, 4°C), twice, and placed at −20°C for 1 h to precipitate the proteins. The samples were ﬁltered with a ﬁlter tube and centrifuged at 4°C for 20 min, the supernatant was freeze-dried and kept at −80°C. The prepared samples were dissolved in 100 uL acetonitrile/water (1:1,v/v) solvent and analyzed by LC-MS(Gao et al., 2021). Histopathological Examination s opa o og ca a a o Histopathological examination of the skin tissues is shown in Figure 1. The epidermal squamous epithelium layer was signiﬁcantly thickened, and the epithelial cells in the funnel- shaped part of the hair follicle were increased. Follicle pores were blocked due to excessive keratinization of the hair follicle and we observed dermis hyperemia, neutrophilic cell inﬁltration, and enlarged sebaceous glands in the model control (MC) group (Figure 1 MC) compared with the blank control (BC) group (Figure1 BC). MC rats were treated with CPT (Figure1 CPT) and showed skin tissue similar to the BC group (Figure1 BC) with reduced keratinization. The inﬂammatory cells were decreased, and the sebaceous glands were normal (Figure1 CPT). A series of parameters were set as follows: Gas1: 60, Gas2: 60, CUR: 30, source temperature: 600°C, ISVF: ±5500 V (positive and negative). For single MS acquisition, the m/z range of the instrument was set as 60–1000 Da, and the scanning cumulative time of TOF MS was set as 0.20 s/spectra. For automatic MS/MS acquisition, the m/z range of the instrument was set as 25–1000 Da, and the scanning cumulative time of the product ions was set as 0.05 s/spectra. The information-dependent acquisition tech was applied in the product ion scan, and the mode was set as high sensitivity. The parameters were set as follows: collision energy: 35 V with ±15 eV; declustering potential: ± 60 V; exclude isotopes: ≤4 Da, and the candidate ion to be monitored for each period was set to 6. Metabolomic Analysis The p-values of proteins and metabolites were obtained with the t-test. Signiﬁcantly changed proteins (fold changes >2.0, or <0.5, and p < 0 0.05) were considered DEPs. In the same way, SDEPs had fold changes >2.0, or <0.5, and p < 0.01 (O’Connell et al., 2018). Signiﬁcantly altered metabolites (VIP value >1.0, and p < 0.05) were considered DEMs. In the same way, SDEMs had VIP value >1.0, and p < 0.01 (O’Connell et al., 2018). All data are presented as mean ± SD. One-way analysis of variance (ANOVA) was used to analyze the data between BC, MC and CPT groups. GraphPad Prism software (version 8.02) was used to perform this statistical analysis. Differences were considered to be statistically signiﬁcant when p < 0.05. Metabolomic analysis was performed by a UHPLC (1290 Inﬁnity LC, Agilent Technologies) coupled to a quadrupole time-of-ﬂight (AB Sciex Triple TOF 6600) in HILIC separation. The samples were analyzed with a water column of Ireland (ACOUIY UPLC BEH, 2.1 mm × 100 mm, 1.7 um). The positive and negative modes were set. The mobile phase A was 25 mM ammonium acetate and hydroxide (1:1,v/v) in water, and the mobile phase B was acetonitrile. The elution gradient was set as follows: 0–0.5 min: 95% B; 0.5–7 min: 95% B- 65% B; 7–7.1 min: from 65% B to 40% B; 7.1–8.1 min: 40% B; 8.1–8.2 min: 40% B-95% B; 8.2–12.2 min: 95% B re-balanced time for employing. An Ireland water column was used (ACQUIY UPLC HSS T3, 2.1 mm × 100 mm, 1.8 um) for sample RPLC separation. In the positive ion mode, mobile phase A was 0.1%-formic acid aqueous solution, and phase B was acetonitrile: 0.1% formic acid. In the negative ion mode, mobile phase A was 0.5 mM ammonium ﬂuoride aqueous solution, and phase B was acetonitrile. The elution gradient was set as follows: 0–1.5 min: 1% B; 1.5–11.5 min: 1% B-99% B; 11.5–15 min: 99% B; 15–15.1 min: 1% B; 15.1–18.5 min: 1% B re- balanced time for employing. The ﬂow rate was set at 0.3 ml/min, then the temperature of the column was set at 25°C. The injection volume of every sample was 2 μL. The sample was placed at a 4°C automatic sampler throughout the analysis process (Yi et al., 2019; Wang et al., 2021). KEGG Pathway Analysis The DEMs were searched and the KEGG enrichment pathways were obtained with the online KEGG database (http:// geneontology.org/). The annotation and enrichment results of KEGG were used in R 3.5.1 version. Next, the Venn diagram and bar plot were drawn. Metabolomics Analysis Chemicals Ammonium acetate (NHAC), ammonium hydroxide (NH4OH), ammonium ﬂuoride (NHaF), and formic acid (FA) were obtained from Sigma Aldrich. Acetonitrile was obtained from Merck. September 2021 \| Volume 12 \| Article 700696 Frontiers in Pharmacology \| www.frontiersin.org 3 Mechanisms of Cryptotanshinone Intervene Acne Zhu et al. Data Processing The raw MS data were converted to MzXML ﬁles with Proteo Wizard MS Convert before importing into freely available XCMS software. Compound identiﬁcation of metabolites was September 2021 \| Volume 12 \| Article 700696 Frontiers in Pharmacology \| www.frontiersin.org 4 Mechanisms of Cryptotanshinone Intervene Acne Zhu et al. FIGURE 1 \| The skin tissues of CPT treatment in acne (HE, magniﬁcation ×200, ×100 and ×40) (A), (B) and (C): magniﬁcation ×200 (100 μm) (D) (E) and (F): magniﬁcation ×100 (200 μm) (G), (H) and (I): magniﬁcation ×40 (500 μm) (A), (D) and (G): BC (blank control) group; (B), (E) and (H): MC (model) group; (C), (F) and (I): CPT (treatment) group; the triangles in MC group represent neutrophilic inﬂammatory cell inﬁltration. FIGURE 1 \| The skin tissues of CPT treatment in acne (HE, magniﬁcation ×200, ×100 and ×40) (A), (B) and (C): magniﬁcation ×200 (100 μm) (D) (E) and (F): magniﬁcation ×100 (200 μm) (G), (H) and (I): magniﬁcation ×40 (500 μm) (A), (D) and (G): BC (blank control) group; (B), (E) and (H): MC (model) group; (C), (F) and (I): CPT (treatment) group; the triangles in MC group represent neutrophilic inﬂammatory cell inﬁltration. MC group were signiﬁcantly separated from those in the BC group and the CPT treatment group, and that the CPT group was signiﬁcantly closer to the BC group (Figure 3). Analysis of Differentially Expressed Proteins (DEPs) and Signiﬁcantly Differential Proteins (SDEPs) Altogether, 3,127 proteins and 25,216 peptides were identiﬁed, and 2,869 proteins were quantitatively analyzed (Supplementary Tables 1,2). A total of 231 DEPs (fold change >2, or <0.5, and p < 0.05) and 89 SDEPs (fold change >2, or <0.5, and p < 0.01) (Yan et al., 2020), were identiﬁed of which 214 DEPs (82 SDEPs) in MC were up-regulated and 17 DEPs (7 SDEPs) were down- regulated compared to BC (Supplementary Table 3). A total of 189 DEPs and 103 SDEPs were changed in MC and CPT groups, respectively. A total of 189 DEPs, 72 DEPs (36 SDEPs) were up- regulated and 117 DEPs (67 SDEPs) were down-regulated in MC and CPT group, respectively (Supplementary Table 4). The DEPs were presented by a cluster heat map. Interestingly, the hierarchical clustering ﬁgure showed that the skin proteins in the KEGG Pathway Enrichment Analysis y y A total of 271 protein pathways were enriched through the KEGG analysis, of which 28 pathways were changed (p < 0.05) in the MC and CPT treatment groups (Supplementary Table 5). As shown in Figure 5, the most enriched pathways in CPT versus MC group were glycolysis/gluconeogenesis, spinocerebellar ataxia, galactose metabolism, histidine metabolism, IL-17 signaling pathway, protein digestion and absorption, estrogen signaling pathway, thyroid hormone signaling pathway, thyroid hormone synthesis, arginine and proline metabolism (consisting of 13, 14, 7, 5, 8, 9, 16, 9, 8 and 8 proteins, respectively). Interestingly, the IL-17 signaling pathway, which is closely related to the pathogenesis of acne (Bernardini et al., 2020), included seven DEPs enriched in CPT versus the MC group. GO Enrichment Analysis The top 20 of GO enrichment analysis are shown in Figure 4. The result showed that DEPs were signiﬁcantly enriched in several biological processes, molecular functions and cellular component categories (p < 0.05) in CPT compared to those of the MC group. DEPs from CPT participated in the biological processes of peptide biosynthesis and metabolic process regulation, translation regulation, amide biosynthesis process, cellular amide metabolic process regulation, cytoplasmic translation, organonitrogen compound biosynthetic process and rRNA processing (Figure 4). These DEPs were related to the regulation of molecular functions, such as structural September 2021 \| Volume 12 \| Article 700696 Frontiers in Pharmacology \| www.frontiersin.org 5 Mechanisms of Cryptotanshinone Intervene Acne Zhu et al. Zhu et al. FIGURE 2 \| (A) Bar graph of stratum corneum (SC) thickness and half of long diameter and short diameter of sebaceous glands (SG); original magniﬁcation, ×200 (B) Cytokine levels in serum of IL-6, IL-8, TNF-α and IL-1β in the BC, MC and CPT groups. , p < 0.01, compared with the BC group; ##, p < 0.01, compared with the CPT group. FIGURE 2 \| (A) Bar graph of stratum corneum (SC) thickness and half of long diameter and short diameter of sebaceous glands (SG); original magniﬁcation, ×200 (B) Cytokine levels in serum of IL-6, IL-8, TNF-α and IL-1β in the BC, MC and CPT groups. *, p < 0.01, compared with the BC group; ##, p < 0.01, compared with the CPT group. signiﬁcantly separated in the positive and negative modes. The skin samples of each group were closely clustered in the PCA score plots. The MC group was signiﬁcantly separated from both the BC and the CPT groups, and the skin samples in the BC group were closer to the CPT group. According to the OPLS-DA score plots in Figure 6B and Figure 6E, the skin metabolic proﬁles in the MC and CPT groups were signiﬁcantly separated. Also, the PLS-DA plots illustrated clear differentiation in metabolomics proﬁles between the MC and CPT groups as showed in Figure 6C and Figure 6F. constituent of ribosome, structural molecule activity, rRNA and RNA binding, heterocyclic compound binding and organic cyclic compound binding. The main enriched cellular components in CPT versus model group were the cytosol, cytosolic ribosome, ribosomal subunit, ribosome, cytosolic small and large ribosomal subunit. There was a signiﬁcant difference in GO enrichment between MC and CPT groups, which was related to acne. Analysis of Differentially Expressed Metabolites (DEMs) The MS/MS total ion chromatography (TIC) and mass spectrometry (MS/MS) of the metabolites were in the Supplementary Materials 2, 3. Altogether, 484 metabolites were identiﬁed (Supplementary Table 6). A total of 77 signiﬁcantly changed metabolites (VIP >1, and p < 0.05, DEMs) were identiﬁed. A total of 55 DEMs were up-regulated (fold change >0.67) and 22 DEMs were down-regulated (fold change <0.67) in MC compared to BC (Supplementary Table 7). A total of 76 DEMs were identiﬁed for MC and CPT. Among these DEMs, 43 metabolites in the CPT group were up-regulated, whereas 33 metabolites were down-regulated versus the MC group (Supplementary Table 8). y Principle Component Analysis (PCA) Score of Skin Samples A total of 58 SDEMs (VIP >1, and p < 0.01) were identiﬁed in MC and CPT groups. Thirty-three metabolites were signiﬁcantly increased, while 25 were signiﬁcantly decreased in the MC and A total of 58 SDEMs (VIP >1, and p < 0.01) were identiﬁed in MC and CPT groups. Thirty-three metabolites were signiﬁcantly increased, while 25 were signiﬁcantly decreased in the MC and The PCA score plots were plotted and the results (Figure 6A and Figure 6D) showed that the BC, MC and CPT groups were September 2021 \| Volume 12 \| Article 700696 Frontiers in Pharmacology \| www.frontiersin.org 6 Mechanisms of Cryptotanshinone Intervene Acne Zhu et al. FIGURE 3 \| Hierarchical clustering heat map of DEPs of the skin. Changed proteins (right side) and the samples in different groups (bottom). The color from red to blue shows the relative intensity of the DEPs. FIGURE 3 \| Hierarchical clustering heat map of DEPs of the skin. Changed proteins (right side) and the samples in different groups (bottom). The color from red to blue shows the relative intensity of the DEPs. pathways in CPT and MC group were closely related to the pathogenesis of acne (Figure 8). CPT groups (Supplementary Table 8). The hierarchical clustering dendrogram indicated that the skin metabolites of the MC group were signiﬁcantly separated from both the BC and CPT groups, whereas the skin metabolites of BC and CPT groups were clustered together (Figure 7A). Metabolites with similar abundance were clustered together, representing the degree of metabolic proximities and the inner relation among the DEMs (Figure 7B). Besides, a multiple comparisons analysis was conducted in the three groups. These results indicated that, compared to the MC group, metabolites of the CPT group were similar to those of the BC group. Combination of Proteomics and Metabolomics Through the KEGG database, a total of 156 shared pathways of proteins and metabolites are shown in a Venn plot (Figure 9 and Supplementary Table 10). Altogether ﬁve pathways were signiﬁcantly altered in both their proteins and metabolites contents and played important roles in acne rats and CPT-treated rats (Figure 10). These pathways included galactose metabolism, histidine metabolism, glycolysis/gluconeogenesis, spinocerebellar ataxia and protein digestion and absorption. There are regulatory relationships between these DEPs and DEMs. The regulation networks of signiﬁcantly altered metabolic pathways in response to CPT treatment were assessed (Figure 11). As shown for the glycolysis/ gluconeogenesis pathway and galactose metabolism, CPT regulated DEPs such as hexokinase 2(Hk2), enolase 1 (Eno1), dihydrolipoamide dehydrogenase (Dld), phosphofructokinase, platelet (Pfkp), phosphoglycerate mutase 1 (Pgam1), aldehyde dehydrogenase 3 family, member A2 (Aldh3a2) and aldo-keto reductase family 1, Metabolic Pathway (KEGG) Enrichment A total of 179 metabolic pathways were enriched by the KEGG analysis, and 51 of these pathways were signiﬁcantly changed (p < 0.05) in MC and CPT groups (Supplementary Table 9). The major metabolites participated in protein digestion and absorption, biosynthesis of unsaturated fatty acids metabolism, arginine biosynthesis, glycerophospholipid metabolism, galactose metabolism, glycine, serine and threonine metabolism, linoleic acid metabolism, glycolysis/gluconeogenesis, histidine metabolism and spinocerebellar ataxia. The metabolic September 2021 \| Volume 12 \| Article 700696 Frontiers in Pharmacology \| www.frontiersin.org 7 Mechanisms of Cryptotanshinone Intervene Acne Zhu et al. Zhu et al. FIGURE 4 \| GO enrichment of DEPs in CPT and MC. The x-axis represents the Biological Process (BP), Cellular Component (CC), and Molecular Function (MF). The y-axis represents gene number, the numbers represent the enrichment factor of DEPs ratio. FIGURE 4 \| GO enrichment of DEPs in CPT and MC. The x-axis represents the Biological Process (BP), Cellular Component (CC), and Molecular Function (MF). The y-axis represents gene number, the numbers represent the enrichment factor of DEPs ratio. FIGURE 4 \| GO enrichment of DEPs in CPT and MC. The x-axis represents the Biological Process (BP), Cellular Component (CC), and Molecular Function (MF). The y-axis represents gene number, the numbers represent the enrichment factor of DEPs ratio. hierarchical clustering ﬁgure showed that the skin proteins and metabolites in the MC group were signiﬁcantly separated from those in the BC and CPT treatment groups, and that the CPT group was signiﬁcantly closer to the BC group. The skin samples of each group were closely clustered in the PCA score plots. The MC group was signiﬁcantly separated from both the BC and the CPT groups, and the skin samples in the BC group were closer to the CPT group. This indicated that the skin metabolic proﬁles were signiﬁcantly changed in acne rats and that treatment with CPT led to a metabolic proﬁle similar to that of BC rats. By the KEGG enrichment analysis, CPT rats had ﬁve pathways that played an important role in acne and were signiﬁcantly altered for both their protein and metabolite contents. These pathways included galactose metabolism, glycolysis/gluconeogenesis, histidine metabolism, spinocerebellar ataxia and protein digestion and absorption. Metabolic Pathway (KEGG) Enrichment CPT regulated differential proteins (DEPs) such as Hk2, Eno1, Dld, Pfkp, Pgam1, Aldh3a2, Akr1b8, Hal and Cndp2, and the DEPs regulated member B8 (Akr1b8), and DEMs such as Phosphoenolpyruvate (PEP), Dihydroxyacetone phosphate, DL-lactate, α-D-Glucose, Glycerate-2P, sucrose, Myo-Inositol, D-Mannose, α-D-Galactose1- phosphate, Galactinol and Stachyose. CPT also regulated DEPs relevant for histidine metabolism such as histidine ammonia lyase (Hal), carnosine dipeptidase 2 (Cndp2) and Aldh3a2, and regulated DEMs such as L-Glutamate, L-Aspartate, L-Histidine, Histamine and Urocanic acid. Frontiers in Pharmacology \| www.frontiersin.org DISCUSSION In the present study, through the integration of proteomics and metabolomics, the mechanisms of CPT treatment in a rat acne model induced by oleic acid were examined. Differentially expressed proteins and metabolites were identiﬁed, and the mechanisms of CPT treatment in acne were illuminated. The In the present study, through the integration of proteomics and metabolomics, the mechanisms of CPT treatment in a rat acne model induced by oleic acid were examined. Differentially expressed proteins and metabolites were identiﬁed, and the mechanisms of CPT treatment in acne were illuminated. The September 2021 \| Volume 12 \| Article 700696 Frontiers in Pharmacology \| www.frontiersin.org 8 Mechanisms of Cryptotanshinone Intervene Acne Zhu et al. FIGURE 5 \| KEGG pathway enrichment of DEPs in CPT and MC groups. The x-axis represents the amount of DEPs, the y-axis represents the pathway and the numbers represent the enrichment factor ratio of DEPs. FIGURE 5 \| KEGG pathway enrichment of DEPs in CPT and MC groups. The x-axis represents the amount of DEPs, the y-axis represents the pathway and the numbers represent the enrichment factor ratio of DEPs. differentially expressed metabolites (DEMs) such as Phosphoenolpyruvate, DL-lactate, Dihydroxyacetone phosphate, DL- 2-Phosphoglycerate, sucrose, α-D-Glucose, Myo-Inositol, D-Mannose, Galactinol, Stachyose, α-D-Galactose1-phosphate, L-Glutamate, L-Aspartate, L-Histidine, Histamine and Urocanic acid. In this study, changes in upstream proteins could lead to changes in downstream metabolites, such as Eno1 regulating PEP, Pgam1 regulating DL-2-Phosphoglycerate (Glycerate-2P), Glycerate-2P regulating Eno1, Hal regulating Urocanic acid and Cndp2 regulating L-Histidine. In parallel, changes in upstream metabolites might lead to changes in downstream proteins, such as α-D-Glucose regulating Hk2 and L-Histidine regulating Hal. These proteins were signiﬁcantly up-regulated in acne rat induced by oleic acid, however they were down-regulated after CPT treatment. Our data suggested that CPT could inhibit the disordered metabolism of acne rats, especially within the glycolysis/gluconeogenesis, galactose and histidine metabolisms, restoring these pathways back to normal. increased in MC rats, while these were signiﬁcantly decreased after CPT treatment. Indeed, CPT rats had a negatively regulated glycolysis/ gluconeogenesis pathway, decreased keratinocyte differentiation and improved keratinization and cellular barrier function (Figure 1, Figure 2A and Figure 11). Through transcriptomics, Eckhart, et al. showed that Hal was signiﬁcantly up-regulated during keratinocyte differentiation (Eckhart et al., 2008). This is consistent with our study that Hal was up- regulated in MC rats, leading to excessive keratinocyte differentiation. This phenomenon was improved after CPT treatment. DISCUSSION Samples in BC and CPT groups were separated from the MC group; ESI+: 2X 0.548 (B) OPLS-DA score plots in positive modes of the MC and CPT groups; ESI+: R2X 0.559, R2Y 0.989, Q2 0.944 (C) PLS-DA score plots in positive odes of the MC and CPT groups; ESI+: R2X 0.559, R2Y 0.989, Q2 0.962 (D) PCA score plots in negative modes of the BC, MC and CPT groups; ESI−: R2X 638 (E)OPLS-DA score plots in negative modes of the MC and CPT groups; ESI−: R2X 0.689, R2Y 0.986, Q2 0.970 (F) PLS-DA score plots in negative modes of e MC and CPT groups; ESI−: R2X 0.729, R2Y 0.997, Q2 0.980. GURE 7 \| (A) Hierarchical clustering heat map of skin DEMs. Cluster of the DEMs (right side) and the samples of each group (bottom) are shown. The color from d to blue showed the relative intensity of the DEMs (B) Pearson’s correlation of DEMs in CPT and MC groups. Red and blue represent the positive and negative orrelation of DEMs, respectively. ntiers in Pharmacology \| www.frontiersin.org September 2021 \| Volume 12 \| Article 700696 10 FIGURE 6 \| The multivariate analysis of skin metabolites in positive (ESI+) and negative (ESI−) modes (n 8); 1-XP (the BC group), 2-XP (the MC group), 4-XP (the CPT group) (A) PCA score plots in positive modes of the BC, MC and CPT groups. Samples in BC and CPT groups were separated from the MC group; ESI+: R2X 0.548 (B) OPLS-DA score plots in positive modes of the MC and CPT groups; ESI+: R2X 0.559, R2Y 0.989, Q2 0.944 (C) PLS-DA score plots in positive modes of the MC and CPT groups; ESI+: R2X 0.559, R2Y 0.989, Q2 0.962 (D) PCA score plots in negative modes of the BC, MC and CPT groups; ESI−: R2X 0.638 (E)OPLS-DA score plots in negative modes of the MC and CPT groups; ESI−: R2X 0.689, R2Y 0.986, Q2 0.970 (F) PLS-DA score plots in negative modes of the MC and CPT groups; ESI−: R2X 0.729, R2Y 0.997, Q2 0.980. DISCUSSION Aldehydal dehydrogenase is produced by Aldh3a2, which improves fatty decomposition. A previous study reported that if the function of Aldh3a2 is disrupted, it may cause excessive accumulation of intracellular fat. This process affects both the physiological function of the cell protective membrane and the nutrients necessary to maintain the physiological function of the body (Kumar et al., 2020). The DEPs of histidine metabolism such as Hal, Aldh3a2 and Cndp2 were over-expressed in the MC group, while they were reduced in the CPT group. In our study, histidine metabolism was negative regulated by CPT in acne rats. A previous study reported that regulation of glycolysis might control keratinocyte differentiation by lowering the expression of Eno1. Indeed, Eno1 has a high expression in keratinocytes with accelerated dysfunction of tight junction, which reduces the integrity of the cellular barrier (Tohgasaki et al., 2018; Sutter et al., 2019). Glycolysis is a key metabolic pathway that provides energy for cellular activities and consumes equivalents to sustain cell division and cell proliferation (Sutter et al., 2019). Decreased glycolysis is related to cell differentiation, particularly in keratinocytes (Li et al., 2020). This is in agreement with our ﬁnding that the DEPs of glycolysis including Eno1, Hk2, Dld, Pfkp, Pgam1 and Aldh3a2 were signiﬁcantly Rats with acne induced by oleic acid had signiﬁcantly up-regulated pathways, such as IL-17 signaling pathway, glycolysis/ gluconeogenesis, galactose metabolism, gap junction, histidine metabolism, spinocerebellar ataxia, protein digestion and absorption, estrogen signaling pathway, biosynthesis of unsaturated fatty acids metabolism, glycerophospholipid metabolism and linoleic acid metabolism. The results of the KEGG enrichment showed that DEMs were mainly related to glycolysis metabolism, histidine September 2021 \| Volume 12 \| Article 700696 Frontiers in Pharmacology \| www.frontiersin.org 9 Mechanisms of Cryptotanshinone Intervene Acne Zhu et al. GURE 6 \| The multivariate analysis of skin metabolites in positive (ESI+) and negative (ESI−) modes (n 8); 1-XP (the BC group), 2-XP (the MC group), 4-XP (the PT group) (A) PCA score plots in positive modes of the BC, MC and CPT groups. DISCUSSION FIGURE 6 \| The multivariate analysis of skin metabolites in positive (ESI+) and negative (ESI−) modes (n 8); 1-XP (the BC group), 2-XP (the MC group), 4-XP (the CPT group) (A) PCA score plots in positive modes of the BC, MC and CPT groups. Samples in BC and CPT groups were separated from the MC group; ESI+: R2X 0.548 (B) OPLS-DA score plots in positive modes of the MC and CPT groups; ESI+: R2X 0.559, R2Y 0.989, Q2 0.944 (C) PLS-DA score plots in positive modes of the MC and CPT groups; ESI+: R2X 0.559, R2Y 0.989, Q2 0.962 (D) PCA score plots in negative modes of the BC, MC and CPT groups; ESI−: R2X 0.638 (E)OPLS-DA score plots in negative modes of the MC and CPT groups; ESI−: R2X 0.689, R2Y 0.986, Q2 0.970 (F) PLS-DA score plots in negative modes of the MC and CPT groups; ESI−: R2X 0.729, R2Y 0.997, Q2 0.980. FIGURE 7 \| (A) Hierarchical clustering heat map of skin DEMs. Cluster of the DEMs (right side) and the samples of each group (bottom) are shown. The color from red to blue showed the relative intensity of the DEMs (B) Pearson’s correlation of DEMs in CPT and MC groups. Red and blue represent the positive and negative correlation of DEMs, respectively. FIGURE 7 \| (A) Hierarchical clustering heat map of skin DEMs. Cluster of the DEMs (right side) and the samples of each group (bottom) are shown. The color from red to blue showed the relative intensity of the DEMs (B) Pearson’s correlation of DEMs in CPT and MC groups. Red and blue represent the positive and negative correlation of DEMs, respectively. FIGURE 7 \| (A) Hierarchical clustering heat map of skin DEMs. Cluster of the DEMs (right side) and the samples of each group (bottom) are shown. The color from red to blue showed the relative intensity of the DEMs (B) Pearson’s correlation of DEMs in CPT and MC groups. Red and blue represent the positive and negative correlation of DEMs, respectively. September 2021 \| Volume 12 \| Article 700696 Frontiers in Pharmacology \| www.frontiersin.org 10 Mechanisms of Cryptotanshinone Intervene Acne Zhu et al. FIGURE 8 \| KEGG pathways of the DEMs. (A): BC and MC groups, (B): MC and CPT groups. DISCUSSION The x-axis represents protein number, the y-axis represents the KEGG pathway and the numbers represent the enrichment factor ratio of DEMs. s of the DEMs. (A): BC and MC groups, (B): MC and CPT groups. The x-axis represents protein number, the y-axis represents the mbers represent the enrichment factor ratio of DEMs. FIGURE 8 \| KEGG pathways of the DEMs. (A): BC and MC groups, (B): MC and CPT groups. The x-axis represents protein number, the y-axis represents the KEGG pathway and the numbers represent the enrichment factor ratio of DEMs. FIGURE 9 \| The Venn plot of common pathways of DEPs and DEMs in MC and CPT groups. The blue and yellow circles represent proteomics and metabolomics, respectively. The overlap was the number of pathways shared by the two omics analyzes. The sum of all numbers in the circle represents the sum of the number of pathways involved in DEPs and DEMs. metabolism, biosynthesis of unsaturated fatty acids, linoleic acid metabolism and glycerophospholipid metabolism (Figure 5 and Figure 8). The metabolites S100 calcium binding protein A8 (S100a8), S100 calcium binding protein A9 (S100a9), heat shock protein 90 alpha family class B member 1 (Hsp90ab1), heat shock protein 90 beta family member 1 (Hsp90b1) and serine and arginine rich splicing factor 1(Srsf1) of the IL-17 signaling pathway were signiﬁcantly up-regulated in the MC group, while these were signiﬁcantly down-regulated after CPT treatment (Figure 3). CPT was shown to down-regulate IL-17 signaling pathway and down- regulate acne-driven immune activation of sebum cells (Oules et al., 2020). The metabolite Gpd1 of the glycerophospholipid metabolism was signiﬁcantly decreased in the model group, and signiﬁcantly increased after CPT treatment (Figure 3). Gpd1 regulated DEMs such as glycerol 3-phosphate and dihydroxyacetone phosphate (Figure 7A), which were signiﬁcantly down-regulated in the MC group and up-regulated in the CPT group. The glycerophospholipid metabolism pathway was affected by CPT treatment. The DEPs hydroxysteroid (17-beta) dehydrogenase 12 (Hsd17b12), sterol carrier protein 2 (Scp2) and hydroxysteroid (17-beta) dehydrogenase 4(Hsd17b4) of the biosynthesis of unsaturated fatty acids metabolism pathway were signiﬁcantly up-regulated by oleic acid; however these were down-regulated by CPT treatment. The DEMs arachidonic acid (AA), oleic acid, stearic acid, all cis-(6,9,12)- linolenic acid, alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), erucic acid and nervonic acid were part of the unsaturated fatty acids metabolism pathway and were signiﬁcantly up-regulated in the MC group. CPT treatment down-regulated these metabolites. DISCUSSION The DEMs of the unsaturated fatty acids metabolism, namely palmitic acid, linoleic acid (LA), docosahexaenoic acid (DHA) and tetracosanoic acid were signiﬁcantly down-regulated in the MC group, whereas they were signiﬁcantly up-regulated after CPT treatment. Hsd17b12 was regulated by the DEMs ALA, EPA, all cis-(6,9,12)-Linolenic acid, LA, AA, palmitic acid, stearic acid, FIGURE 9 \| The Venn plot of common pathways of DEPs and DEMs in MC and CPT groups. The blue and yellow circles represent proteomics and metabolomics, respectively. The overlap was the number of pathways shared by the two omics analyzes. The sum of all numbers in the circle represents the sum of the number of pathways involved in DEPs and DEMs. FIGURE 9 \| The Venn plot of common pathways of DEPs and DEMs in MC and CPT groups. The blue and yellow circles represent proteomics and metabolomics, respectively. The overlap was the number of pathways shared by the two omics analyzes. The sum of all numbers in the circle represents the sum of the number of pathways involved in DEPs and DEMs. tetracosanoic acid, oleic acid, erucic acid and nervonic acid. In parallel, DHA was regulated by Scp2 and Hsd17b4. The DEMs of the linoleic acid metabolism pathway, namely AA, 1- Stearoyl-2-oleoyl-sn-glycerol 3-phosphocholine (SOPC) and all cis-(6,9,12)-Linolenic acid were signiﬁcantly up-regulated in the MC group, whereas CPT treatment led to a down-regulation of these metabolites. Linoleic acid (LA) is an essential unsaturated fatty acid that plays a crucial role in inﬂammation (Burns et al., 2018). Research has shown that down-regulation of LA led to disordered LA metabolism on inﬂammatory rats (Liu et al., 2020). Indeed, LA was down- September 2021 \| Volume 12 \| Article 700696 Frontiers in Pharmacology \| www.frontiersin.org 11 Mechanisms of Cryptotanshinone Intervene Acne Zhu et al. FIGURE 10 \| Histogram of common KEGG pathways of the DEPs and DEMs through multiple comparisons of MC and CPT groups. The blue and orange columns represent proteomics and metabolomics, respectively. The y-axis represents the pathway, and the x-axis represents the p-value. FIGURE 10 \| Histogram of common KEGG pathways of the DEPs and DEMs through multiple comparisons of MC and CPT groups. The blue and orange columns represent proteomics and metabolomics, respectively. The y-axis represents the pathway, and the x-axis represents the p-value. FIGURE 11 \| Network for a mechanistic explanation of the proteomics and metabolomics analyzes. DISCUSSION versus the MC group, the purple and blue represent up- regulation and down-regulation, respectively. The yellow color represents the metabolism pathway. The DEPs and DEMs are represented by rectangles and circles, respectively. FIGURE 11 \| Network for a mechanistic explanation of the proteomics and metabolomics analyzes. versus the MC group, the purple and blue represent up- regulation and down-regulation, respectively. The yellow color represents the metabolism pathway. The DEPs and DEMs are represented by rectangles and circles, respectively. homeostasis of this layer (Martin et al., 2014). In this study, the gap junctions were disturbed in the MC group, while CPT treatment turned the skin barrier back to normal. regulated in acne rats, suggesting oleic acid might disrupt LA metabolism, causing inﬂammation. LA was up-regulated in the CPT treatment group. This is in accord with our histopathological analysis showing that acne rats turned to back normal after CPT treatment. The biosynthesis of unsaturated fatty acids metabolism, glycerophospholipid metabolism and linoleic acid metabolism pathway could signiﬁcantly alter sebum production and control sebaceous gland secretion after CPT treatment (Clayton et al., 2019). The effective epidermal physical barrier primarily in the stratum corneum (SC) requires a structural and functional combination of adherent junctions, tight junctions, gap junctions (GJ) and desmosomes (Cohen-Barak et al., 2020). The gap junction network of the epidermis contributes to keeping the integrity and Ces1d (Carboxylesterase 1D) is a glycoprotein involved in lipid metabolism and catalyzes the hydrolysis of triglycerides and monoglycerides (Zheng et al., 2020). cytochrome c oxidase subunit 5B(Cox5b) affects mitochondrial activity and also is involved in lipid synthesis (Soro-Arnaiz et al., 2016). Compared to the BC group, Ces1d in the MC group was signiﬁcantly down- regulated, while Cox5b was up-regulated. After CPT treatment, Ces1d signiﬁcantly increased, while Cox5b signiﬁcantly decreased, when compared to the MC group. Our analysis suggested that CPT could improve the metabolism and biosynthesis of lipids, and September 2021 \| Volume 12 \| Article 700696 Frontiers in Pharmacology \| www.frontiersin.org 12 Mechanisms of Cryptotanshinone Intervene Acne Zhu et al. mitochondrial function in acne rats. These results are in agreement with our team’s previous study (Ruan et al., 2020). in rats. CPT could inhibit the disordered metabolism of several pathways in rats with acne, especially glycolysis/ gluconeogenesis, galactose metabolism and histidine metabolism, which play an important role in acne development. These results can aid to explain the mechanism of action of CPT to treat acne. DISCUSSION In conclusion, CPT might be a safe and potential drug to treat acne. Keratin 16 (Krt16) and Keratin 17 (Krt17) are the type I intermediate ﬁlament proteins, which are primarily presented in the base cells of hair follicles and sebaceous glands of the epithelium. Krt17 can regulate various biological processes of skin cells, such as cell growth, cell proliferation, inﬂammation and hair follicle circulation, as well as immune responses (Jin and Wang, 2014; Yang et al., 2019). Krt16 has a close relationship with genes that participate in the maintenance of the skin barrier and natural immunity (Lessard et al., 2013). Keratin 14 (Krt14) is a major structural component of keratinocytes in the epidermal base, affecting biological processes such as cell mechanics, cell homeostasis, and epidermal barrier function (Guo et al., 2020). Krt 14, Krt 16 and Krt 17 were signiﬁcantly up-regulated in the MC group and down-regulated after CPT treatment (Figure 3). These results and the histopathological and lesions analysis agree with previous research that reported that the expression of Krt 14, Krt 16 and Krt 17 were up- regulated in acne rats and could promote keratinocyte proliferation leading to excessive keratinization (Yang et al., 2017). ETHICS STATEMENT The animal study was reviewed and approved by Laboratory Animal Ethics Committee of Southern Medical University. Tubulin alpha-4A chain (Tuba4a) and Moesin (Msn) were found to be DEPs in MC and CPT groups. Msn is regulated by tight junction assembly in the tight junction barrier, while Tuba4a was found to be signiﬁcantly up-regulated in acne rat. CPT treatment decreased Tuba4a levels, indicating that the tight junction barrier was restored after CPT treatment. Previous studies have shown that Msn can become activated and induce the inﬁltration of inﬂammatory cells in tissues (Liu et al., 2015). We found that Msn in our model group was notably up-regulated. After CPT treatment, Msn was decreased when compared to the levels found for the MC group. The results of the histopathological analysis showed that there was inﬂammatory cell inﬁltrationintheskin tissueofacne rats,however this phenomenonwas improved after treatment with CPT. These results are consistent with a previous study of our group (Ruan et al., 2020). ACKNOWLEDGMENTS We thank the Shanghai Applied Protein Technology Co., Ltd. for technical assistance. FUNDING The biological process of leukocyte transendothelial migration is related to the estrogen signaling pathway. Studies have shown that estrogen treatment could attenuate leukocyte inﬁltration in rat (Schneider et al., 2012). Krt 14, Krt 16, Krt 17, Hsp90b1 and Hsp90ab1 are proteins of the estrogen signaling pathway that were signiﬁcantly up-regulated in the MC group and decreased in the CPT group (Figure 5). As shown in the cytokine levels in serum of IL-6, IL- 8, TNF-α and IL-1β, the CPT could inhibit the release of these cytokines, thus improving inﬂammation in acne rats (Figure 2B). This indicated that the skin inﬂammation was controlled after CPT treatment. Further exploration is needed in the future. This study was supported by the National Natural Science Foundation of China (81874346) and the Science and Technology Program of Guangzhou (201907010018). This study was supported by the National Natural Science Foundation of China (81874346) and the Science and Technology Program of Guangzhou (201907010018). This study was supported by the National Natural Science Foundation of China (81874346) and the Science and Technology Program of Guangzhou (201907010018). AUTHOR CONTRIBUTIONS QL, HZ designed this study, writing review and editing, funding acquisition. ZZ writing original draft, performed experiments and analyzed the data. TC, ZW, YX, WW and YW provided techniques assistance in this study. QD, YFW and QZ performed several experiments. CJ, CS and LL reviewed the article. DATA AVAILABILITY STATEMENT The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: PXD027219 via ProteomeXchange. Cryptotanshinone: A Mechanistic Review. Phytother Res. 35, 155–179. doi:10.1002/ptr.6815 Bernardini, N., Skroza, N., Tolino, E., Mambrin, A., Anzalone, A., Balduzzi, V., et al. (2020). IL-17 and its Role in Inﬂammatory, Autoimmune, and Oncological Skin Diseases: State of Art. Int. J. Dermatol. 59, 406–411. doi:10.1111/ijd.14695 CONCLUSION The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar.2021.700696/ full#supplementary-material Through proteomics and metabolomics studies, we showed that CPT may regulate multiple biological processes to improve acne REFERENCES Cryptotanshinone: A Mechanistic Review. Phytother Res. 35, 155–179. doi:10.1002/ptr.6815 Bernardini, N., Skroza, N., Tolino, E., Mambrin, A., Anzalone, A., Balduzzi, V., et al. (2020). IL-17 and its Role in Inﬂammatory, Autoimmune, and Oncological Skin Diseases: State of Art. Int. J. Dermatol. 59, 406–411. doi:10.1111/ijd.14695 Cryptotanshinone: A Mechanistic Review. Phytother Res. 35, 155–179. doi:10.1002/ptr.6815 Bernardini, N., Skroza, N., Tolino, E., Mambrin, A., Anzalone, A., Balduzzi, V., et al. (2020). IL-17 and its Role in Inﬂammatory, Autoimmune, and Oncological Skin Diseases: State of Art. Int. J. 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https://openalex.org/W3199383601	https://europepmc.org/articles/pmc8493797?pdf=render	English	null	Clinical Significance of the Head-Up Tilt Test in Improving Prognosis in Patients with Possible Neurally Mediated Syncope	Biology	2,021	cc-by	8,564	Article Clinical Signiﬁcance of the Head-Up Tilt Test in Improving Prognosis in Patients with Possible Neurally Mediated Syncope e 1,* , Tomoyoshi Komiyama 2,* , Misaki Hasegawa 1 , Tetsuri Sakai 1, Masahiro Morise 1, ama 1, Atsuhiko Yagishita 1, Mari Amino 1, Yuji Ikari 1 and Koichiro Yoshioka 1 Kengo Ayabe 1,* , Tomoyoshi Komiyama 2,* , Misaki Hasegawa 1 , Tetsuri Sakai 1, Masahiro Morise 1, Susumu Sakama 1, Atsuhiko Yagishita 1, Mari Amino 1, Yuji Ikari 1 and Koichiro Yoshioka 1 1 Department of Cardiology, Tokai University School of Medicine, Isehara 259-1193, Kanagawa, Japan; 299947@cc.u-tokai.ac.jp (M.H.); st219820@tsc.u-tokai.ac.jp (T.S.); m-morise@tokai.ac.jp (M.M.); ss014572@tsc.u-tokai.ac.jp (S.S.); ayagishita@tsc.u-tokai.ac.jp (A.Y.); mariam@is.icc.u-tokai.ac.jp (M.A.); ikari@is.icc.u-tokai.ac.jp (Y.I.); ko1@is.icc.u-tokai.ac.jp (K.Y.) 1 Department of Cardiology, Tokai University School of Medicine, Isehara 259-1193, Kanagawa, Japan; 299947@cc.u-tokai.ac.jp (M.H.); st219820@tsc.u-tokai.ac.jp (T.S.); m-morise@tokai.ac.jp (M.M.); ss014572@tsc.u-tokai.ac.jp (S.S.); ayagishita@tsc.u-tokai.ac.jp (A.Y.); mariam@is.icc.u-tokai.ac.jp (M.A.); ikari@is.icc.u-tokai.ac.jp (Y.I.); ko1@is.icc.u-tokai.ac.jp (K.Y.) jp jp 2 Department of Clinical Pharmacology, Tokai University School of Medicine, Isehara 259-1193 Kanagawa Japan jp jp 2 Department of Clinical Pharmacology, Tokai University School of Medicine, 2 Department of Clinical Pharmacology, Tokai University School of Medicine, Isehara 259-1193, Kanagawa, Japan epa t e t o C ca a aco ogy, oka U ve s ty Sc oo o Med c e, Isehara 259-1193, Kanagawa, Japan g p * Correspondence: kengo.ayabe@fuji.tokai-u.jp (K.A.); komiyama@tokai-u.jp (T.K.) Simple Summary: Several diseases can cause syncope, which is commonly known as fainting; however, syncope triggered by a reﬂex mechanism, also termed neurally mediated syncope (NMS), is one of the most common forms. While NMS is considered a benign disease, it can cause critical clinical events, such as severe trauma due to syncope. The head-up tilt test (HUTT) is one of the modalities for the diagnosis of NMS. The clinical signiﬁcance of HUTT in the prognosis of NMS, such as recurrence rate of syncope and mortality, are still to be elucidated. This research aimed to clarify the value of HUTT for the diagnosis of NMS, and to investigate the prognosis of patients with NMS by analyzing the data of 101 patients with syncope and their close long-term (four years at the longest) follow-up. Furthermore, as insertable cardiac monitors (ICMs) are considered effective in patients with syncope, this study also aimed to evaluate the usefulness of ICM in patients with negative HUTT results. Finally, our research contributes to the improvement of the clinical management for patients with syncope. biology biology biology biology 1. Introduction Syncope is a common chief complaint encountered in daily clinical practice. It is deﬁned as the transient loss of consciousness due to global cerebral hypoperfusion [1], which is characterized by a rapid onset and spontaneous recovery. Depending on the underlying etiology (benign or life-threatening conditions or environmental triggers), syncope can be neurally mediated (reﬂex), cardiac, or orthostatic. Furthermore, some neurologic diseases can cause symptoms that mimic syncope [2]. Although the severity and clinical signiﬁcance of syncope might vary according to the patient’s background, cardiogenic syncope was found to be associated with poor prognosis [3,4]. The main causes of cardiogenic syncope are ischemic heart diseases, valvular diseases, and life-threatening arrhythmias, such as ventricular tachycardia, ventricular ﬁbrillation, and conduction disorders (complete heart block and sick sinus syndrome). On the contrary, neurally mediated syncope (NMS) is non-life-threatening and represents a benign disorder with a better prognosis [5]. NMS comprises a relatively wide variety of types, such as vasovagal, situational, carotid sinus, and others [3]. To clarify the cause of syncope, circulatory dynamics are usually investigated in patients with NMS using high-resolution Holter electrocardiography and correlation analysis of changes in adenylate cyclase activity, blood pressure, and pulse during the head-up tilt test (HUTT) [6,7]. Additionally, the mechanism of the molecular interaction and the polymorphisms of the alpha-2 adrenoreceptor (α2B- AR) gene as the potential psychiatric cause of incentive stress are analyzed [8]. Despite its benign nature, NMS prevents patients from performing their regular daily activities due to the unanticipated onset of the disease and a paucity of treatment options [9]. Therefore, the categorization of NMS as a benign disorder is questionable, and further research is warranted. While obtaining the details of clinical history is very important, the HUTT is one of the standard diagnostic procedures for NMS [10]. According to the guidelines on syncope published by the Japanese Society of Cardiology (2012), European Society of Cardiology, and American College of Cardiology and American Heart Association, NMS is classiﬁed into four types (vasopressor, cardioinhibitory, mixed, and orthostatic hypotension types) based on the results of this test [2,11]. The clinical importance of the HUTT has been studied widely. However, the clinical sensitivity and speciﬁcity of the HUTT have not yet been adequately established [12,13]. Herein, we focus on the clinical signiﬁcance of HUTT in patients with syncope and whose probable diagnosis was NMS. Citation: Ayabe, K.; Komiyama, T.; Hasegawa, M.; Sakai, T.; Morise, M.; Sakama, S.; Yagishita, A.; Amino, M.; Ikari, Y.; Yoshioka, K. Clinical Signiﬁcance of the Head-Up Tilt Test in Improving Prognosis in Patients with Possible Neurally Mediated Syncope. Biology 2021, 10, 919. https://doi.org/10.3390/ biology10090919 Abstract: Syncope is commonly encountered in daily clinical practice. Depending on its etiology (benign or life-threatening conditions or environmental triggers), syncope can be neurally mediated (reﬂex), cardiac, or orthostatic. Furthermore, neurologic disease can cause symptoms that mimic syncope. However, there is limited research on neurally mediated syncope (NMS), which is consid- ered a benign disorder, and close follow-ups are rarely performed. NMS can cause serious clinical events, including severe trauma and car accidents. The head-up tilt test (HUTT) is the gold standard for diagnosing NMS; however, its clinical signiﬁcance remains unknown, and its relevance to NMS prognosis requires further research. This retrospective study aimed to assess the clinical signiﬁcance of the HUTT for NMS. We reviewed the charts of 101 patients who underwent HUTT at Tokai University Hospital in Japan between January 2016 and March 2019. During the HUTT, 72 patients (69.2%) experienced syncope. Patients were followed up for 886.1 ± 457.7 days (interquartile range: 518–1293 days). The syncope recurrence rate was 16.9%; however, no signiﬁcant difference was observed between the two groups (HUTT positive vs. negative) (13.8% vs. 18.1%, p = 0.772). Four of 29 (13.9%) and two of 72 (2.8%) patients in the negative and positive HUTT groups, respectively, experienced cardiac events (p = 0.019). Negative HUTT results may assist in anticipating unexpected clinical events within a few years. A negative HUTT result may allow us to reconsider the NMS diagnosis based on clinical information. Close outpatient follow-up of patients with negative HUTT results is warranted. Academic Editor: Vincenzo Lionetti Received: 6 August 2021 Accepted: 8 September 2021 Published: 15 September 2021 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/). Keywords: atrial ﬁbrillation; atrioventricular block; hypotension; sinoatrial node; tilt-table test; vasovagal syncope https://www.mdpi.com/journal/biology Biology 2021, 10, 919. https://doi.org/10.3390/biology10090919 2 of 11 Biology 2021, 10, 919 2.1. Ethics Statement The Institutional Review Board of Tokai University School of Medicine approved our study (approval number: 14R-053). 1. Introduction Limited studies concerning the long-term follow-up of patients with syncope have been reported [14]. The prognosis of NMS remains to be elucidated. Therefore, we followed up our patients for a maximum of 3 years. The speciﬁcity of the HUTT has been reported to be as high as 90% [3,15]. Hence, we also focused on the clinical importance of negative HUTT results because close follow-up of these patients is occasionally not performed due to the belief that NMS is a benign disorder. Negative HUTT results suggest that the etiology of syncope remains to be determined by more detailed examination. Insertable cardiac monitors (ICMs) are widely used in patients with syncope of unknown etiologies [16]. In addition, ICMs are effective in detecting atrial ﬁbrillation (AF), one of the many causes of syncope due to sinus node pause [17]. Although we did not perform ICM implantation in our patients, we also aimed to evaluate the usefulness of ICM in patients with syncope whose HUTT results were negative. 2.3. Head-Up Tilt Test (HUTT) 2 3 Head Up Tilt Test (HUTT) The HUTT was performed as previously described [6]. All study patients were admitted to our hospital a day before the test. We performed the test in the morning after sufﬁcient night rest. The tilt table was set to a standing position at 70 degrees (70◦). The patient initially lay on the table and wore belts to prevent accidental injury if syncope occurred. We then placed a continuous blood pressure monitoring and ECG electrode patch on the patient, using the tilt test monitoring device developed by Nihon Koden (Nihon Kohden, Tokyo, Japan). Blood pressure, heart rate, and ECGs were monitored continuously. The room light and air conditioner were turned off during the test. After conﬁrming that the patient was in a stable condition with his/her security belts on, we moved up the study patient to 70◦. The patient was instructed to stand for 20 min before administering nitroglycerin sublingually. Afterwards, the patient was instructed to stand for another 10 min. When the patient complained of presyncopal symptoms or developed syncope, we deﬁned the HUTT result as positive. If syncope occurred, the tilt bed was leveled immediately, and vital signs were maintained. The protocol of the HUTT is shown in Figure 1. 2.3. Head-Up Tilt Test (HUTT) The HUTT was performed as previously described [6]. All study patients were admitted to our hospital a day before the test. We performed the test in the morning after sufficient night rest. The tilt table was set to a standing position at 70 degrees (70°). The patient initially lay on the table and wore belts to prevent accidental injury if syncope occurred. We then placed a continuous blood pressure monitoring and ECG electrode patch on the patient, using the tilt test monitoring device developed by Nihon Koden (Nihon Kohden, Tokyo, Japan). Blood pressure, heart rate, and ECGs were monitored continuously. The room light and air conditioner were turned off during the test. After confirming that the patient was in a stable condition with his/her security belts on, we moved up the study patient to 70°. The patient was instructed to stand for 20 min before administering nitroglycerin sublingually. Afterwards, the patient was instructed to stand for another 10 min. When the patient complained of presyncopal symptoms or developed syncope, we defined the HUTT result as positive. If syncope occurred, the tilt bed was leveled immediately, and vital signs were maintained. 2.2. Patient Information and Data Collection We performed a retrospective chart review of 101 patients who underwent the HUTT at Tokai University Hospital in Japan between January 2016 and March 2019. 3 of 11 Biology 2021, 10, 919 All patients presented with syncope once or more times at our hospital, and were initially evaluated by a cardiologist. Laboratory analyses, electrocardiography (ECG), echocardiography, and chest radiography were also performed to rule out cardiac diseases, such as coronary artery disease and cardiomyopathy. Echocardiography was performed to rule out systolic and diastolic dysfunctions and structural abnormalities using standard methods. Additionally, electroencephalography and computed tomography of the head were performed in some patients to rule out epilepsy. NMS was diagnosed based on systemic inquiry, thoughtful clinical history taking, and clinical tests. HUTT was performed in patients whose most likely diagnosis was NMS. Patients with any possibility of cardiac or neurological causes were excluded from the study. Patients’ information, including age, past medical history, history related to syncope, and social history, were collected from their medical charts. at Tokai University Hospital in Japan between January 2016 and March 2019. All patients presented with syncope once or more times at our hospital, and were initially evaluated by a cardiologist. Laboratory analyses, electrocardiography (ECG), echocardiography, and chest radiography were also performed to rule out cardiac diseases, such as coronary artery disease and cardiomyopathy. Echocardiography was performed to rule out systolic and diastolic dysfunctions and structural abnormalities using standard methods. Additionally, electroencephalography and computed tomography of the head were performed in some patients to rule out epilepsy. NMS was diagnosed based on systemic inquiry, thoughtful clinical history taking, and clinical tests. HUTT was performed in patients whose most likely diagnosis was NMS. Patients with any possibility of cardiac or neurological causes were excluded from the study. Patients’ information, including age, past medical history, history related to syncope, and social history were collected from their medical charts 2.3. Head-Up Tilt Test (HUTT) 2 3 Head Up Tilt Test (HUTT) The protocol of the HUTT is shown in Figure 1. Figure 1. HUTT protocol. This figure illustrates the HUTT protocol. HUTT was performed till the end without Figure 1. HUTT protocol. This ﬁgure illustrates the HUTT protocol. HUTT was performed till the end without any syncope If syncope occurred the tilt table was moved back to 0◦immediately If This figure illustrates the HUTT protocol. HUTT was performed till the end without any syncope. If syncope occurred, the tilt table was moved back to 0° immediately. If syncope did not occur for 20 min, nitroglycerin was sublingually administered. The total time for HUTT was 30 min This ﬁgure illustrates the HUTT protocol. HUTT was performed till the end without any syncope. If syncope occurred, the tilt table was moved back to 0◦immediately. If syncope did not occur for 20 min, nitroglycerin was sublingually administered. The total time for HUTT was 30 min. 2.5. Follow-Up in the Patients after the HUTT Patients were instructed to perform the tilt training at home by standing against a wall twice a day for a planned duration of up to 30 min, regardless of the HUTT results. The effectiveness of this tilt training was recommended by a previous study [18]. Patients were followed up for a maximum of 3 years at an outpatient clinic regarding syncope recurrence. Additionally, data of any unexpected events, such as death or unanticipated syncopal problems, were collected. If patients did not present to the outpatient clinic, we contacted them asking the same questions that were asked at the clinic. 2 4 Evaluation of the HUTT Results 2.4. Evaluation of the HUTT Results 2.4. Evaluation of the HUTT Results Blood pressure, heart rate, and ECG were continuously monitored during the test. We classiﬁed the HUTT results into four types according to the latest guidelines for syncope Biology 2021, 10, 919 4 of 11 published by the Japanese Society of Cardiology: vasopressor, cardioinhibitory, mixed, or orthostatic hypotension. The cardioinhibitory type was deﬁned as a heart rate of <40 beats/min that lasted for 10 s, or cardiac arrest lasting >3 s before syncope. The vasopressor type was characterized by a reduction in blood pressure without bradycardia. The mixed type was comprised of both of the above-mentioned characteristics. Classical orthostatic hypotension was deﬁned as a reduction in blood pressure that was sufﬁcient to cause syncope within 3 min of a change in position from supine to standing. A positive test result was deﬁned as the occurrence of presyncope or syncope during the test, regardless of the necessity of sublingual nitroglycerin administration. We calculated each percentage of the four NMS types listed above and analyzed the patients’ data for each NMS group. An unexpected cardiac event was deﬁned as a cardiac issue that was not noticeable at the point of the HUTT, and required an invasive procedure or hospital admission for further treatment. Sudden deaths were also included in this category. 2.6. Statistical Analyses All statistical analyses were performed using the statistical software EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), which is a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria). More precisely, it is a modiﬁed version of R commander designed to add statistical functions frequently used in biostatistics [19]. Log-rank analysis and a Mann–Whitney U test were used to analyze the data. 3.1. Patients’ Characteristics 3.1. Patients’ Characteristics 3.1. Patients’ Characteristics Between March 2016 and June 2019, we performed 101 HUTTs in the patients whose probable diagnosis was NMS based on their clinical history and diagnostic tests, such as blood tests, ECG, 24-h Holter electrocardiography, and echocardiography, performed by cardiologists at our institute (Table 1). Table 1. Baseline clinical characteristics of the study patients. Negative (n = 29) Positive (n = 72) p Value Age 51.8 ± 19.7 years old 48.8 ± 21.6 years old 0.51 Male 72.4% (21) 63.9% (46) 0.49 Hypertension 41.4% (12) 25% (18) 0.15 RAS inhibitor 27.6% (8) 18.1% (13) 0.49 α blocker 3.4% (1) 4.2% (3) 1 β blocker 13.8% (4) 6.9% (5) 0.27 Diuretics 13.8% (4) 7.8% (2) 0.06 Calcium channel blocker 20.7% (6) 15.3% (11) 0.56 CVA 3.4% (1) 0% (0) 0.29 Diabetes mellitus 13.8% (4) 11.1% (8) 0.74 Atrial ﬁbrillation 6.8% (2) 5.5% (4) 1 Ischemic heart disease 13.8% (4) 6.9% (5) 0.27 Non-ischemic heart disease 6.8% (2) 1.4% (1) 0.2 Ejection fraction of Left ventricle 64.9 ± 10.7% 68.8 ± 8.1% 0.05 History of Smoking 51.7% (15) 43.1% (31) 0.31 Epilepsy 0% (0) 1.4% (1) 1 Abbreviations: CVA, Cerebrovascular attack; RAS, renin-angiotensin system. Table 1. Baseline clinical characteristics of the study patients. 5 of 11 Biology 2021, 10, 919 There was no signiﬁcant difference in baseline patient characteristics between the positive HUTT group and the negative HUTT group. The median age was 49.6 ± 21.0 years and there were 67 male patients (66.3%). Patients with cardiac diseases, such as ischemic heart disease and conduction disease that required immediate treatment were excluded. Regarding blood test results of all patients, hemoglobin levels and electrolyte levels, such as sodium, potassium, and calcium, were within normal limits. Of all included patients, 28.9% had hypertension. The study included patients who were on renin-angiotensin system inhibitors (20.2%), α-blockers (3.9%), β- blockers (8.65%), diuretics (5.8%), and calcium channel blockers (16.4%). Twelve patients (11.5%) had diabetes mellitus, which was well controlled with medications. Two patients (2.0%) had a family history of NMS. Before the HUTT, we conﬁrmed that the patients’ cardiac condition was stable enough to tolerate the test. The mean left ventricle ejection fraction measured using echocardio- graphy was 67.7 ± 9.0%. There was no signiﬁcant difference in the electrocardiographic ﬁndings between the positive and negative HUTT groups. One patient had a history of epilepsy. 3.1. Patients’ Characteristics She was evaluated by a neurologist, and no association between syncope and epilepsy was conﬁrmed. All patients presented with syncope without any other provoca- tion. 3.2. HUTT Result During the HUTT, 72 patients (69.2%) experienced syncope. No adverse events (AEs), except for syncope, were documented during the test. Blood pressure, heart rate, or both dropped when the patient experienced syncope. However, the patients were managed appropriately by the bedside physician. The types of syncope are shown in Figure 2. The syncope was classiﬁed as vasopressor, cardioinhibitory, mixed type, and orthostatic hypotension in 30 (27.9%), 10 (9.6%), 27 (25.9%), and ﬁve (4.8%) patients, respectively (Figure 2). There was no signiﬁcant difference between the two groups in terms of the use of medications listed in Table 1, presence of a history of cardiac diseases, diabetes mellitus, hypertension, AF, and stroke. There was no signiﬁcant difference in age (51.8 ± 19.7 vs. 48.8 ± 21.6 years, p = 0.59) and left ventricle ejection fraction (64.9 ± 10.7 vs. 68.8 ± 8.1%, p = 0.13) between the two groups. EW 6 of 12 Figure 2. HUTT results are classified according to the NMS type. Negative included the patients who did not presented with syncope during HUTT. Figure 2. HUTT results are classiﬁed according to the NMS type. Negative included the patients who did not presented with syncope during HUTT. Figure 2. HUTT results are classified according to the NMS type. Negative included the patients who did not presented with syncope during HUTT. Figure 2. HUTT results are classiﬁed according to the NMS type. Negative included the patients who did not presented with syncope during HUTT. 3.3. Details of 12 Lead ECG Analysis 3.3. Details of 12 Lead ECG Analysis 3.3. Details of 12 Lead ECG Analysis 3.3. Details of 12 Lead ECG Analysis f y We performed analysis of 12 lead ECG of each patient who underwent HUTT. The only significant differences were shorter QRS intervals and QT intervals in patients with a positive HUTT compared to that of those with a negative HUTT (Table 2). We performed analysis of 12 lead ECG of each patient who underwent HUTT. The only signiﬁcant differences were shorter QRS intervals and QT intervals in patients with a positive HUTT compared to that of those with a negative HUTT (Table 2). 6 of 11 Biology 2021, 10, 919 Table 2. Analysis of baseline electrocardiogram. 3.4. Recurrence of Syncope after the HUTT and ICM Placement The patients were followed up for 886.1 ± 457.7 days (interquartile range: 518–1293 days). The recurrence rate of syncope was 16.9%, and there was no significant difference between the two groups in recurrence rate (13.8% vs. 18.1%, p = 0.772, Figure 3). We performed log-rank analysis and found no significant difference between the two groups (p = 0.772). EW 7 of 12 The patients were followed up for 886.1 ± 457.7 days (interquartile range: 518–1293 days). The recurrence rate of syncope was 16.9%, and there was no significant difference between the two groups in recurrence rate (13.8% vs. 18.1%, p = 0.772, Figure 3). We performed log-rank analysis and found no significant difference between the two groups (p = 0.772). IEW 7 of 12 Figure 3. Kaplan Meier curves depict the recurrence rate of syncope according to the HUTT results. The black line stands for the patients whose HUTT was negative. The red line stands for the patients whose HUTT was positive. Figure 3. Kaplan Meier curves depict the recurrence rate of syncope according to the HUTT results. The black line stands for the patients whose HUTT was negative. The red line stands for the patients whose HUTT was positive. Figure 3. Kaplan Meier curves depict the recurrence rate of syncope according to the HUTT results. The black line stands for the patients whose HUTT was negative. The red line stands for the patients whose HUTT was positive. Figure 3. Kaplan Meier curves depict the recurrence rate of syncope according to the HUTT results. The black line stands for the patients whose HUTT was negative. The red line stands for the patients whose HUTT was positive. 3.5. Unexpected Cardiac Events after the HUTT Regular follow-up of patients revealed six unexpected cardiac events (5.9%). Four out of 29 (13.9%) patients in the negative HUTT group and two out of 72 (2.8%) in the positive HUTT group experienced cardiac events. Of the four patients in the negative Two patients (6.9%) in the negative HUTT group underwent ICM placement. However, no signiﬁcant arrhythmia was detected to explain the cause of syncope. None of the patients in the positive HUTT group underwent ICM placement, and none of the patients received any prescription for the treatment of NMS. 3.2. HUTT Result HUTT p Value Negative (n = 29) Positive (n = 71) HR 68 ± 14 62 ± 14 0.06 PR 163 ± 30.6 162 ± 23.8 0.88 Axis 65.2 ± 79.2 46.3 ± 39.0 0.1 QRS 113 ± 61.9 94.4 ± 19.3 0.03 QTc 430 ± 33.9 413 ± 26.9 0.01 RBBB 3 (10.3%) 10 (14.1%) 0.75 LBBB 2 (6.9%) 0 (0%) 0.08 Brugada 1 (3.4%) 2 (2.8%) - DP 0 (0%) 1 (1.4%) - ERP 3 (10.3%) 13 (18.3%) 0.547 Abbreviations: HR, heart rate (beats per minute); PR, PR interval (ms); Axis, electronic axis (degree); QRS, QRS interval (ms); RBBB, right bundle branch block; LBBB, left bundle branch block; DP, delayed potentials; ERP, early repolarization. Abbreviations: HR, heart rate (beats per minute); PR, PR interval (ms); Axis, electronic axis (degree); QRS, QRS interval (ms); RBBB, right bundle branch block; LBBB, left bundle branch block; DP, delayed potentials; ERP, early repolarization. 3.4. Recurrence of Syncope after the HUTT and ICM Placement HUTT group, one patient required a pace atrioventricular conduction block One pat 3.5. Unexpected Cardiac Events after the HUTT atrioventricular conduction block. One patient required an implantable cardioverter defibrillator for syncope associated with ventricular tachycardia due to possible sarcoidosis. One patient underwent catheter ablation for AF with a rapid ventricular response (RVR). Sudden death occurred in the last patient 417 days after the HUTT. Among the two patients in the positive HUTT group, one patient required pacemaker Regular follow-up of patients revealed six unexpected cardiac events (5.9%). Four out of 29 (13.9%) patients in the negative HUTT group and two out of 72 (2.8%) in the positive HUTT group experienced cardiac events. Of the four patients in the negative HUTT group, one patient required a pacemaker implantation due to an advanced atrioventricular con- 7 of 11 Biology 2021, 10, 919 duction block. One patient required an implantable cardioverter deﬁbrillator for syncope associated with ventricular tachycardia due to possible sarcoidosis. One patient underwent catheter ablation for AF with a rapid ventricular response (RVR). Sudden death occurred in the last patient 417 days after the HUTT. Among the two patients in the positive HUTT group, one patient required pacemaker implantation due to sick sinus syndrome, and the other required hospital admission because of AF with RVR. Details of all events are listed in Table 3. We performed log-rank analysis and found signiﬁcant differences in the occurrence rate of cardiac events between the two groups (p = 0.019, Figure 4). Table 3. Details of cardiac events in each patient. Positive HUTT Patient Sex Age The Type of NMS Days after HUT Test The Details of Cardiac Event 1 Female 70 Vasopressor 549 Although no AF was detected prior to HUTT, the patient presented with syncope due to AF with rapid ventricular response. She underwent catheter ablation because her AF was refractory to antiarrhythmic medications. 2 Female 73 Vasopressor 1111 Although no AF was detected prior to HUTT, the patient presented with syncope due to AF with rapid ventricular response. She was told to undergo catheter ablation, however, she refused the procedure. Negative HUT test 3 Female 75 N/A 23 The patient presented with syncope and found to have 5 sec of long pause with faintness at the emergency room. The patient required pacemaker implantation due to sick sinus syndrome. 4 Male 82 N/A 516 The patient presented with bradycardia and syncope and was brought to the emergency room. He was found out to have advanced atrioventricular conduction block and required pacemaker implantation. 4. Discussion This retrospective clinical study assessed the clinical signiﬁcance of the HUTT in patients with NMS. The results demonstrated that vasopressors and mixed types of NMS were more common than the cardioinhibitory type. While the pre-test probability was high, the rate of positive test results was not as high as anticipated. Although we ruled out possible diseases that could cause syncope before the test, the patients’ condition on the test date might have affected the test results. There are several studies on the HUTT, and our results are comparable to previous research data [20]. Sublingual nitroglycerin administration during the HUTT has also made the results obtained more accurate [21] g [ ] NMS is sometimes underestimated in daily clinical settings because its prognosis is believed to be benign. Outpatient follow-up is sometimes discontinued at the patient’s or physician’s discretion [22]. A limited variety of treatment options, such as medications and tilt training, have also contributed to inadequate outpatient follow-up [23]. Therefore, there was a paucity of studies of syncope recurrence after the HUTT. We followed up our patients for a relatively long period and found that the recurrence rate was 16.9%. It has been reported that syncope recurrence in the elderly was as high as 32.5% [22]. Since our patients had a median age of 49.6 ± 21.0 years, the recurrence rate was lower than the average rate reported in previous studies [24]. Of note, there was no signiﬁcant difference between the negative and positive HUTT result groups. We did not compare our patients with syncope to patients who did not undergo the HUTT. However, the test itself and education for syncope may help patients avoid unexpected syncope recurrence in their daily life. We educated all included patients to perform the tilt training after they were discharged home from the hospital. Although there are conﬂicting results regarding the efﬁcacy of the tilt training, a high compliance rate with this training in our patients may have contributed to the low recurrence rate of syncope [18,22]. More research is needed to elucidate how a high compliance rate with tilt training can improve the rate of syncope recurrence. None of our patients received pharmacological treatment because there is a lack of evidence for the efﬁcacy of pharmacological treatment in patients with NMS [25]. HUTT group, one patient required a pace atrioventricular conduction block One pat 3.5. Unexpected Cardiac Events after the HUTT 5 Male 55 N/A 417 The patient had history of old myocardial infarction (ejection fraction of 44%) of left anterior descending artery. He was found to be dead at home due to unknown etiology. After the investigation, we decided that the most likely cause of death was cardiogenic disease. 6 Female 61 N/A 1 The patient presented with syncope and the monitor electrocardiogram demonstrated unstable ventricular tachycardia with heart rate was 180 beats per minute. The syncope occurred on the day when we performed HUTT. She was diagnosed as cardiac sarcoidosis and required ICD placement. Abbreviations: HUTT, head-up tilt test; NMS, neurally mediated syncope; AF, Atrial Fibrillation, ICD, Implantable Cardioverter Deﬁbrillator. Table 3. Details of cardiac events in each patient. 8 of 11 bl Biology 2021, 10, 919 Abb Figure 4. KAPLAN Meier curves depict the occurrence rate of unanticipated cardiac events classified according to the HUTT results. The black line stands for the patients whose HUTT was negative. The red line stands for the patients whose HUTT was positive. Figure 4. KAPLAN Meier curves depict the occurrence rate of unanticipated cardiac events classiﬁed according to the HUTT results. The black line stands for the patients whose HUTT was negative. The red line stands for the patients whose HUTT was positive. Figure 4. KAPLAN Meier curves depict the occurrence rate of unanticipated cardiac events classified according to the HUTT results. The black line stands for the patients whose HUTT was negative. The red line stands for the patients whose HUTT was positive. Figure 4. KAPLAN Meier curves depict the occurrence rate of unanticipated cardiac events classiﬁed according to the HUTT results. The black line stands for the patients whose HUTT was negative. The red line stands for the patients whose HUTT was positive. 4. Discussion Although there have been several reports regarding the usefulness of the HUTT in the last decade, we suggest that two important ﬁndings regarding the clinical signiﬁcance of the test should be noted [20,26]. First, if the test is positive, we could conﬁrm that the cause of syncope is NMS. It is very important to rule out other etiologies of syncope before the test. It has been reported that 15.5% of all patients with syncope admitted to the emergency department (ED) experienced AEs within a year [27]. It should be noted that the etiology itself was unknown when patients present with syncope at the ED for the ﬁrst time and that patients who are brought to the ED tend to be in a more critical condition than those who visit the outpatient clinic. Therefore, it is reasonable that the occurrence rate of AEs 9 of 11 Biology 2021, 10, 919 was higher in patients who presented to the emergency room than in our study patients. If a patients HUTT results are negative, such a patient should not be diagnosed with NMS. However, we found that patients with negative HUTT results could encounter unexpected cardiac events more frequently than those with positive HUTT results. Therefore, close outpatient monitoring, including 24-h Holter electrocardiography and blood tests, should be recommended. In addition, ICM is also recommended for unexplained syncope. was higher in patients who presented to the emergency room than in our study patients. If a patients HUTT results are negative, such a patient should not be diagnosed with NMS. However, we found that patients with negative HUTT results could encounter unexpected cardiac events more frequently than those with positive HUTT results. Therefore, close outpatient monitoring, including 24-h Holter electrocardiography and blood tests, should be recommended. In addition, ICM is also recommended for unexplained syncope. All current guidelines published in the United States and Europe are in general agree- ment that the decision to use an ICM for syncope should consider patients’ characteristics, frequency of syncope, and pre-test probability of arrhythmic cause [28]. Several studies re- ported that ICM placement led to a 36.6–51.8% of diagnostic yield during the 17–39 months of follow-up period [29,30]. Our ﬁndings suggest that a negative HUTT requires further investigation to determine the etiology of syncope. All our patients presented with unex- pected cardiac events. Arrhythmia related to AF is a common cause of syncope among the elderly [31]. Limitations This was a single-center study with a small number of patients. Due to the retrospec- tive nature of the study, selection bias could not be excluded. It has been reported that female patients might develop NMS more frequently than their male counterparts [32]. Thus, our study does not reﬂect the real-world scenario. Further research with a larger number of patients is required to validate our ﬁndings. Closer attention should have been paid to the characteristics of patients with syncope. Our patients were selected because they had a high likelihood of NMS to take the HUTT. Therefore, selection bias cannot be ignored. In other words, although the initial diagnosis appears to be NMS, we should endeavor to determine the etiology of syncope when the HUTT is negative. We found that negative HUTT result is associated with more cardiac events; however, further studies are needed to support this ﬁnding. While ICM is recommended as per the current guidelines for unknown etiology syncope, only two patients in our study underwent ICM placement. This may affect the analysis of the recurrence rate of NMS. 4. Discussion Therefore, patients with negative HUTT results and older age should undergo ICM implantation for possible intracardiac conduction disorder and AF. 5. Conclusions Negative HUTT results may assist in anticipating unexpected clinical events within a few years in patients with syncope. A negative HUTT result may allow us to reconsider the diagnosis of NMS based on clinical information. However, close outpatient follow-up for patients with negative HUTT results is warranted. Author Contributions: Conceptualization, K.A. and T.K.; methodology, K.A. and T.K.; validation, K.A. and K.Y.; formal analysis, K.A.; investigation, K.A. and T.K.; resources, K.A., M.H., T.S., M.M., S.S., A.Y., M.A., Y.I. and K.Y.; data curation, K.A.; writing—original draft preparation, K.A. and T.K.; writing—review and editing, K.A. and T.K.; supervision, Y.I. and K.Y; project administration, K.A. and T.K.; funding acquisition, K.A. and T.K. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by KAKENHI, grant number 20K08498 and 21K16037. Institutional Review Board Statement: This study was conducted under the approval of the Ethics Review Committee and the Medical Ethics Committee of Tokai University School of Medicine, Clinical Research Review Board (No. 14R-53) [5] and was in accordance with the Declaration of Helsinki. Institutional Review Board Statement: This study was conducted under the approval of the Ethics Review Committee and the Medical Ethics Committee of Tokai University School of Medicine, Clinical Research Review Board (No. 14R-53) [5] and was in accordance with the Declaration of Helsinki. Informed Consent Statement: Written informed consent has been obtained from the patients to publish this paper. None of the patients had brain or heart disease. Informed Consent Statement: Written informed consent has been obtained from the patients to publish this paper. None of the patients had brain or heart disease. Data Availability Statement: Not applicable. Acknowledgments: We thank the Support Center for Medical Research and Education of Tokai University for the technical assistance. Acknowledgments: We thank the Support Center for Medical Research and Education of Tokai University for the technical assistance. 10 of 11 Biology 2021, 10, 919 Conﬂicts of Interest: The authors declare no conﬂict of interest. References Hasegawa, M.; Komiyama, T.; Ayabe, K.; Sakama, S.; Sakai, T.; Lee, K.H.; Morise, M.; Yagishita, A.; Amino, M.; Sasaki, A.; et al. Diagnosis and prevention of the vasodepressor type of neurally mediated syncope in Japanese patients PLoS ONE 2021 16 y p 6. Komiyama, T.; Nagata, E.; Hashida, T.; Sakama, S.; Ayabe, K.; Kamiguchi, H.; Sasaki, A.; Yoshioka, K.; Kobayashi, H. Neurally mediated syncope diagnosis based on adenylate cyclase activity in Japanese patients. PLoS ONE 2019, 14, e0214733. [CrossRef] mediated syncope diagnosis based on adenylate cyclase activity in Japanese patients. PLoS ONE 2019, 14, e0214733. 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Incidence and prognosis of syncope. N. Engl. J. Med. 2002, 347, 878–885. [CrossRef] g 5. Walsh, C.A. Syncope and sudden death in the adolescent. Adolesc Med. 2001, 12, 105–132. 5. Walsh, C.A. Syncope and sudden death in the adolescent. Adolesc Med. 2001, 12, 105–132. 6. Komiyama, T.; Nagata, E.; Hashida, T.; Sakama, S.; Ayabe, K.; Kamiguchi, H.; Sasaki, A.; Yoshioka, K.; Kobayashi, H. Neurally mediated syncope diagnosis based on adenylate cyclase activity in Japanese patients. PLoS ONE 2019, 14, e0214733. [CrossRef] 7. 27. Henderson, S.S.; Reed, M.J.; Newby, D.E.; Gray, A.J. 018 One-year prognosis after syncope and the failure of the ROSE decision instrument to predict 1-year adverse events. Emerg. Med. J. 2011, 28, A8–A9. [CrossRef] 28. Giancaterino, S.; Lupercio, F.; Nishimura, M.; Hsu, J.C. Current and future use of insertable cardiac monitors. JACC Clin. Electrophysiol. 2018, 4, 1383–1396. [CrossRef] p y 29. Li, Y.; Nantsupawat, T.; Olson, M.; Tholakanahalli, V.; Adabag, S.; Wang, Z.; Benditt, D.G.; Li, J.-M. A single center experience on the clinical utility evaluation of an insertable cardiac monitor. J. Electrocardiol. 2018, 51, 583–587. [CrossRef] [PubMed] 29. Li, Y.; Nantsupawat, T.; Olson, M.; Tholakanahalli, V.; Adabag, S.; Wang, Z.; Benditt, D.G.; Li, J.-M. A single center experience on the clinical utility evaluation of an insertable cardiac monitor. J. Electrocardiol. 2018, 51, 583–587. [CrossRef] [PubMed] 30. Burkowitz, J.; Merzenich, C.; Grassme, K.; Brüggenjürgen, B. Insertable cardiac monitors in the diagnosis of syncope and the detection of atrial ﬁbrillation: A systematic review and meta-analysis. Eur. J. Prev. Cardiol. 2016, 23, 1261–1272. [CrossRef] 31. Dalgaard, F.; Pallisgaard, J.L.; Numé, A.K.; Lindhardt, T.B.; Gislason, G.H.; Torp-Pedersen, C.; Ruwald, M.H. Rate or rhythm control in older atrial ﬁbrillation patients: Risk of fall-related injuries and syncope. J. Am. Geriatr. Soc. 2019, 67, 2023–2030. the clinical utility evaluation of an insertable cardiac monitor. J. Electrocardiol. 2018, 51, 583–587. [CrossRef] [PubMed] 30. Burkowitz, J.; Merzenich, C.; Grassme, K.; Brüggenjürgen, B. Insertable cardiac monitors in the diagnosis of syncope and the detection of atrial ﬁbrillation: A systematic review and meta-analysis. Eur. J. Prev. Cardiol. 2016, 23, 1261–1272. [CrossRef] p y 29. Li, Y.; Nantsupawat, T.; Olson, M.; Tholakanahalli, V.; Adabag, S.; Wang, Z.; Benditt, D.G.; Li, J.-M. A single center experience on the clinical utility evaluation of an insertable cardiac monitor. J. Electrocardiol. 2018, 51, 583–587. [CrossRef] [PubMed] 30. Burkowitz, J.; Merzenich, C.; Grassme, K.; Brüggenjürgen, B. Insertable cardiac monitors in the diagnosis of syncope and the p y 29. Li, Y.; Nantsupawat, T.; Olson, M.; Tholakanahalli, V.; Adabag, S.; Wang, Z.; Benditt, D.G.; Li, J.-M. 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Effectiveness of ﬂudrocortisone and salt in preventing syncope recurrence in children: A double-blind, l b t ll d d i d t i l J Am Coll Cardiol 2005 45 484 488 [C R f] 25. Salim, M.A.; Di Sessa, T.G. Effectiveness of ﬂudrocortisone and salt in preventing syncope recurren placebo-controlled, randomized trial. J. Am. Coll. Cardiol. 2005, 45, 484–488. [CrossRef] 26. Ninni, S.; Kouakam, C.; Szurhaj, W.; Baille, G.; Klug, D.; Lacroix, D.; Derambure, P. Usefulness of head-up tilt test combined with video electroencephalogram to investigate recurrent unexplained atypical transient loss of consciousness. Arch. Cardiovasc. Dis. 2019, 112, 82–94. [CrossRef] 11 of 11 11 of 11 Biology 2021, 10, 919 27. Henderson, S.S.; Reed, M.J.; Newby, D.E.; Gray, A.J. 018 One-year prognosis after syncope and the failure of the ROSE decision instrument to predict 1-year adverse events. Emerg. Med. J. 2011, 28, A8–A9. [CrossRef] p y g J 28. Giancaterino, S.; Lupercio, F.; Nishimura, M.; Hsu, J.C. Current and future use of insertable cardiac monitors. JACC Clin. Electrophysiol. 2018, 4, 1383–1396. [CrossRef] p y 29. Li, Y.; Nantsupawat, T.; Olson, M.; Tholakanahalli, V.; Adabag, S.; Wang, Z.; Benditt, D.G.; Li, J.-M. A single center experience on the clinical utility evaluation of an insertable cardiac monitor. J. Electrocardiol. 2018, 51, 583–587. [CrossRef] [PubMed] y 30. Burkowitz, J.; Merzenich, C.; Grassme, K.; Brüggenjürgen, B. Insertable cardiac monitors in the diagnosis of syncope and the detection of atrial ﬁbrillation: A systematic review and meta-analysis. Eur. J. Prev. Cardiol. 2016, 23, 1261–1272. [CrossRef] y y 31. Dalgaard, F.; Pallisgaard, J.L.; Numé, A.K.; Lindhardt, T.B.; Gislason, G.H.; Torp-Pedersen, C.; Ruwald, M.H. Rate or rhythm control in older atrial ﬁbrillation patients: Risk of fall-related injuries and syncope. J. Am. Geriatr. Soc. 2019, 67, 2023–2030. [CrossRef] 32. Park, J.; Jang, S.Y.; Yim, H.R.; On, Y.K.; Huh, J.; Shin, D.H.; Kim, J.H.; Kim, J.S. Gender difference in patients with recurrent neurally mediated syncope. Yonsei Med. J. 27. Henderson, S.S.; Reed, M.J.; Newby, D.E.; Gray, A.J. 018 One-year prognosis after syncope and the failure of the ROSE decision instrument to predict 1-year adverse events. Emerg. Med. J. 2011, 28, A8–A9. [CrossRef] 28. Giancaterino, S.; Lupercio, F.; Nishimura, M.; Hsu, J.C. Current and future use of insertable cardiac monitors. JACC Clin. Electrophysiol. 2018, 4, 1383–1396. [CrossRef] 29. Li, Y.; Nantsupawat, T.; Olson, M.; Tholakanahalli, V.; Adabag, S.; Wang, Z.; Benditt, D.G.; Li, J.-M. A single center experience on the clinical utility evaluation of an insertable cardiac monitor. J. Electrocardiol. 2018, 51, 583–587. [CrossRef] [PubMed] 30. Burkowitz, J.; Merzenich, C.; Grassme, K.; Brüggenjürgen, B. Insertable cardiac monitors in the diagnosis of syncope and the detection of atrial ﬁbrillation: A systematic review and meta-analysis. Eur. J. Prev. Cardiol. 2016, 23, 1261–1272. [CrossRef] 31. Dalgaard, F.; Pallisgaard, J.L.; Numé, A.K.; Lindhardt, T.B.; Gislason, G.H.; Torp-Pedersen, C.; Ruwald, M.H. Rate or rhythm control in older atrial ﬁbrillation patients: Risk of fall-related injuries and syncope. J. Am. Geriatr. Soc. 2019, 67, 2023–2030. [CrossRef] 32. Park, J.; Jang, S.Y.; Yim, H.R.; On, Y.K.; Huh, J.; Shin, D.H.; Kim, J.H.; Kim, J.S. Gender difference in patients with recurrent neurally mediated syncope. Yonsei Med. J. 2010, 51, 499–503. [CrossRef] [PubMed] y 30. Burkowitz, J.; Merzenich, C.; Grassme, K.; Brüggenjürgen, B. Insertable cardiac monitors in the diag detection of atrial ﬁbrillation: A systematic review and meta-analysis. Eur. J. Prev. Cardiol. 2016, 23, 12 instrument to predict 1 year adverse events. Emerg. Med. J. 2011, 28, A8 A9. [CrossRef] 28. Giancaterino, S.; Lupercio, F.; Nishimura, M.; Hsu, J.C. Current and future use of insertable cardiac monitors. JACC Clin. Electrophysiol. 2018, 4, 1383–1396. [CrossRef] 29. Li, Y.; Nantsupawat, T.; Olson, M.; Tholakanahalli, V.; Adabag, S.; Wang, Z.; Benditt, D.G.; Li, J.-M. A single center experience on the clinical utility evaluation of an insertable cardiac monitor. J. Electrocardiol. 2018, 51, 583–587. [CrossRef] [PubMed] 30 B k it J M i h C G K B ü jü B I t bl di it i th di i f d th References 2010, 51, 499–503. [CrossRef] [PubMed] 32. Park, J.; Jang, S.Y.; Yim, H.R.; On, Y.K.; Huh, J.; Shin, D.H.; Kim, J.H.; Kim, J.S. Gender difference in patients with recurrent neurally mediated syncope. Yonsei Med. J. 2010, 51, 499–503. [CrossRef] [PubMed] 32. Park, J.; Jang, S.Y.; Yim, H.R.; On, Y.K.; Huh, J.; Shin, D.H.; Kim, J.H.; Kim, J.S. Gender difference in patients with recurrent neurally mediated syncope. Yonsei Med. J. 2010, 51, 499–503. [CrossRef] [PubMed]
https://openalex.org/W3091876434	https://ojs.aut.ac.nz/nzjer/article/download/16/10	English	null	On the Bright Side: Covid-19, A Perverse Once-in-One-Hundred Year Opportunity	New Zealand journal of employment relations	2,020	cc-by	5,820	Abstract The Covid-19 pandemic, challenging as it is, provides an opportunity to rethink employment relations arrangements in New Zealand in a broader context of economic policy change, in circumstances which echo the challenges of the 1930s. Major transformation will require a combination of political imagination and will, novel economic policy initiatives, and strong commitment from the social partners. Keywords: New Zealand elections, economic policy, employment relations, pandemic New Zealand Journal of Employment Relations, 45(1): 47-58 New Zealand Journal of Employment Relations, 45(1): 47-58 Commentary: On the Bright Side: Covid-19, a Perverse Once-in- One-Hundred Year Opportunity NIGEL HAWORTH* * Emeritus Professor of Human Resource Development, Department of Management and International Business, University of Auckland, New Zealand New Zealand Journal of Employment Relations, 45(1): 47-58 Employment relations is always a political consideration, for it lies at the heart of economic arrangements and political voice in the liberal democracies. It has the potential to contribute significantly to radical transformation as it will be the focus of reform and reconfiguration in the alternative model. It follows from this that a new Labour-led government, intent on radical change, would make labour market policy and employment relations a key facet of its interventions, and that the parties – employers’ representatives and trade unions – would be actively involved, by choice and by encouragement, in the formulation of a new accommodation. As I discuss below, this expectation emphasises the combination of factors required to configure the labour market and employment relations dimensions of the accommodation – clear and well-developed policy, strong and energetic political leadership (especially at ministerial level), employer and union parties staffed and sufficiently grounded in the policy to make tripartism function effectively, institutions for engagement and dialogue that command loyalty and respect. Introduction As we approach the October 2020 General Election, the spectre of the Covid-19 pandemic hovers over New Zealand’s politics and economic performance. Employment relations might seem to be small beer in this context, with labour market disruption and multiple emergency interventions notionally crowding out any interest in employment relations and bargaining arrangements. In terms of short term measures to mitigate the effects of Covid-19, support for the labour market is most important, but, equally, employment relations arrangements and labour market interventions will go hand-in-hand in the long-term post-Covid-19 recovery. The corollary of Covid-19’s impact is the opportunity that it creates for radical transformation of economic arrangements in general, and of employment relations in particular. This is the focus of this commentary. It suggests that a Labour-led government, should it be returned strongly to power in the 2020 election, might undertake simultaneously both the short-term recovery measures required by the Covid-19 pandemic, and the medium-to-long-term piloting of the economy, away from the siren calls of a renewed neo-liberal model, to a contemporary version of the 1930s “Keynesian Accommodation”. Both phases require major innovation in labour market and employment relations settings. The sub-text of this argument, though not developed in detail here, is that the alternative to a new accommodation is likely to be recourse to a renewed and stringent neo-liberalism, that is, a pervading emphasis on cutting state expenditures and reinforcing market-based measures for economic stimulus and employment relations. This discussion assumes, for historical and analytical reasons, a binary reality in current policy settings. In other words, the recent history, ideological strength and institutional power of neo-liberalism, domestically and internationally, is unlikely to permit “third ways”. This view differs in some ways from that of, for example, Piketty, but draws on his and other recent analysts of contemporary Capitalism (Piketty, 2014). 47 New Zealand Journal of Employment Relations, 45(1): 47-58 New Zealand Journal of Employment Relations, 45(1): 47-58 New Zealand Journal of Employment Relations, 45(1): 47-58 attempt to recast employment relations in New Zealand in a modern form of industry bargaining derived substantially from the efforts of the late Helen Kelly, then President of the New Zealand Council of Trade Unions (CTU), supported by affiliates. The Greens, in general, supported the idea; New Zealand First was much less enthusiastic. National and ACT were implacably opposed. A paucity of urgency and agreement across the governing parties resulted in the proposal languishing. If the flagship policy drifted, there was important work undertaken elsewhere. Overall, global conditions and macroeconomic policy have contrived to keep unemployment low. Business advice has been given a nod in a Business Advisory Council. Labour market development has been promoted by tax credits for research and development, regionalisation of skills strategy, and abatement thresholds to promote the move from unemployment to work. The Equal Pay Amendment Act is a major initiative, as was the attempted reintroduction of collective bargaining for screen workers and contractors. Meal and rest breaks have been restored and the ‘90-day trial’ provision has been significantly limited. Before the Great Disruption: the lacuna around Industry Standards Agreements The outcome of the 2017 General Election, understandably, surprised many commentators. The upsurge in Labour’s fortunes, following its change of leadership close to the election date, was striking, as was the assuredness with which the new Labour leader came to the role. Whilst New Zealand First’s decision to ally with Labour was more likely than not, given the relationship between New Zealand First and National, it could not be taken for granted. Equally, National’s performance in the election shocked its supporters and pundits. The outcome was a government, at best, modestly prepared for power, with relatively few senior politicians with experience of ministerial office, and simultaneously grappling with the complexities of a challenging three-party MMP arrangement. Here one must also recognise the limitations of the three year term, especially in the context of an inconsistent policy and implementation capability in central government, variable expertise (particularly in employment relations) among public servants, and, in some cases, resistance to change they consider “radical”. Even a highly experienced, homogeneous and well-prepared government, coming to power after nine years in opposition, must work extraordinarily hard and successfully to make major legislative changes before the demands of the next general election impinge. Given the election outcome, and the three-party MMP arrangement that it permitted, the workload expectations of the incoming government were most challenging. It is fair to suggest, also, that contemporary Labour-in-power has moved significantly from the democratic socialist perspective of its founders and Constitution. Its current broad configuration is less class-based, more interest-based, in line with a modern Social Democrat perspective. A view exists that modern Labour is, today, perhaps less a Social Democrat, and more a Social Interest, party. It may follow from this that employment relations issues no longer capture fully the attention and imagination of Labour’s policy makers, reflecting also a decline of influence of organised labour in the party. In the complex post-election environment, labour market and employment relations issues were relatively small beer. The flagship employment relations policy for Labour was a commitment to Industry Standards Agreements, renamed, in a tetchy debate, Fair Pay Agreements. This 48 New Zealand Journal of Employment Relations, 45(1): 47-58 For government, there was no blue-print for a response. The comprehensive challenge posed by Covid-19 required rapid and comprehensive action in a constantly evolving inchoate situation. The scope of the crisis marginalised significantly other government initiatives, which though continuing, faced reprioritisation in terms of primacy and budgets. It also recast the structure of political leadership in New Zealand, focussing de facto authority in a relatively small group of ministers, officials and advisers. We should recognise the pressure brought to bear by a crisis of this magnitude on leadership and decision-making. Short-term Responses: Saving the Labour Market Here, I address rather narrowly, and briefly, the immediate labour market responses to Covid- 19 undertaken by the government. These nest within a wider range of health, social, educational and other interventions, some of which have indirect labour market consequences (NZ Treasury, 2020a). At the heart of the labour market response has been expenditure of over $12 billion on wage subsidies designed to cushion the employment impact of Covid-19 by sustaining the relationship between employer and employee. This includes sick leave provisions designed to support workers who might be forced to self-isolate (New Zealand Work and Income, 2020). Accompanying the wage subsidy have been measures to support leave taken from work, workers displaced as a result of Covid-19, and migrant workers. Business support, indirectly supporting employment, includes $2.8 billion committed to tax changes to support business activity; $600 million to support crucial supply chains and $900 million to support Air New Zealand; $6.25 billion in a Business Finance Guarantee programme to reduce debt pressure on companies; measures to support potentially insolvent enterprises. There appears to be a consensus that the government has acted promptly and decisively in intervening to support the labour market and business operations. Fortunately, government books were healthy when the pandemic arose, government is spending heavily and holds considerable further spending potential in reserve. It remains to be seen whether the course of the pandemic, in terms of both epidemiological and vaccine development, will allow a “v- shaped” economic recovery (the most positive scenario modelled by Treasury) or will entail a much longer and potentially far more difficult adjustment. Current government modelling across three scenarios sees GDP declining between 13.5 per cent and 34 per cent in 2021; unemployment sitting between 4.4 per cent and 6.4 per cent by 2024; nominal GDP cumulatively down between 2020 and 2024, by between $121 billion and $150-217 billion. At the time of writing, thinking is towards the lower end of the estimates, but with government debt (core Crown debt as a percentage of GDP) forecast to grow from an actual 19 per cent in 2019 to a daunting 53 per cent in 2023. The Great Disruption Government is a dialectic between what the ruling party (parties) seek(s) to achieve, and the contingent crises and attacks that seek to derail well-made plans. There is a traditional management problem of strategy and tactics, exacerbated by MMP. MMP tends to dilute ideological impact on policy implementation, making the agreed policy programme more contingent and piecemeal that perhaps was the case historically. Other factors drive this contingency, such as, in Labour’s case, its evolution into a post-modern party of interests, rather than class. Notwithstanding this evolutionary change, government usually has a programme and, in any three year term, understands that it is likely to achieve a handful of major changes, and a penumbra of the minor. Depending on the balance of forces in parliament, government is more or less challenged by the opposition, which, as recent political cycles show, is often distracted and frustrated by its distance from effective power. Since 2017, numbers in the House have been tight, and the government alliance has required careful management. That challenge has a small silver lining, as tight numbers in the House permit the “we wanted to do X, but were stymied by our partners” argument to be used sparingly. The 2017-2020 government has faced normal management challenges – ministerial performance, MP behaviour, rifts in the government alliance and so on – and has emerged reasonably unscathed. However, the terrorist attack in Christchurch was a major disruptor, as was the White Island eruption. These two events have now been complemented by the Covid- 19 pandemic, a once-in-a-hundred-year crisis, the full ramifications of which are still to be understood. Covid-19 is a comprehensive crisis, in scope as yet indefinable and fluid. It challenges the performance of New Zealand’s political apparatus – government and Civil Service – in a way not seen since the Great Depression or the Second World War. It disrupts economic activity – production, distribution and international trade – at national and international levels in ways still emerging. It strikes at the fabric of social order in terms of security of employment, social cohesion and order, physical and mental wellbeing, and provision of support services (especially medical services). 49 New Zealand Journal of Employment Relations, 45(1): 47-58 The Longer Term: a New Accommodation or Neo-Liberalism “on Steroids” The economic consequences of the Covid-19 pandemic, generally, and its impact on government books particularly, attracted ab initio significant attention in political and policy circles. Government’s application to the short-term human consequences was accompanied by a growing concern about the post-Covid-19 economy and its recuperation. 50 New Zealand Journal of Employment Relations, 45(1): 47-58 We can see the Covid-19 crisis in terms of two phases. The first is the immediate response to mitigate the impact of lockdowns, enterprise difficulties, unemployment and faltering global trade and connections. As the core Crown debt figures suggest, this has been addressed by massive expenditure, fortunately in an economy in good heart at the beginning of the crisis. Despite the usual search for political advantage, the current consensus seems to be that government acted swiftly and proportionately. However, the focus will, in time, turn to a second phase – the rebuild phase. This has national and international dimensions. The international aspect relates primarily to rebuilding global trade performance, from transport systems to market recuperation. Competitiveness will become a key feature determining post-Covid19 success in international trade and must therefore play its part in the domestic rebuild. The configuration of the domestic rebuild is heavily contested and quite different from the context in which domestic economies rebuilt after the Great Depression. Putting aside the argument that the Second World War was a key catalyst for growth between the 1950s and 1970s, the “package” of measures employed from the mid-1930s to mitigate economic crisis and provide a platform for growth, and its ideological underpinning (Keynesianism and the Welfare State), developed in a context different from current conditions. The defining difference is the contemporary strength and “hold” of neo-liberalism in politics and policy making after over 40 years of primacy. Neo-liberal orthodoxy has not been weakened by Covid-19, which can be presented, not as an economic crisis challenging that orthodoxy, but as an Act of God requiring the more fulsome application of neo-liberal precepts. Covid-19, unlike the Great Depression, does not challenge directly the neo-liberal position, and, indeed, for some may strengthen it. Moreover, the hold of neo-liberalism over key policy-makers and institutions cannot be underestimated. It may be that the leadership of the Reserve Bank displays greater heterodoxy than has been the case in recent years, but the staff cadre of both Treasury and the Reserve Bank is, in general, inured to orthodoxy by training and experience. New Zealand Journal of Employment Relations, 45(1): 47-58 New Zealand Journal of Employment Relations, 45(1): 47-58 at some point, all major parties in parliament seeing some merit in its future. In sum, in the binary clash in train, an effective alternative to the resurgence of neo-liberalism “on steroids” is a challenge. at some point, all major parties in parliament seeing some merit in its future. In sum, in the binary clash in train, an effective alternative to the resurgence of neo-liberalism “on steroids” is a challenge. One should also note that the challenge is made all the more difficult because, in practical political terms, it may be better to pretend that the accommodation per se does not exist, and, instead, seek coherent, long-term change by means of apparently unrelated, contingent measures, which over time aggregate to the required outcome. This “change by stealth” approach mirrors the way the 1930s Accommodation was introduced, but differs in two ways. The first has already been mentioned – the contemporary undiminished power of neo-liberal orthodoxy. The second is more prosaic. Reading contemporary documents about Labour-in- power from 1935, there was a simple contingency at play. Labour did not innovate with reference to a guiding blueprint, for, then, there was no Keynesian Accommodation. Equally, Treasury was less ideologically driven and more open to supporting what seemed to work, and inter-party political antipathies under first-past-the-post arrangements were less pronounced and more amenable to consensual “national” solutions. The Longer Term: a New Accommodation or Neo-Liberalism “on Steroids” From a political and policy perspective, this poses a binary choice for government. The approach to rebuilding of national and global economies will be a contest between a neo- liberalism arguing for “more market” solutions, especially where government debt has grown massively. Pressure will be exerted to reduce that debt by cuts in public expenditure. The anticipated size of that debt will tend to inflate the proposed cuts. The dislocation faced by the business sector as a result of Covid-19 will be seen as a rationale to promote efficiency and competition, which in New Zealand has, since the 1980s, translated into a real decline in the labour share. The implications of this outcome for employment relations and labour market dynamics need little exposition. Hence, the striking question for the future of economic and social development in New Zealand, and particularly for its employment relation system, is whether or not there is a commitment by government to an alternative to neo-liberal orthodoxy. One cannot stress enough the challenges involved in making that commitment. There are five challenges that arise immediately. First, it requires the intellectual foundation able to challenge nearly a half century of neo-liberal orthodoxy. Second, it requires the political will to challenge that orthodoxy and, also steer an alternative through the democratic process. And MMP is a model for compromise, not radical change. Third, populist voices broadly consistent with neo-liberal orthodoxy are, in the era of social media and opinion-based journalism, powerful opponents. Fourth, it requires important sectors of Capital to see the value in a new accommodation. Fifth, it will require several consecutive terms in government to establish a new accommodation, and, 51 A New Accommodation: Key Elements The idea of a comprehensive, indeed radical, overhaul of economic thinking in New Zealand has emerged as a result of Covid-19 (McCrone, 2020). Here are, laid out simply, the six key elements of an alternative to neo-liberalism, before considering their implications for employment relations. Figure 1 The components of accommodation. One might agree that we are in an epoch in which economic recovery can no longer be considered outwith its environmental context. For now, let us accept that any alternative economic settings must conform to environmental standards that sustain life on Earth. The implications of this commitment permeate production, consumption and trade. Environment Taxation High Performance Tripartism Collective Bargaining Trade Figure 1 The components of accommodation. One might agree that we are in an epoch in which economic recovery can no longer be considered outwith its environmental context. For now, let us accept that any alternative economic settings must conform to environmental standards that sustain life on Earth. The implications of this commitment permeate production, consumption and trade. One might agree that we are in an epoch in which economic recovery can no longer be considered outwith its environmental context. For now, let us accept that any alternative economic settings must conform to environmental standards that sustain life on Earth. The implications of this commitment permeate production, consumption and trade. 52 New Zealand Journal of Employment Relations, 45(1): 47-58 New Zealand Journal of Employment Relations, 45(1): 47-58 The final building block is high performance work and a wider suite of productivity-related measures, involving up-skilling in the delivery of both goods and services, and support from training and active labour market policies. The final building block is high performance work and a wider suite of productivity-related measures, involving up-skilling in the delivery of both goods and services, and support from training and active labour market policies. Underpinning these key elements of an accommodation would be other macroeconomic measures that, particularly, deal with the accumulated debt created by Covid-19 related government expenditures. Contemporary heterodox economic thinking on policy responses to the long-term adverse consequences of the pandemic takes many forms, but is broadly united in rejecting neo-liberal arguments for austerity and “small government” responses. For example, the economic consequences of Covid-19 have given life to the debate around Modern Monetary Theory, which inverts much contemporary economic thinking, and makes a strong case for increased government spending whilst there is under-used economic capacity and unemployment. In the same vein, Adair Turner (2016) has argued strongly for quantitative easing based on direct transfers to private or government consumption (rather than into private sector financial or commercial operations). Such arguments are challenging, yet demand attention in a situation in which economic orthodoxy appears increasingly ineffectual. New Zealand Journal of Employment Relations, 45(1): 47-58 New Zealand Journal of Employment Relations, 45(1): 47-58 Wealth and taxation are a second fundamental focus of a new accommodation. Growth in wealth inequalities, especially since the early 1970s, has been noted by a shift from Labour to Capital in the Capital-Labour share data. Figure 2: New Zealand Wage and Salary Share of GDP compared to OECD (Rosenberg, 2018) Figure 2: New Zealand Wage and Salary Share of GDP compared to OECD (Rosenberg, 2018) Wealth inequality not only represents a growing unfairness in rewards distribution, but also may threaten social cohesion in potentially dangerous fashion. A sustainable accommodation would include taxation arrangements that reverse the post-1970s oncome advantage assumed by Capital. Wealth taxes, carefully positioned, are the obvious means to achieve this rebalancing. Tripartism is a fundamental aspect of any accommodation. It transcends bargaining arrangements, in contemporary terms now involving partnership from the enterprise to national economic strategy, as well as its extension into social and trade settings. Important is the reassertion of collective bargaining – traditional and innovative - to rebalance rewards to Capital and Labour. The Employment Contracts Act (ECA) triggered the shift of rewards from Labour to Capital seen in the 1990s, a shift that has not been rectified since (see later discussion). This is why the standing commitment to Fair Pay Agreements, and the anticipation of their enactment, are important. Just as the Keynesian Accommodation of the 1930s was intimately linked to a commitment to growth in trade, so must a modern accommodation be associated with a continued commitment to fair trade. Fairness in trade is a powerful modern condition of acceptable trade relations, as is trade conducted to appropriate environmental standards. 53 New Zealand Journal of Employment Relations, 45(1): 47-58 level with employers and employer associations, but has not taken advantage of the opportunity for strategic tripartite arrangements. In this situation, the reemergence of tripartism as an effective form of decision-making and enforcement depends on government taking a strong lead. Again, the proposed Industry Transformation Plans are a promising initiative as is the Future of Work programme. The reemergence of collective bargaining, central to a new accommodation, should be considered in conjunction with a complementary focus on a wider industrial democracy. As mentioned previously, Labour has a model for reformed employment relations in the Fair Pay Agreements, but it has found it difficult to progress this initiative. This blockage has to be overcome if an accommodation is to be achieved. However, such agreements are perhaps a necessary but not sufficient condition for a sustainable accommodation. There is an opportunity, even necessity, to develop appropriate forms of industrial democracy, in particular to embed necessary high performance production arrangements. New Zealand’s dalliance with partnership since the early 2000s has been unconvincing. Clearly, a renewed interest in industrial democracy would have to be fostered energetically, in the context of an extant employment relations model with little previous experience of modern, high-performing industrial democracy. A renewed focus on industrial democracy and workplace voice speaks also to the need for continuing support for workplace reform. High performance initiatives in New Zealand have been attempted for at least two decades, with Labour-led governments taking the lead with the creation of agencies such as the Partnership Resource Centre. Since 2016, Labour currently has had a central policy theme in the “Future of Work”, with an associated Future of Work Tripartite Forum (NZ Treasury, 2020b). This workstream has been sustained since 2017, but impacts consequent on its outputs are more difficult to determine. A post-Covid-19 recovery strategy would, one suggests, place renewed emphasis on high performance systems, if only because of the trade advantages they would foster. There is much to be done in the area of labour market development. It is trite to restate the importance of high-quality training arrangements, and their integration with the compulsory education system. However, given the importance of “green transformation” and “Just Transition” and the emphasis on high-performance work arrangements, active labour market policies associated with multiple pathways into training and retraining will be vital. Tripartism should provide a supportive platform to support the creation of those pathways. A New Accommodation: Implications for Employment Relations The implications of a new accommodation for employment relations and labour market policy are many, and here I will discuss only some of the most salient. Government measures taken post-2017 in relation to Taranaki and its energy sector illustrate the steps along the “green transformation” path, which all modern economies are considering to a greater or lesser extent. Similarly, new approaches to rural land-use are seeking to deal with the politically-sensitive emissions issues associated with rural production. The trade union movement has committed to a “Just Transition” approach to climate issues (Huggard, 2019). Powerful voices within the business community are also speaking clearly about climate change (see, for example, the Sustainable Business Council, 2020). At the heart of this growing pressure for change is the reconfiguration of production, distribution and consumption practices in tune with sustainability goals. This carries with it major long-term implications for investment decisions, production and distribution systems, local and national labour markets, training provisions, work organisation practices and accountabilities. A positive initiative is the proposed industry policy with its commitment to Industry Transformation Plans (MBIE, 2020). These plans are relevant to the subsequent discussion of tripartism and industrial democracy. The magnitude of this challenge, and an attached urgency, confronts the tripartite parties inescapably. One must be concerned about the current scope and quality of thinking about these issues. Taking into account the reducing timeframes for effective change predicted by science, one anticipates increased urgency and application in this policy area. Central to a new accommodation is a renewed commitment to tripartism. This is a looming challenge, for tripartite institutions and practices in New Zealand have been sorely weakened since the 1980s. The three parties are each, in their different ways, inexperienced and tentative about renewed tripartite arrangements. The relative weakness of the trade union movement since the ECA was introduced challenges its authority as a tripartite party, as do some forces within the CTU membership, which are at best chary of such engagement. Employer associations are both inexperienced in and questioning of tripartism. The Labour-led government maintains cordial relationships with the CTU, and engages consistently at high 54 New Zealand Journal of Employment Relations, 45(1): 47-58 What is to be Done? It falls to government to manage both the short-term impacts of Covid-19, in themselves oppressive, whilst also charting the long-term recovery for New Zealand. It is a daunting task, made all the more challenging by little political consensus about how that course should be laid. Unity in recognising the Covid-19 challenge is not immediately reflected in agreed ways forward. Aspects of that course are less contentious. For example, most agree that a renewed focus on trade is a vital component of recovery. But not only is there little agreement about a new accommodation or an alternative thereto, there is also little public debate to date about the options for the future. Government has publicly recognised the need for a long-term response, as it has also highlighted the crushing public debt that is being accrued. Details of that response are sparse. Political savants will have marked the absence of Labour’s usual election manifesto, which might normally be expected to give some clues about Labour’s longer-term thinking. That said, the magnitude of the Covid-19 crisis is such that government might reasonably expect some flexibility in demands made of it. Will a new version of the 1930s Keynesian Accommodation emerge? Much depends on four factors – the will of government, voter choice, employer commitment and trade union recovery. These factors interplay in the difficult context of contemporary populism and weakened liberal democracy. Government will be the first mover on a new accommodation. Its support is fundamental in terms of leadership, resources and political authority. The current government has manifested strong leadership and authority across several crises, and, in the context of its Covid-19 response, has released significant funding. That strength of response, impressive as it is, has still to be translated into a post-Covid-19 vision, in which the vision of an accommodation, explicit or otherwise, is lodged. Noting a point already made, that is, too explicit a formulation of a road to accommodation may threaten politically its emergence, the jury is out on the strategic direction the government is plotting for the next three and more years. However, a note of concern has been sounded above. Accommodation requires strong tripartism and a firm political will to move away from the binary option of intensified neo- liberalism. I do not discount the impact of core civil service ideological preferences, more confident and empowered than, for example, in the 1930s. New Zealand Journal of Employment Relations, 45(1): 47-58 Sovereign states have the responsibility to manage immigration policies in an open and transparent fashion. Reasonably, these policies must enjoy the confidence of the community. This becomes more complex and challenging in periods of economic distress, especially in terms of labour market disruption. Governments must strike a balance between active labour market policies to promote the engagement of domestic labour, and the continued use of migrant labour. Some forms of migrant labour – for example, that which is associated with long-established seasonal work and/or with an aid component – may be unscathed by post- Covid-19 arrangements. Equally, constraints on general migration may be appropriate when major labour market disruption arises. Effective planning – perhaps through Industry Transformation Plans – coupled with an unequivocal, transparent statement of the rationale for regulation are important. 55 New Zealand Journal of Employment Relations, 45(1): 47-58 New Zealand Journal of Employment Relations, 45(1): 47-58 New Zealand Journal of Employment Relations, 45(1): 47-58 Business New Zealand, regional employer organisations and Chambers of Commerce preferences. Trades unions have not recovered from the impact of the ECA. Union density remains at 18 per cent, and is centred in large private sector organisations and in the state sector. In this sense, union voice is doubly unbalanced – in terms of the vast layers of unorganized small and medium enterprises (SMEs) in the New Zealand economy, and in terms of the growth of state sector unions versus the flat-lining of private sector voice. The CTU remains a strong nd creative voice, but, Janus-like, must face inwards to the internal pressures within the union movement, and outwards to the challenges facing New Zealand, whilst managing on a modest budget. In sum, a new accommodation would require the Covid-19 crisis to galvanise both employer and trades union partners into, in the case of the former, very different, pluralist thinking about the oversight of New Zealand’s future, and, in the case of the latter, significantly greater coherence in terms of industrial democracy, broadly defined. One might speculate on the potential role of government in driving those changes. The other important condition is the political process and voter behaviour, in a context in which liberal democracy’s hold over the voter is weakened, in part because of new technology’s capacity to “manage” voter behavior, in part because of a cynical denigration of the liberal democratic process under neo-liberalism. The short three-year electoral cycle plays against the establishment of a new accommodation, requiring a Labour-led government to be in power for at least two further cycles. In a period of instability such as we face, and where Labour’s prominence is markedly dependent on its leader, that requirement is not guaranteed. One should also recognise that the vagaries of MMP may intervene to qualify or derail radical policy directions. What is to be Done? Labour, the dominant party in the government coalition, has in recent decades moved to the political centre from its founding democratic socialist principles. If it has, indeed, become a party of social identity, then it is unlikely that an historic accommodation is imagined. This points unerringly to the stark option facing Labour. It has been both blessed and cursed to be in power in an epochal crisis. To it falls the choice of effective transformational change or the status quo as it plans for the next decade or more. Were that government choice to be truly transformational, then the role of the social partners comes into play. Overall, this is also a challenging prospect. In general, employer associations have for 30 years or more eschewed tripartism as a fundamental economic and social building block. When Labour is in power, a nod to tripartism is usually offered, but that commitment is easily parked when National is in power. There are individual (large) employers, and some employer associations (e.g. in the environmental context) who are more open to tripartism and partnership. I suggest that this is a minority presence in comparison with the general tenor of 56 New Zealand Journal of Employment Relations, 45(1): 47-58 A Final Word The Covid-19 pandemic has delivered, internationally, a body blow to communities and nations. We are still to experience the full extent of its harm. Commentaries already speak of a decade of recovery. New Zealand has, to date, responded successfully to Covid-19’s immediate depredations and must chart its course through an extended recovery phase. This commentary argues, first, that there is a powerful alternative to a return to neo-liberalism available to government and policy makers. That alternative rests on a new, domestic accommodation binding together the social partners. A modern accommodation will be driven by the same motivations as that of the 1930s, but will take a different form, primarily because of the impact of climate change. Second, if that alternative is to be realised, it will be driven by a government committed to fundamental transformation, and willing to forge the bonds that unite the tripartite parties in that endeavor. 57 New Zealand Journal of Employment Relations, 45(1): 47-58 Turner, A. (2016). Between Debt and the Devil: Money, Credit and fixing Global Finance. Princeton University Press. References Huggard, S. (2019). CTU releases agenda to achieve a just transition. New Zealand Council of Trade Unions. https://www.union.org.nz/ctu-releases-agenda-to-achieve-a-just- transition/ McCrone, J. (2020). A kinder, greener, fairer economics to replace neoliberalism? Questioning New Zealand's chances of making the move. Stuff. https://www.stuff.co.nz/national/politics/122379921/a-kinder-greener-fairer- economics-to-replace-neoliberalism-questioning-new-zealands-chances-of-making- the-move MBIE (Ministry of Business, Innovation and Employment). (2020). Industry Policy. https://www.mbie.govt.nz/business-and-employment/economic- development/industry-policy/ NZ Treasury. (2020a). COVID-19 Economic Response Measures. https://www.treasury.govt.nz/information-and-services/new-zealand-economy/covid- 19-economic-response/measures NZ Treasury. (2020b). Future of Work Tripartite Forum. https://treasury.govt.nz/information- and-services/nz-economy/future-work-tripartite-forum NZ Work and Income. (2020), Covid-19. https://workandincome.govt.nz/covid-19/index.html Piketty, T. (2014). Capital in the Twenty-First Century. Harvard University Press. Rosenberg, B. (2018). Workers out of pocket $11500 a year as a result of decades of poor government policy. New Zealand Council of Trade Unions. https://www.union.org.nz/workers-out-of-pocket-11500-a-year-as-a-result-of- decades-of-poor-government-policy/ Sustainable Business Council. (2020). Pre-election Brief. https://www.sbc.org.nz/resources/reports/sbc-reports/pre-election-brief-2020 Turner, A. (2016). Between Debt and the Devil: Money, Credit and fixing Global Finance. Princeton University Press. 58
https://openalex.org/W3012937215	https://europepmc.org/articles/pmc7049176?pdf=render	English	null	Psychometric properties and factor structure of the Korean version of the screen for child anxiety related emotional disorders (SCARED)	BMC psychiatry	2,020	cc-by	9,847	© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. 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Open Access Psychometric properties and factor structure of the Korean version of the screen for child anxiety related emotional disorders (SCARED) Jiyoon Shin1, Kyoung Min Kim2, Kyung Hwa Lee1, Soon-Beom Hong1, Jung Lee3, Chi-Hyun Choi4, Ji Youn Han1, Seong Hae Kim1, Da Eun Suh1, Soo-Churl Cho5 and Jae-Won Kim1* Abstract Background: The aim of this study was to examine the psychometric properties of the Korean version of Screen for Child Anxiety Related Emotional Disorders (SCARED) on a sample of Korean youths and to examine the cross- cultural differences in adolescents’ anxiety. Methods: Our study included 147 adolescents (ages 12–17, 92 girls), 93 with major depressive disorder and 54 as controls. Participants were evaluated using the Kiddie-Schedule for Affective Disorders and Schizophrenia for School-Age Children-Present and Lifetime Version (K-SADS-PL), SCARED, Child Behavior Checklist (CBCL), Disruptive Behavioral Disorder Scale (DBD) and Attention Deficit Hyperactivity Disorder Rating Scale (ADHD-RS). Pearson’s r and Cronbach’s α values of the SCARED were calculated, and exploratory factor analysis was conducted. Results: The Korean SCARED scores were correlated with the total anxiety scores of K-SADS-PL (r = 0.74) and the CBCL anxious/depressed subscale scores (r = 0.35). Results showed a five-factor structure with good internal consistency, in which some items were loaded on different factors compared to previous studies. Conclusions: The Korean SCARED demonstrated promising psychometric properties, and could be a valid scale for screening anxiety symptoms in primary care. The fact that different items comprised the factors may reflect the cultural difference between United States and Korea in experiencing anxiety. Keywords: SCARED, Validity, Reliability, Factor structures, Anxiety disorder [1]. In Korean children and adolescents aged 6 to 18, the one-year prevalence rate of anxiety disorder was 11.7% [2]. Another study of Korean children aged 6 to 12 showed that the past-year prevalence of anxiety disor- ders was 10.3% [3]. Despite these high prevalence rates, anxiety disorders are often underdiagnosed and remain untreated for the following reasons: first, they are not as evident as behavioral disorders [4]; second, they are fre- quently comorbid with other psychiatric disorders that gain more clinical attention, such as depressive disorders and substance abuse [5, 6]. Background Anxiety disorders are the most prevalent psychiatric dis- orders in children and adolescents worldwide. In the United States, the point prevalence rate of adolescents’ anxiety disorders based on DSM-IV diagnosis was 15% and the 12-month prevalence rate was as high as 25% * Correspondence: snuh.may@gmail.com 1Division of Child and Adolescent Psychiatry, Department of Psychiatry, Seoul National University College of Medicine, 103 Daehak-Ro, Chongno-Gu, Seoul 03080, Republic of Korea Full list of author information is available at the end of the article * Correspondence: snuh.may@gmail.com 1Division of Child and Adolescent Psychiatry, Department of Psychiatry, Seoul National University College of Medicine, 103 Daehak-Ro, Chongno-Gu, Seoul 03080, Republic of Korea Full list of author information is available at the end of the article Shin et al. BMC Psychiatry (2020) 20:89 https://doi.org/10.1186/s12888-020-02505-3 Shin et al. BMC Psychiatry (2020) 20:89 https://doi.org/10.1186/s12888-020-02505-3 * Correspondence: snuh.may@gmail.com 1Division of Child and Adolescent Psychiatry, Department of Psychiatry, Seoul National University College of Medicine, 103 Daehak-Ro, Chongno-Gu, Seoul 03080, Republic of Korea Full list of author information is available at the end of the article Page 2 of 12 Shin et al. BMC Psychiatry (2020) 20:89 Page 2 of 12 Shin et al. BMC Psychiatry (2020) 20:89 Korean version of the SCARED to assist better diagnosis and treatment of anxiety disorders in Korean youth. In South Korea, there is a cultural emphasis on aca- demic achievement [7], which creates an excessive aca- demic burden, an overly competitive atmosphere, and university entrance-oriented education [8, 9]. As a result, adolescents’ anxiety and depression tend to increase as the grade level increases in schools [8–10]. According to a report by the Korean National Youth Policy Institute, 20.9% of Korean youth have thought about committing suicide in one year mainly due to academic stress and anxiety [11]. Anxiety is known to be an important pre- dictive factor for suicidality [10]. Thus, early detection and intervention of Korean youths’ anxiety disorders are important, considering the severity and consequences of these disorders. Due to an historical background of Confucianism, there exists a strong sense of community in South Korea [21, 22]. Children are often brought up to be considerate of others and to be attentive to how they are viewed by others, a character that is called ‘Nun-chi’ in Korean [22, 23]. In social atmospheres as seen in Korea and Japan, there is a culture-specific social fear regarding concern at offending others, called ‘Taein Kongpo’ in Korean, or ‘Taijin Kyofusho’ in Japanese [24, 25]. In a study of Ko- rean middle and high school students, 13% of middle school students and 14.8% of high school students thought that they would cause harm to others because of their social anxiety [23]. Children and adolescents’ ex- cessive social anxiety cause difficulties in peer relation- ships and academic ability, school refusal, abandonment of school activities, and deterioration of their psycho- social functions [26, 27]. Due to the cultural specificity of Korea, it is possible that the factor structure of the Korean version of the SCARED might differ from that of the English SCARED. Both the Korean versions of the State-Trait Anxiety Inventory (STAIC) and the Revised Children’s Manifest Anxiety Scale (RCMAS) measure youth anxiety. The STAIC and RCMAS are used to evaluate anxiety in vari- ous aspects. The STAIC includes both the state and trait scales. While the state scale measures the current feeling of anxiety, the trait scale measures a more stable and pervasive tendency to experience anxiety. The three sub- scales of RCMAS assess worry/oversensitivity, fear/con- centration, and physiological anxiety. Although the STAIC and RCMAS measure general anxiety symptoms, both have limitations. First, they do not provide infor- mation regarding specific anxiety disorders. Second, they have some limitations regarding discriminant validity such that they did not distinguish definitely anxiety dis- orders from behavioral disorders such as ADHD [12]. In this study, we aimed to establish the reliability and validity of the Korean SCARED and investigated the fac- tor structure of the scale. We hypothesized that the Ko- rean SCARED would be a valid and reliable scale to assess anxiety symptoms in Korean youth. In addition, we hypothesized that the cross-cultural differences in ex- periencing social anxiety would be reflected in the factor structure of the Korean SCARED. The Screen for Child Anxiety Related Emotional Disor- ders (SCARED) is a screening questionnaire for anxiety disorders in youth. It is a rating scale completed by chil- dren or parents. Although it has been used in some clin- ical studies conducted in Korea [13, 14], the psychometric properties of the Korean version are yet to be determined. The scale has several advantages compared to other anx- iety scales mentioned above. First, the SCARED can assess specific anxiety disorders in addition to assessing general anxiety symptoms. Second, it can be applied to a wider age range of youth, from 6 to 19 years [15, 16], compared to the STAIC, which is indicated for youth ages 9–12. Third, the English SCARED shows good psychometric properties, specifically in terms of discriminant validity, and can differentiate children with an anxiety disorder from depression or disruptive disorder-only groups [17]. Internal consistency and factor structure were also shown to be robust when applied to different cultures and races, including European countries, South Africa, China, non- Hispanic White and African Americans, and Arabia [18, 19]. In addition, the SCARED is a self-report measure, which is more practical compared to diagnostic clinical in- terviews [20]. Given such advantages of the SCARED, it is meaningful to examine the psychometric properties of the Assessments At baseline, all participants were assessed using the K- SADS-PL, SCARED, Child Behavior Checklist (CBCL), Disruptive Behavioral Disorder Scale (DBD) and ADHD Rating Scale (ADHD-RS). At baseline, all participants were assessed using the K- SADS-PL, SCARED, Child Behavior Checklist (CBCL), Disruptive Behavioral Disorder Scale (DBD) and ADHD Rating Scale (ADHD-RS). g The SCARED is a 41-item self-report that screens for anxiety disorders and can be completed by child and parent. In our study, the SCARED child version was used. In a validation study of the Korean SCARED, the researcher team translated the original SCARED items into Korean, back-translated them to English by a bilin- gual researcher, and re-checked the back-translated ver- sion. Finally, the scale was confirmed by a researcher with Ph.D. in clinical psychology [33]. Our sample size satisfied the minimum required sam- ple size for a factor analysis [38], which allowed us to conduct factor analysis to investigate the factor structure of the Korean SCARED. Since the data was not normally distributed [39], principal axis factoring was used to ex- tract factors. Scree test was used to decide the number of factors. Lastly, promax rotation, a method used for analyzing correlated factors [39], was performed to de- termine the factor structure. All primary data were ana- lyzed with SPSS version 22.0. During assessment, a psychiatrist (J.-W.K) who was blind to the SCARED scores administered the K-SADS- PL to the participants and their parents. To investigate the correlation between the SCARED and K-SADS-PL anxiety symptoms, we calculated a total score by adding up the following subscale scores as per screen section: panic disorder (1 item), separation anxiety disorder (5 items), social phobia (2 items) and generalized anxiety disorder (4 items). Finally, a sensitivity analysis was conducted to assess the robustness and reproducibility of the factor structure we elicited, using an independent dataset. From the ‘Valid- ation of depression-related scales in child and adolescent psychiatric outpatients’ study (IRB No. 1908–088-1055), the sample consisted of 464 participants (278 of whom were girls) aged 7 to 19, recruited from the child and ado- lescent psychiatric outpatient clinic of Seoul National Uni- versity Hospital between August 2015 and July 2019. Inclusion criteria were to have completed a semi- structured interview and the same questionnaires as men- tioned above in the Assessment section for the intake as- sessment. Demographic and clinical data were collected in the same manner. Methods Participants Our study included 95 depressed participants between the ages of 12–17 years. They were part of a study that investigated the biomarkers of the antidepressant re- sponse and suicidal events in depressed youth (NRF- 2015R1A2A2A01004501). Major depressive disorder (MDD) diagnoses were made using the Kiddie-Schedule for Affective Disorders and Schizophrenia for School- Age Children-Present and Lifetime Version (K-SADS- PL) [7, 28, 29], which is based on Diagnostic and Statis- tical Manual of Mental Disorders-5th Edition (DSM-5) [30] criteria. The participants were recruited from the child and adolescent psychiatric outpatient clinic at the Seoul National University Hospital between August 2015 and June 2018. Participants were included in the study if they were di- agnosed with MDD, had symptoms lasting for at least 4 weeks or more without psychotic features, scored 40 or higher on the Children’s Depression Rating Scale- Revised (CDRS-R) [31], and scored 4 or higher points on the Clinical Global Impression-Severity scale [32]. Exclu- sion criteria were intelligence quotient (IQ) under 70, a Page 3 of 12 Shin et al. BMC Psychiatry (2020) 20:89 Shin et al. BMC Psychiatry (2020) 20:89 items), ODD (8 items) and conduct disorder (CD) (15 items) symptoms [35, 36]. In our study, we used the CD subscale to assess behavioral symptoms only. The ODD subscale also shows behavioral symptoms, however, they might not be prominent to the parents if the behavioral problems are only confined to the school or outside the home. Thus, we selected CD subscale, which would be more evident to parents. Each item is rated on a 4-point Likert scale. The ADHD-RS is an 18-item self-report used to screen for ADHD [37]. The scale measures two features of ADHD: inattention and hyperactivity- impulsivity. Each item is rated on a 4-point Likert scale. The ADHD-RS is known to specifically evaluate ADHD symptoms, which can be used to screen, diagnose, and evaluate the effect of treatment for ADHD [37]. For this reason, we chose the ADHD-RS over the ADHD sub- scales of the CBCL and the DBD. history of chronic medical and neurological diseases, diagnosis of psychotic disorders such as schizophrenia, diagnosis of bipolar disorders and developmental disor- ders such as autism, substance abuse within the past 6 months, a diagnosis of bipolar I disorder among immedi- ate relatives, and taking any psychiatric medication. To- tally, 57 healthy controls were recruited via school flyers. Statistical analyses Demographic variables were analyzed using ANOVA or Chi-square tests. To examine the criterion and discrim- inant validity of the SCARED, Pearson’s correlation coef- ficients were measured. ANOVA was used to compare the clinical variables among anxiety + depression (A + D), depression only (D only) and control groups. A Tukey’s honest significance test or Dunnett T3, depend- ing on whether the equality of variances was met or not, was done as a post hoc analysis, to examine the differ- ences in the Korean SCARED total and subscale scores among A + D, D only and control groups. Methods Participants They visited our clinic and were initially screened using the K-SADS-PL. Among the initially recruited 152 subjects, 2 depressed participants and 3 healthy controls withdrew their par- ticipation consent and therefore were excluded from the study. Thus, a total of 147 subjects (92 of which were girls) were included in the analyses; these were divided into depression and anxiety comorbid (n = 44), depres- sion only (n = 49) and control (n = 54) groups based on the psychiatric diagnoses from the K-SADS-PL. The study was approved by the Institutional Review Board for Human Subjects of Seoul National University Hospital. All the parents and youth were provided with detailed information about the study. Written informed consent was obtained from all participants before enter- ing the study. Criterion validity y As shown in Table 2, the Korean SCARED showed sig- nificant correlation with the K-SADS-PL anxiety sub- scale total scores (r = 0.74, p < 0.001). We calculated a total score of the K-SADS-PL anxiety subscales by sum- ming up panic disorder (PD), Separation Anxiety Dis- order (SAD), social phobia (SP), and generalized anxiety disorder (GAD) subscale scores. The Korean SCARED scores were correlated with all K-SADS-PL anxiety sub- scales (rs > 0.214, p-values ≤0.009), of which the GAD Assessments Among the sample, subjects included in Parents completed the CBCL, DBD, and ADHD-RS. The CBCL is a 113-item scale measuring six DSM ori- ented scales- affective, anxiety, ADHD, conduct, oppos- itional defiant disorder (ODD), and somatic problems [34]. In our study, we used the anxious/depressed sub- scale, internalizing and externalizing scales of CBCL. The DBD is a 41-item scale used to measure ADHD (18 Page 4 of 12 Shin et al. BMC Psychiatry (2020) 20:89 groups in scores on the Korean SCARED, CBCL intern- alizing scales and the K-SADS-PL anxiety subscale. Post hoc analyses revealed that CBCL anxious/depressed, CBCL externalizing scales, ADHD and CD scores dif- fered significantly between the D only and control groups, but not between the A + D and D only groups (Table 1). the primary factor analyses were excluded. After applying the inclusion and exclusion criteria, the sample consisted of 431 participants (257 of whom were girls) aged 7 to 19. Among them, 262 were diagnosed with MDD, 49 with bi- polar disorder, 69 with anxiety disorder, 11 with ADHD, 5 with conduct disorder, 2 with tic disorder, 3 with autism, and 29 with eating disorder. One participant had no psy- chiatric disorder. Confirmatory factor analysis (CFA) using the maximum-likelihood method was performed on the independent dataset. CFA was conducted with R version 1.2.5019, Lavaan package and internal consistencies were calculated with SPSS version 22.0. Demographics and clinical characteristics No significant difference was found in age, gender and IQ between the A + D, D only, and control groups. How- ever, there were significant differences between three Table 1 Demographic and clinical characteristics of primary dataset § Anxiety + Depression (n = 44) §§ Depression only (n = 4 Table 1 Demographic and clinical characteristics of primary dataset § Anxiety + Depression (n = 44) §§ Depression only (n = 49) Control (n = 54) statistics p-value Gender, n (%) χ2 = 1.88 0.39 Male 14 (31.8) 17 (34.7) 24 (44.4) Female 30 (68.2) 32 (65.3) 30 (55.6) Age, mean (SD) 14.8 (1.7) 14.7 (1.6) 14.4 (1.6) F = 0.80 0.45 IQ, mean (SD) 104.6 (14.0) 106.1 (14.2) 108.5 (11.6) F = 1.09 0.34 Anxiety diagnosis Social phobia 15 (34.1) Agoraphobia 3 (6.8) GAD 28 (63.6) Panic 5 (11.4) Separation anxiety 3 (6.8) PTSD 4 (9.1) Specific phobia 1 (2.3) †K-SADS-PL_anxiety total 21.0 (4.0) 16.3 (2.4) 12.7 (1.6) F = 105.47 < 0.001 †SCARED 43.6 (15.1) 32.4 (14.9) 10.6 (9.7) F = 79.17 < 0.001 CBCL †CBCL_anx/dep 58.8 (16.5) 56.2 (13.3) 43.9 (10.6) F = 17.62 < 0.001 †CBCL_ext 50.1 (11.8) 50.3 (12.7) 41.8 (9.6) F = 9.40 < 0.001 †CBCL_int 67.4 (8.7) 62.0 (9.8) 45.0 (10.5) F = 72.27 < 0.001 †ADHD 8.9 (8.4) 12.1 (11.4) 3.7 (5.2) F = 12.40 < 0.001 †CD 1.8 (3.0) 2.5 (4.0) 0.4 (0.9) F = 7.01 0.001 § Anxiety + Depression: participants with co-morbid anxiety disorders and MDD §§ Depression only: participants with MDD only GAD generalized anxiety disorder; PTSD post traumatic stress disorder SCARED The Screen for Child Anxiety Related Emotional Disorders K-SADS-PL Kiddie-Schedule for Affective Disorders and Schizophrenia-Present and Lifetime version CBCL Child Behavior Checklist CBCL_anx/dep CBCL anxious/depressed subscale CBCL ext CBCL externalizing scale Shin et al. BMC Psychiatry Page 5 of 12 Shin et al. Factor structure Factor analysis showed that the Korean version of the SCARED was comprised of five factors, which reflected generalized anxiety (GAD), panic/somatic symptoms (PD), social anxiety (SP), separation anxiety (SAD) and school avoidance (SA) (Table 3). We determined the number of the factors using Cattell’s scree test, which di- rects the dropping of components after showing a gradi- ent decline in the scree plot. Our scree test suggested retention of five or six factors; thus, analyses were car- ried out to assess the interpretability and factor structure of both five- and six-factor structures. The six-factor so- lution resulted in a factor which contained only two var- iables, which did not satisfy the general principle that a Demographics and clinical characteristics BMC Psychiatry (2020) 20:89 Table 2 Correlations among SCARED, CBCL_anx/dep, CBCL_int, CBCL_ext, CD and ADHD SCARED K-SADS-PL_anxiety CBCL_anx/dep CBCL_int CBCL_ext CD ADHD SCARED 1 K-SADS-PL_anxiety .74 1 CBCL_anx/dep .35 .37 1 CBCL_int .57 .60 .78 1 CBCL_ext .12 .21* .68 .59 1 CD .10 .12 .25 .32 .48 1 ADHD .15 .14 .46 .49 .54 .55 1 p < 0.01, * p < 0.05 SCARED: The Screen for Child Anxiety Related Emotional Disorders K-SADS-PL: Kiddie-Schedule for Affective Disorders and Schizophrenia-Present and Lifetime version CBCL Child Behavior Checklist CBCL_anx/dep CBCL anxious/depressed subscale CBCL_ext CBCL externalizing scale CBCL_int CBCL internalizing scale ADHD Attention Deficit Hyperactivity Disorder Rating Scale (ADHD-RS) CD conduct disorder assessed by Disruptive Behavioral Disorder Scale (DBD) subscale (r = 0.75, p < 0.001) showed the strongest correlation. factor is more stable when it contains at least 3 items or more [39]. In addition, the factor did not yield any sig- nificant independent factor based on K-SADS-PL nor DSM-V [40]. Therefore, a five-factor structure was chosen for better interpretation [39]. Every item with the exception of item 4, which had a factor loading of 0.23, exceeded the factor loading of 0.30, the minimum re- quired loading of an item [41]. Two items, items 10 and 15, showed cross loading, which means that two or more items had factor loading of more than 0.32 [41], for the GAD factor (0.34 and 0.33, respectively) and the PD fac- tor (0.66 and 0.42, respectively). The explained variance of the factor structure was 58.9%. The Korean SCARED and CBCL anxious/depressed subscale also showed significant correlation, although it was relatively low (r = 0.35, p < 0.001). Discriminant validity The correlation between the Korean SCARED and the externalizing problem of CBCL was not significant (r = 0.13, p = 0.14). On the contrary, the Korean SCARED and the internalizing problem of CBCL showed moder- ate correlation (r = 0.57, p < 0.001). The Korean SCARED scores did not correlate with CD of the DBD (r = 0.29, p = 0.25), as well as with the ADHD-RS (r = 0.15, p = 0.07) (Table 2). The GAD factor consisted of 12 items, and of these items, ‘I worry about people liking me’ had the highest factor loading (1.01). Factor loadings for all items ranged from 0.37 to 1.01. The PD factor consisted of 11 items, and the item ‘When I get frightened, I feel like I am choking’ scored the highest factor loading (0.99). Factor loadings for PD ranged from 0.42 to 0.99. The SP factor was composed of 8 items, and the factor loadings ranged from 0.23 to 0.93. The item ‘I feel shy with people I don’t know well’ showed the highest factor loading. SAD included 6 items and their factor loadings ranged from 0.53 to 0.79. ‘I am afraid to be alone in the house’ showed the greatest factor loading. The SA factor was composed of 4 items with factor loadings of these items ranging from 0.36 to 0.90. (Table 3). Internal consistency As shown in Table 3, internal consistencies of five fac- tors (for details, see 5. Factor structure) ranged from 0.89 to 0.94. Overall internal consistency was good (Cronbach’s α =0.96). Comparison of SCARED scores between anxiety group and non-anxiety groups The comparison of the SCARED scores based on our five-factor structures among the A + D, D only and con- trol groups is presented in Table 4. There were signifi- cant group differences in the Korean SCARED scores for generalized anxiety, social anxiety, separation anxiety Page 6 of 12 Shin et al. BMC Psychiatry (2020) 20:89 Table 3 Factor loadings and factor internal consistency (N = 147) Factor Item Factor loading Cronbach α Generalized anxiety 0.91 5 I worry about other people liking me 1.01 0.90 21 I worry about things working out for me 0.83 0.90 33 I worry about what is going to happen in the future 0.80 0.90 14 I worry about being as good as other kids 0.78 0.90 23 I am a worrier 0.78 0.90 28 People tell me that I worry too much 0.74 0.90 35 I worry about how well I do things 0.74 0.90 37 I worry about things that have already happened 0.73 0.90 20 I have nightmares about something bad happening to me 0.61 0.91 9 People tell me I look nervous 0.55 0.91 7 I am nervous 0.49 0.91 19 I get shaky 0.37 0.91 Panic/somatic symptoms 0.89 27 When I get frightened, I feel like I am choking 0.99 0.86 6 When I get frightened, I feel like passing out 0.83 0.90 12 When I get frightened, I feel like I am going crazy. 0.83 0..86 1 When I feel frightened, it is hard to breathe 0.82 0.86 38 When I get frightened, I feel dizzy. 0.73 0.86 34 When I get frightened, I feel like throwing up 0.69 0.86 24 I get really frightened for no reason at all 0.64 0.87 30 I am afraid of having anxiety (or panic) attacks 0.62 0.87 18 When I get frightened, my heart beats fast 0.47 0.87 22 When I get frightened, I sweat a lot. 0.46 0.87 15 When I get frightened, I feel like things are not real. Comparison of SCARED scores between anxiety group and non-anxiety groups 0.42 0.87 Social anxiety 0.88 32 I feel shy with people I don’t know well 0.93 0.87 26 It is hard for me to talk with people I don’t know well 0.83 0.87 41 I am shy 0.80 0.87 3 I don’t like to be with people I don’t know well 0.69 0.87 40 I feel nervous about going to parties, dances, or any place where there will be people that I don’t know well 0.67 0.86 10 I feel nervous with people I don’t know well 0.66 0.86 39 I feel nervous when I am with other children or adults and I have to do something while they watch me (e.g. read aloud, speak, play a game, play a sport) 0.53 0.88 4 I get scared if I sleep away from home 0.23 0.89 Separation anxiety 0.78 25 I am afraid to be alone in the house 0.79 0.73 29 I don’t like to be away from my family 0.74 0.74 13 I worry about sleeping alone 0.72 0.73 16 I have nightmares about something bad happening to my parents 0.67 0.76 31 I worry that something bad might happen to my parents 0.64 0.74 Shin et al. BMC Psychiatry (2020) 20:89 Page 7 of 12 Page 7 of 12 Shin et al. BMC Psychiatry (2020) 20:89 Table 3 Factor loadings and factor internal consistency (N = 147) (Continued) Factor Item Factor loading Cronbach’s α 8 I follow my mother or father wherever they go 0.53 0.75 School avoidance 0.86 36 I am scared to go to school 0.90 0.78 17 I worry about going to school 0.88 0.78 2 I get headaches when I am at school 0.40 0.83 11 I get stomachaches at school 0.36 0.87 Total Cronbach’s alpha 0.95 p < 0.01 and school avoidance. The scores of these factors were in the following order: A + D, D only, and control groups. However, A + D and D only group did not show any differences in scores of panic symptoms. English SCARED done in clinical samples, in which Cronbach’s α of subscales ranged from 0.79 to 0.89 [6]. The Korean SCARED also demonstrated good criter- ion validity. The Korean SCARED total scores had a high correlation with K-SADS-PL anxiety subscales, and a moderate correlation with the CBCL anxious/de- pressed subscale and the CBCL internalizing scale. Comparison of SCARED scores between anxiety group and non-anxiety groups These findings may imply the discrepancies between the reports of the participants and their parents because the CBCL was completed by parents. Parents tend to under- report their children’s internalizing symptoms such as depression and anxiety compared to externalizing symp- toms because the latter are more evident to parents [44]. In addition, the CBCL anxious/depressed subscale not only measures anxiety, but also depressive symptoms, which may have contributed to the weakened correlation between the SCARED and the CBCL anxious/depressed subscale. Moreover, the Korean SCARED was not asso- ciated with the CBCL externalizing scale, CD of the DBD or ADHD-RS, which supports high discriminant validity of the Korean SCARED. Taken together, the Ko- rean SCARED would be a reliable and valid screening tool for anxiety symptoms in youth. Sensitivity analysis The absolute fit of the five-factor SCARED we elicited was tested on the independent clinical sample. The model yielded χ2/df value of 2.63, (χ2 = 2021.13, df = 769, p-value < 0.001) which is acceptable [42]. Root Mean Square Error of Approximation (RMSEA) was 0.063 and Standardized Root Mean Square Residual (SRMR) was 0.078, which showed adequate fit to the data [42]. However, relative fit indices showed a moder- ate fit. The comparative fit index (CFI) was 0.86 and the Tucker-Lewis index (TLI) showed 0.85, which did not reach the optimal cutoff of 0.95 [42, 43]. The standard- ized factor loadings of the Korean SCARED for the sec- ond sample are presented in Table 5. Factor loadings were all higher than 0.36 (Table 5). Discussion As expected, our results showed that the Korean SCARED showed high internal consistency with each subscale demonstrating high internal consistency. This finding is consistent with other validation studies of the In our sample, exploratory factor analysis produced five factors: GAD, PD, SP, SAD, and SA. This factor structure was comparable to the factor structures pro- duced in other cultures, which generally showed four Table 4 Comparison of SCARED among Anxiety + Depression(A + D), Depression only(D only), and Control groups Factor § Anxiety + Depression (n = 44) §§ Depression only (n = 49) Control (n = 54) statistics p-value Total† 43.57 (15.08) 32.35 (14.85) 10.61 (9.73) F = 79.17 < 0.001 Generalized anxiety† 16.91 (5.12) 13.31 (6.42) 4.26 (4.19) F = 75.77 < 0.001 Panic/somatic symptoms†† 6.55 (5.78) 4.47 (4.44) 0.96 (1.45) F = 22.73 < 0.001 Social anxiety† 10.61 (4.04) 7.94 (4.51) 3.83 (3.27) F = 36.93 < 0.001 Separation anxiety† 4.18 (3.30) 2.65 (2.66) 1.06 (1.86) F = 17.31 < 0.001 School avoidance† 5.32 (2.31) 3.63 (2.52) 0.48 (0.97) F = 73.74 < 0.001 § Anxiety + Depression: participants with co-morbid anxiety disorders and MDD §§ Depression only: participants with MDD only † A + D > D only > Control group †† A + D = D only > Control group Shin et al. Discussion BMC Psychiatry (2020) 20:89 Page 8 of 12 Table 5 Standardized factor loadings and factor internal consistency on independent dataset (N = 431) Factor Item Factor loading Cronbach α Generalized anxiety 0.91 5 I worry about other people liking me 0.71 0.90 21 I worry about things working out for me 0.67 0.90 33 I worry about what is going to happen in the future 0.72 0.90 14 I worry about being as good as other kids 0.73 0.90 23 I am a worrier 0.75 0.90 28 People tell me that I worry too much 0.69 0.90 35 I worry about how well I do things 0.74 0.90 37 I worry about things that have already happened 0.75 0.90 20 I have nightmares about something bad happening to me 0.60 0.91 9 People tell me I look nervous 0.58 0.91 7 I am nervous 0.58 0.91 19 I get shaky 0.63 0.91 Panic/somatic symptoms 0.89 27 When I get frightened, I feel like I am choking 0.81 0.86 6 When I get frightened, I feel like passing out 0.36 0.90 12 When I get frightened, I feel like I am going crazy. 0.77 0.86 1 When I feel frightened, it is hard to breathe 0.75 0.86 38 When I get frightened, I feel dizzy. 0.76 0.86 34 When I get frightened, I feel like throwing up 0.71 0.86 24 I get really frightened for no reason at all 0.69 0.86 30 I am afraid of having anxiety (or panic) attacks 0.62 0.87 18 When I get frightened, my heart beats fast 0.64 0.87 22 When I get frightened, I sweat a lot. 0.55 0.87 15 When I get frightened, I feel like things are not real. 0.63 0.87 Social anxiety 0.88 32 I feel shy with people I don’t know well 0.73 0.86 26 It is hard for me to talk with people I don’t know well 0.71 0.86 41 I am shy 0.73 0.86 3 I don’t like to be with people I don’t know well 0.69 0.87 40 I feel nervous about going to parties, dances, or any place where there will be people that I don’t know well 0.80 0.86 10 I feel nervous with people I don’t know well 0.81 0.86 39 I feel nervous when I am with other children or adults and I have to do something while they watch me (e.g. Discussion The result might reflect that the factor structure of the Korean SCARED explains anxiety disorders in Korea youth well. However, the prominent difference in the explained vari- ance value can be due to our sample characteristics, which was a clinical sample with a high proportion of the youth having mood problems, whereas the majority of previous SCARED factor analyses were conducted with a community sample. There were differences in some items that comprise the factors compared to Bir- maher’s factor structure [5]. Item 4, ‘I get scared when I sleep away from home’, belonged to social anxiety rather than separation anxiety as identified in Birmaher’s study [5]. Item 9, ‘People tell me I look nervous’, and 19, ‘I get shaky’ were included in the generalized anxiety factor, not panic disorders as in Birmaher’s analysis. Finally, item 20, ‘I have nightmares about something bad hap- pening to me’ belonged to generalized anxiety, not sep- aration anxiety as in the original study. (GAD, PD, SP, and SAD) to five (GAD, PD, SP, SAD, and SA) factors [5, 6, 18, 45–47]. The explained variance in our factor structure (58.9%) was higher than the ex- plained variances from other five-factor solution studies, which ranged between 36.3 and 39.5% [18]. The result might reflect that the factor structure of the Korean SCARED explains anxiety disorders in Korea youth well. However, the prominent difference in the explained vari- ance value can be due to our sample characteristics, which was a clinical sample with a high proportion of the youth having mood problems, whereas the majority of previous SCARED factor analyses were conducted with a community sample. There were differences in some items that comprise the factors compared to Bir- maher’s factor structure [5]. Item 4, ‘I get scared when I sleep away from home’, belonged to social anxiety rather than separation anxiety as identified in Birmaher’s study [5]. Item 9, ‘People tell me I look nervous’, and 19, ‘I get shaky’ were included in the generalized anxiety factor, not panic disorders as in Birmaher’s analysis. Finally, item 20, ‘I have nightmares about something bad hap- pening to me’ belonged to generalized anxiety, not sep- aration anxiety as in the original study. Another example showing the cultural difference is that item 9, ‘people tell me I look nervous’, can be thought of as a question of how adolescents perceive others’ view of them. Discussion read aloud, speak, play a game, play a sport) 0.66 0.87 4 I get scared if I sleep away from home 0.49 0.89 Separation anxiety 0.76 25 I am afraid to be alone in the house 0.72 0.73 29 I don’t like to be away from my family 0.57 0.74 13 I worry about sleeping alone 0.66 0.73 16 I have nightmares about something bad happening to my parents 0.54 0.76 31 I worry that something bad might happen to my parents 0.58 0.74 Shin et al. BMC Psychiatry (2020) 20:89 Page 9 of 12 Page 9 of 12 Shin et al. BMC Psychiatry (2020) 20:89 Table 5 Standardized factor loadings and factor internal consistency on independent dataset (N = 431) (Continued) Factor Item Factor loading Cronbach’s α 8 I follow my mother or father wherever they go 0.54 0.75 School avoidance 0.86 36 I am scared to go to school 0.93 0.78 17 I worry about going to school 0.93 0.78 2 I get headaches when I am at school 0.65 0.83 11 I get stomachaches at school 0.56 0.87 Total Cronbach’s alpha 0.95 p < 0.01 factor loadings and factor internal consistency on independent dataset (N = 431) (Continued) western sports camps in that it requires obligatory par- ticipation. At the school retreat, Korean children are often required to collaborate with unfamiliar classmates. In this context, it is possible that a Korean child may in- terpret item 4 as a situation in which unwanted social interactions are required. This may cause them to have worries about how other people view them, ‘Nun-chi’ in Korean, and social fear, ‘Taein Kongpo’ in Korean. Con- sistent with this interpretation, item 4 was included in the social phobia factor, highly correlating with items such as ‘I am shy’, ‘I feel shy with people I don’t know well’ and ‘I feel nervous about going to parties, dances, or any place where there will be people that I don’t know well’. In addition, factor loading was relatively low (0.23) compared to other items, which might partially explain the different result. (GAD, PD, SP, and SAD) to five (GAD, PD, SP, SAD, and SA) factors [5, 6, 18, 45–47]. The explained variance in our factor structure (58.9%) was higher than the ex- plained variances from other five-factor solution studies, which ranged between 36.3 and 39.5% [18]. Discussion However, we in- cluded a control group that can mitigate selection bias. Additionally, participants’ ages were between 12 to 17 years, which can limit the generalizability to younger chil- dren. However, sensitivity analysis suggests that the factor structure can be applied to a wider age range of children and adolescents. Another limitation is that parents com- pleted all the questionnaires with the exception of the Ko- rean SCARED, and their reports may significantly differ from the participants’ report regarding the internalizing symptoms such as anxiety and depression [44]. Neverthe- less, there are some studies about child and parent agree- ment that revealed concordance between child and parent reports to be moderate to high [56, 57]. This study was also limited in that the anxiety only group was not in- cluded and only the anxious/depressed comorbid group was included in the analysis. Despite this limitation, it is important to note that anxiety disorders are highly comor- bid with other psychiatric disorders in adolescents, and thus discriminating anxiety only subjects may be a chal- lenging task. Lastly, the small sample size was another limitation of our study. However, our sample size met the minimum sample size for factor analysis as suggested by Mundfrom [38] and other studies, which state that sample size should be at least 3 times the number of variables or a minimum size of 100 [58, 59]. We further investigated how the Korean SCARED can be used to discriminate the A + D, D only and control groups, according to the elicited factor structures. All of the anxiety subscales, excluding PD, significantly distin- guished the three groups: scores were the highest in the A + D group and the lowest in the control group. The PD subscale score also followed the same order, but the difference between the A + D and the D only group was not significant. Although the panic disorder is catego- rized as an anxiety disorder according to DSM-5 [30], depressive disorder is also highly comorbid with panic symptoms [52]. In this sense, the Korean SCARED could not clearly distinguish the A + D comorbid group from the D only group in terms of PD. Nevertheless, the PD subscale distinguished the A + D and the D only groups from the control group. Discussion GAD worries tend to strongly focus on interpersonal difficulties [50] and prominent self-consciousness [51]. The fact that item 4 and 9 belonged to a different factor compared to Birmaher’s original study [5] may reflect the Korean cultural ten- dency to worry about other people’s views and assess- ments rather than their own sense of the self, panic or somatic symptoms. Therefore, these differences in the items comprising the factors might reflect the cultural differences between South Korea and the U.S. As for item 4, the difference in factor structure may be due to the different age range of participants. In contrast to Birmaher’s study which included children from the age of 9 to 18 years old, our participants were all adoles- cents, with the age range between 12 to 17 years [5]. Rates of separation anxiety are highest in younger chil- dren [48]. However, the SCARED factor structures found in the research of different countries with similar age ranges as our study, resemble the original study by Birmaher. In those studies, item 4 was included in SAD [45, 49]. Another reason for the different results may be related to the cultural differences between South Korea and the United States. For example, in South Korea, sleeping away from home could be because of school re- treats (Suhak Yeohaeng in Korean), an educational activ- ity that students are required to attend once or twice a year. This event is conceptually different from the The social anxiety scores prominently differed between our sample and the US clinical sample. The mean scores of A + D group (10.6) and D only group (7.9) in our sample were higher than those from a study with a cor- responding US clinical sample (3.7 in anxiety only group and 2.7 in depression only group) [5]. This indicates that Korean adolescents tend to feel more anxious in inter- personal and social situations. This result is in line with the above-mentioned result that Korean adolescents tend to understand item 4 as a social interaction related anxiety rather than separation-related anxiety. Shin et al. BMC Psychiatry (2020) 20:89 Page 10 of 12 Page 10 of 12 Shin et al. BMC Psychiatry (2020) 20:89 Shin et al. BMC Psychiatry (2020) 20:89 population, and depressed patients were recruited from an outpatient clinic. This may obscure the generalizability of the results to the general population. Discussion In this respect, the Korean SCARED might be useful as an anxiety assessing tool that can fairly discriminate between the subcategories of anxiety disorders. y Sensitivity analyses demonstrated that our factor struc- ture had a good fit measured by absolute fit indices, SRMR and RMSEA, but a moderate fit by relative fit indices, CFI and TLI. Several factors may have influenced the inad- equacy of relative fit indices. First, limitations of our factor structure may have influenced the results, because there were items with cross-loadings (e.g. item 10 and 15) and low factor loading (e.g. item 4). Second, sample size of our primary sample may have affected the results, which met the minimal sample size needed for the factor analysis, but not the optimal size [38]. In addition, composition dif- ference between our primary sample and the independent clinical sample may have affected the results. Our sample was composed of youths with mood disorders and healthy controls, whereas all but one of the independent clinical sample had psychiatric disorders. Our results of low CFI/ TLI values and adequate SRMR/RMSEA of our factor structure may be due to the fact that the CFI/TLI are rela- tive fit indices, while the SRMR/RMSEA are absolute indi- ces. Relative fit indices compare the model with null hypothesis, which assumes that all variables are not corre- lated [53]. Lower than optimal value of relative fit indices may reflect the moderate correlations between inter-factor items, although intra-factor items were highly correlated [54]. However, absolute fit indices are determined by how far the model is from the perfect fit, which is presumed to have a fit of zero [42, 53, 55]. The adequate SRMR sug- gested that our factor structure captures the data well, and the good RMSEA index indicated that our model fits well relative to degree of freedom [54], which indicates that our factor structure is adaptable to clinical samples. Dif- ferent modeling formulas seem to have caused the differ- ence in the results, lowering the relative fit indices. h l l h d h l Despite the above-noted limitations, our study showed that the Korean SCARED is a reliable and valid screen- ing tool for assessing adolescent anxiety with high criter- ion and discriminant validity. Discussion Taking into account the high prevalence and the negative influence on the daily function of adolescents’ anxiety disorders, the Korean SCARED can serve as a utilitarian method to screen anxiety disorders and provide a diagnostic impression about the types of anxiety disorders. Furthermore, inves- tigation of the factor structure suggested [5] a cultural difference of anxiety symptoms between Korean and Western adolescents. Future studies that utilize the SCARED to explore sub-anxiety differences among countries are needed, which might reveal additional cul- tural differences. Acknowledgements Not applicable. 6. Birmaher B, Brent DA, Chiappetta L, Bridge J, Monga S, Baugher M. Psychometric properties of the screen for child anxiety related emotional disorders (SCARED): a replication study. J Am Acad Child Adolesc Psychiatry. 1999;38(10):1230–6. Funding Th 10. Kim MY, Kim JM. A study on the influencing mental health factors to suicidal ideation in adolescents. Korean J Youth Stud. 2013;20(12):267–93. g This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF-2015R1A2A2A01004501) and was supported by Promising-Pioneering Research Program through Seoul Na- tional University (SNU) in 2015. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 11. Mo SH, Kim HJ, Lee SY. A study on mental health improvement policy for children and adolescents : general report. National Youth Policy Institute; 2013. 12. Perrin S, Last CG. Do childhood anxiety measures measure anxiety? J Abnorm Child Psychol. 1992;20(6):567–78. 13. Lim YB, Kim JW, Hong S-B, Kim J-W. Association between BDNF and antidepressant effects of exercise in youth: A preliminary study. J Korean Acad Child Adolesc Psychiatry. 2016;27(1):72–81. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. 14. Kim JY, Lee CH, Kim H-M. Behavioral consequences of children with sleep- disordered breathing after adenotonsillectomy. World J Pediatr. 2018;14(1): 57–65. Received: 16 September 2019 Accepted: 20 February 2020 22. Song JY, Park KH. The sociocultural variables influencing social anxiety with concern for offending others: focusing on Che-myeon, nun-chi, and self- construal. Korean J Health Psychol. 2009;14(2):329–43. Conclusion Our study suggests that the Korean SCARED is a prom- ising screening tool for assessing adolescent anxiety to be used practically in community settings as well as clin- ical environments including primary care. The fact that different items comprised the factors may reflect cultural differences in experiencing anxiety between youth in the United States and Korea; Korean youth report higher so- cial fears and worry about how other people view them. There are several limitations to this study. The sample of the study was not randomly selected from the general Page 11 of 12 Page 11 of 12 Page 11 of 12 Shin et al. BMC Psychiatry (2020) 20:89 Shin et al. BMC Psychiatry Shin et al. BMC Psychiatry (2020) 20:89 Author’s contribution JS, KK., SC, and JK contributed to conception and design of the study. JL, CC, JH, SK., DS, and JK were involved in data acquisition. JS, KK, KL, SH, and JK participated in data analysis. JS, KK, KL, SH, JL, CC, SC and JK contributed to interpretation of the data. JS, KK, KL, JL, CC, DS and JK drafted the manuscript or substantively revised it. All authors agreed to the manuscript for submission and publication. All authors have agreed both to be personally accountable for the author’s own contributions and to ensure that questions related to the accuracy or integrity of any part of the work. The author(s) read and approved the final manuscript. 7. Kim KM, Nam S, Choi JW, Jung AH, Hong SB, Kim JW, et al. Psychometric properties and factor structures of the Korean version of Children's depression rating scale-revised. J Child Adolesc Psychopharmacol. 2018; 28(4):285–92. 8. Wu Y, Bang MY. Analysis of variables influencing social anxiety of at-risk adolescents. J Emotional Behav Disord. 2012;28(2):235–53. 9. Kim J-B. “A study of influencing factors to teenagers’ maladjustment at school life”- the main focus on maladjustment teenagers. Korean J Clin Soc Work. 2009;6(2):25–48. p g No conflict of interest. 18. Hale WW, Crocetti E, Raaijmakers QA, Meeus WH. A meta-analysis of the cross-cultural psychometric properties of the screen for child anxiety related emotional disorders (SCARED). J Child Psychol Psychiatry. 2011;52(1):80–90. Consent for publication Not applicable. Consent for publication Not applicable. 17. Monga S, Birmaher B, Chiappetta L, Brent D, Kaufman J, Bridge J, et al. Screen for child anxiety-related emotional disorders (SCARED): convergent and divergent validity. Depress Anxiety. 2000;12(2):85–91. Ethics approval and consent to participate 15. Weitkamp K, Romer G, Rosenthal S, Wiegand-Grefe S, Daniels J. German screen for child anxiety related emotional disorders (SCARED): reliability, validity, and cross-informant agreement in a clinical sample. Child Adolesc Psychiatry Ment Health. 2010;4:19. Ethics approval and consent to participate The study was approved by the Institutional Review Board for Human Subjects of Seoul National University Hospital (IRB No. 1504–113-668). Detailed information about the study was provided to the parents and youth and written informed consent was obtained from all participants before entering the study. The study was approved by the Institutional Review Board for Human Subjects of Seoul National University Hospital (IRB No. 1504–113-668). Detailed information about the study was provided to the parents and youth and written informed consent was obtained from all participants before entering the study. 16. Hale WW, Raaijmakers QAW, García-López LJ, Fernández LE, Muela JA, MdMD C. Psychometric Properties of the Screen for Child Anxiety Related Emotional Disorders for Socially Anxious and Healthy Spanish Adolescents. Span J Psychol. 2013;16:E25. Author details 1 1Division of Child and Adolescent Psychiatry, Department of Psychiatry, Seoul National University College of Medicine, 103 Daehak-Ro, Chongno-Gu, Seoul 03080, Republic of Korea. 2Department of Psychiatry, Dankook University College of Medicine, Cheonan 31116, Republic of Korea. 3Integrative Care Hub, Seoul National University Children’s Hospital, Seoul 03080, Republic of Korea. 4Department of Psychiatry, Seoul National University Boramae Medical Center, Seoul 07061, Republic of Korea. 5Department of Psychiatry, Korea Armed Forces Capital Hospital, Bundang, Republic of Korea. 19. Hariz N, Bawab S, Atwi M, Tavitian L, Zeinoun P, Khani M, et al. Reliability and validity of the Arabic screen for child anxiety related emotional disorders (SCARED) in a clinical sample. Psychiatry Res. 2013;209(2):222–8. 20. Caporino NE, Sakolsky D, Brodman DM, McGuire JF, Piacentini J, Peris TS, et al. Establishing clinical cutoffs for response and remission on the screen for child anxiety related emotional disorders (SCARED). J Am Acad Child Adolesc Psychiatry. 2017;56(8):696–702. 21. Kim EJ, Cho YR. The relationship of social phobia with the concern for offending others, anger-related variables, and individualism-collectivism. Korean J Clin Psychol. 2008;27(3):613–32. Received: 16 September 2019 Accepted: 20 February 2020 Received: 16 September 2019 Accepted: 20 February 2020 Received: 16 September 2019 Accepted: 20 February 2020 Abbreviations disorders in the national comorbidity survey replication adolescent supplement. Arch Gen Psychiatry. 2012;69(4):372–80. disorders in the national comorbidity survey replication adolescent supplement. Arch Gen Psychiatry. 2012;69(4):372–80. A + D (Anxiety + Depression): Participants with co-morbid anxiety disorders and MDD; ADHD-RS: ADHD Rating Scale; CBCL: Child Behavior Checklist; CDRS-R: Children’s Depression Rating Scale-Revised; CFI: Comparative fit index; D only: Participants with MDD only; DBD: Disruptive Behavioral Disorder Scale; DSM-5: Diagnostic and Statistical Manual of Mental Disorders- 5th Edition; GAD: Generalized anxiety; IQ: Intelligence quotient; K-SADS- PL: Kiddie-Schedule for Affective Disorders and Schizophrenia for School-Age Children-Present and Lifetime Version; MDD: Major depressive disorder; PD: Panic/somatic symptoms; RCMAS: Revised Children’s Manifest Anxiety Scale (RCMAS); RMSEA: Root Mean Square Error of Approximation; SA: School avoidance; SAD: Separation anxiety; SCARED: Screen for Child Anxiety Related Emotional Disorders; SP: Social anxiety; SRMR: Standardized Root Mean Square Residual; STAIC: State-Trait Anxiety Inventory; TLI: Tucker-Lewis index 2. Park S, Kim B-N, Cho S-C, Kim J-W, Shin M-S, Yoo H-J. Prevalence, correlates, and comorbidities of DSM-IV psychiatric disorders in children in Seoul, Korea. Asia Pac J Public Health. 2015;27(2):NP1942–NP51. Korea. Asia Pac J Public Health. 2015;27(2):NP1942–NP51. 3. Kim B-N, Park E-J, Choi T-Y, Kwak Y-S. Prevalence and risk factors of psychiatric disorders in child and adolescent population-shool based research. Ministry of Health and Welfare, Korean mental health technology R&D project; 2018. 4. Wren FJ, Berg EA, Heiden LA, Kinnamon CJ, Ohlson LA, Bridge JA, et al. Childhood anxiety in a diverse primary care population: parent-child reports, ethnicity and SCARED factor structure. J Am Acad Child Adolesc Psychiatry. 2007;46(3):332–40. 5. Birmaher B, Khetarpal S, Brent D, Cully M, Balach L, Kaufman J, et al. The screen for child anxiety related emotional disorders (SCARED): scale construction and psychometric characteristics. J Am Acad Child Adolesc Psychiatry. 1997;36(4):545–53. 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https://openalex.org/W2903947771	https://www.biorxiv.org/content/biorxiv/early/2018/12/13/495887.full.pdf	English	null	Neural activity in a hippocampus-like region of the teleost pallium are associated with navigation and active sensing	bioRxiv (Cold Spring Harbor Laboratory)	2,018	cc-by	21,550	Title: Neural activity in a hippocampus-like region of the teleost pallium are associated with navigation and active sensing Fotowat H1, Lee C2, Jun JJ3, Maler L2 Fotowat H1, Lee C2, Jun JJ3, Maler L2 Fotowat H1, Lee C2, Jun JJ3, Maler L2 1Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA. 2Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Canada. 3 Center for Computational Biology, Flatiron Institute, New York, NY, USA. Fotowat H , Lee C , Jun JJ , Maler L 1Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA. 2Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Canada. 3 Center for Computational Biology, Flatiron Institute, New York, NY, USA. 1Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA. 2Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Canada. 3 Center for Computational Biology, Flatiron Institute, New York, NY, USA. 3 Center for Computational Biology, Flatiron Institute, New York, NY, USA. . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint Title: Neural activity in a hippocampus-like region of the teleost pallium are associated with navigation and active sensing Abstract Neural mechanisms underlying spatial navigation in fish are unknown and little is known, for any vertebrate, about the relationship between active sensing and the formation of spatial maps. The weakly electric fish, Gymnotus Carapo, uses their active electric sense for spatial navigation. The electric organ discharge rate (EODr) undergoes transient increases during navigation to enhance electrosensory sampling. Gymnotus also uses stereotyped forward/ backward swimming as a second form of active sensing that brings objects towards the electroreceptor-dense head region. We wirelessly recorded neural activity from the pallium of freely swimming Gymnotus. Spiking activity was sparse and occurred only during swimming. Notably, some units exhibited significant place specificity and/or association with both forms of active sensing. Our results provide the first characterization of neural activity in a hippocampal- like region of a teleost fish brain and connects active sensing via sensory sampling rate and directed movements to higher order encoding of spatial information. . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint Introduction It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: ioRxiv preprint range active electrosensory system. This study further demonstrated that, in the process of learning, Gymnotus use several active sensing strategies. These include increasing the rate of their electric organ discharge (EOD), which results in an increased rate of sensory sampling, as well as back and forth swimming (B-scans) past landmarks (Jun et al 2016). These fish therefore provide an excellent model system for reading out the dynamics of sensory sampling and “attentive state” and relating this information to neuronal activity associated with landmarks during spatial navigation. Interestingly, once the fish have learned about the location of food, they appear to identify landmarks they encounter along their current trajectory, and then move towards the food location despite not being able to electrosense other landmarks or food from afar. Based on this finding, Jun et al (2016) hypothesized that fish’s trajectories from a learned landmark to food location were based on path integration. Changes in electric field caused by the presence of landmarks are sensed by thousands of electroreceptors located on the fish’s skin. Primary afferents relay this information to the electrosensory lateral line lobe which then projects to the mid-brain torus semicircularis that provides electrosensory input to tectum (Krahe and Maler 2014, Carr et al 1982). Cells in the fish’s tectum respond to both electrosensory and visual object motion (Bastian 1982) and would therefore be expected to discharge as the fish swims past landmarks. The tectal cells responsive to object motion project to the preglomerular nucleus (PG), an analog or homolog of the mammalian thalamus (Giassi et al 2012b; Wallach et al. 2018), which in turn projects to DL. Based on its connectivity and gene expression, DL has characteristics of both mammalian cortex and the hippocampus (Elliott et al 2017). Introduction Neural mechanisms underlying spatial navigation has been intensely studied for decades in mammals and, especially, in rodents (Eichenbaum 2017, Moser et al 2017, Chersi and Burgess 2015). Various cell types in different areas of the hippocampal formation have been identified to encode various self, environmental, and social cues that enable the animal to successfully navigate towards a goal. These cells include place cells (O’Keefe and Dostrovsky 1971), boundary cells (Saveli et al 2008, Solstad et al 2008), grid cells (Saveli et al 2017), head direction cells (Taub 2007), goal direction cells (Sarel et al 2017), and more recently social place cells (Danjo et al 2018, Omer et al 2018). Remarkably, there is a large degree of similarity in the neural representation of space among phylogenetically distant mammals (e.g. between echolocating bats and rodents: Ulanovsky and Moss 2007). It is known that teleost fish, whose common ancestor with mammals lived approximately 450 million years ago are also capable of spatial learning (Rodriguez et al 2002, Jun et al 2016). Whether these fish utilize the same neural mechanisms for spatial learning as mammals do remains completely unknown. In goldfish, studies based on lesion and cytochrome oxidase histochemistry have identified the dorsolateral pallium (DL) as selectively essential for spatial learning (Ocana et al 2017, Uceda et al 2015, Rodriguez 2002, Duran et al 2010, Broglio et al 2010). Based on these studies as well as patterns of connectivity and gene expression, it has been hypothesized that DL has similar connectivity and perhaps is even homologous to the mammalian hippocampus (Elliot et al 2017; Harvey-Girard et al 2012). Although one study reported recordings from one putative place cell near the medial edge of DL of gold fish (Canfield and Mizumori 2004), to our knowledge no studies have quantitatively demonstrated the presence of place-associated cells in the teleost pallium. We chose to study the neural activity of the pallium of pulse-type weakly electric fish Gymnotus sp. in the context of spatial navigation. Through detailed analysis of their spatial learning behavior, Jun et al (2016) have shown that these fish can learn the location of food relative to landmarks in complete darkness relying mainly on their short- 2 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Introduction DL projects massively and in a highly convergent manner to the intermediate subdivision of dorsal pallium (DDi); DDi then provides strong feedback to DL via a magnocellular component of DD (DDmg, Giassi et al 2012c, Trinh et al 2016, Elliott et al 2017). DL has vastly more neurons than either DDi or DDmg (Supplementary Figure 1, Trinh et al 2016), and they are far more densely packed (Giassi et al 2012c). We therefore decided that sampling DDi would be far more efficient than sampling DL and would still give us an idea of the kind of spatial information that might be extracted by the teleost pallium. 3 3 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: ioRxiv preprint A recent paper has suggested that DDi has similar connectivity, and might be homologous to, the mammalian CA3 region and might therefore be expected to contain cells responsive to spatial and/or motion features of the environment (Elliott et al 2017). We therefore used a wireless transmitter system to record neural activity from DDi as the fish swam freely in the dark in an experimental tank (open maze) containing differently shaped landmarks – we used the same tank and landmarks as in the Jun et al (2016) study so that we could directly compare behavioral and physiological data. We simultaneously tracked the fish’s position and recorded its EOD signal using electrodes placed inside the tank (Jun et al 2014a). Introduction We asked: what are the neural dynamics of cells in DDi and how do they relate to the rate of sensory sampling, the fish’s location and scanning movements relative to landmarks. 4 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint Spikes were sparse and occurred mainly during movement It is known that at very low speeds, that is during moments of behavioral quiescence or “down-states”, the EOD rate is lower than when the fish is active (generally less than 50 Hz down states in Gymnotus sp., Jun et al 2014b), resulting in a reduction in sensory sampling rate. Consistently, we found that spikes were highly unlikely during down-states (Figure 3C, notice the absence of spikes at EODr < 50 Hz) indicating that spikes in this region are likely linked to both sensory and motor activity. Spikes were sparse and occurred mainly during movement Figure 1 shows the wireless transmitter, the tetrode and their assembly prior to (A-C), and after implantation (D). Extracellular recordings from neurons within the dorsal pallium (DD) were wirelessly transmitted as the fish freely swam in a large circular tank that contained differently shaped landmarks (Figure 1E, see Methods). We aimed our tetrodes to the intermediate subdivision of DD: DDi (see Methods, Supplementary Figure 1). Figure 2A shows an example extracellular recording together with the raster plot for the four units that could be sorted based on their shape. Figure 2B shows the shape of the sorted units and their first three principle components that were used for clustering units (see Methods). The EOD rate (bottom red trace, Fig. 2A) was calculated using the EOD signal that was simultaneously recorded by the tank electrodes (see Methods). Figure 3A shows the distribution of the average firing rates of all units recorded from all the fish and across all trials, highlighting sparsity of spiking in this region (25 units, 5 fish, 23 trials, 20 hours and 36 min of recording). We found that that units in DDi fired at strikingly low rates, and that it was very unlikely to encounter a spike at low swimming speeds or during periods of quiescence. Figure 3B shows the histogram of swim speeds calculated across all time bins and trials (purple) and those in time bins where there was a spike (green). Notice a peak at low swim speeds in the purple histogram, which is missing in the green one. It is known that at very low speeds, that is during moments of behavioral quiescence or “down-states”, the EOD rate is lower than when the fish is active (generally less than 50 Hz down states in Gymnotus sp., Jun et al 2014b), resulting in a reduction in sensory sampling rate. Consistently, we found that spikes were highly unlikely during down-states (Figure 3C, notice the absence of spikes at EODr < 50 Hz) indicating that spikes in this region are likely linked to both sensory and motor activity. Notice a peak at low swim speeds in the purple histogram, which is missing in the green one. . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint Relation between spiking activity, EOD rate, swim speed and sampling density The electric organ discharge rate (EODr) continuously varies in time (Figure 2, red trace). Higher EODrs indicate higher sensory acquisition rates, and large, fast transients in the 5 5 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: oRxiv preprint EODr has been previously reported to occur near landmarks in the context of spatial learning (Jun et al 2016). Fish swim speed is variable and is also associated with proximity to landmarks and changes during learning (Jun et al 2016). Sampling density, the number of EOD pulses per unit length, depends on both EODr and swim speed and therefore also varies strongly with the fish’s location near landmarks (Jun et al 2016). Below, we describe the relation between DDi spiking and EODr, swim speed and, consequently, with sampling density. EODr: To examine the relation between sensory acquisition rate and spiking activity of DDi units we calculated spike-triggered-EODr (stEODr) averages for all units that fired more than 10 spikes in a trial (see example in Figure 4A). We found that for the majority of units (15 out of 21), the mean EODr in an 8 second window around the spike time was significantly higher than that calculated for randomly time-shifted spikes (Fig 4A black trace). Moreover, we found that for many units the mean EODr increased prior to spike time and peaked within a few hundred milliseconds around the time of the spike. 13 out of the 21 units showed a significant peak in the stEODr average. Figure 4A shows an example unit with a significant peak in its stEODr average (stEODr for all the other units are presented in Supp. Fig 2). Figure 4B shows the probability density histogram of timing of peaks in EODr relative to individual spikes from all units and all fish (orange bars). Relation between spiking activity, EOD rate, swim speed and sampling density The peaks were more likely to occur around the timing of the spike; within a second prior to and 0.5 s after spike time (median peak time was zero). The histogram for the timing of stEODr calculated using randomly time-shifted spike trains did not show such a peak around the spike time (grey bars). Swim speed: We next considered the correlation between swim speed and spiking of the units. Interestingly, we observed that the fish’s spike-triggered-swim-speed (stSpeed) often decreased to a minimum (dip) before the time of spike, followed by a peak after (Figure 4C, blue and orange arrows, respectively. see also Supp Fig 2). 14 out of 21 units showed a significant peak in stSpeed average after the spike time. 10 out of these 14 units also had a significant peak in their stEODr average, with all stEODr average peaks except one occurring after the spike time (Supp. Fig 2, Fish 5, 1st unit). Figure 4D shows the distribution of the timing of speed dips (blue bars) and peaks (orange bars) across all 6 6 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: ioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint units and fish (median stSpeed dip time =-0.56 s median stSpeed peak time = 0.2 s). Unlike the stEODr, the timing of the dips and peaks in stSpeed were significantly shifted to negative and positive values i.e. before and after the timing of the spike, respectively (for dips: p= 4.4 e-24, for peaks: p=8.5e-11, non-parametric sign test). This pattern was not evident for the timing of peaks and dips of stSpeed calculated for randomly time- shifted spikes (grey bars show the peak times, similar pattern was observed for the dips, data not shown). Relation between spiking activity, EOD rate, swim speed and sampling density CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: oRxiv preprint with the results of Jun et al (2016), which shows that sampling density increases in the vicinity of landmarks. The net effect of these coordinated changes in EODr and swim speed leads to strongly enhanced electrosensory sampling (SmpD) over the trajectory traversed ~1-3 s before a spike followed by reduced sampling for up to 3 s after the occurrence of the spike. Spiking occurred near boundaries and landmarks Units in DDi tended to fire in multiple locations within the experimental tank often close to the tank boundaries and landmarks. Figure 5 shows examples of swimming trajectories, spiking locations, and firing rate maps for 8 units recorded in 5 fish. To quantify place- specificity of each unit’s activity we calculated the “place information” in bits/spike (see Methods) for the 21 units that fired more than 10 spikes during the trial. The average of information across all units and trials was 1.58 bits/spike (SD= 1.15). For each unit and trial, we tested the statistical significance of place information level by comparing it to that calculated from randomly time-shifting the spike train relative to swim trajectory. Examples of the firing pattern of units that conveyed significant place information, and those that did not are given in Figure 5A and B, respectively. 11 of the 21 recorded units in 4 fish (3,3,3,2 units in each fish respectively) showed significant place specificity. Examples of the firing pattern of units that conveyed significant place information, and those that did not are given in Figure 5A and B, respectively. 11 of the 21 recorded units in 4 fish (3,3,3,2 units in each fish respectively) showed significant place specificity. Interestingly, 8 out of 11 units with significant place selectivity also had significant peaks in their stEODr averages (Supplementary Figures 2 and 3). Four of these units (2 fish) further showed a clear peak in SmpD before spike time. There was no clear correlation between lack of place specificity and absence of a peak in stEODr (5 out of the 10 units that were not place specific, also showed a significant peak in their stEODr average, and the other 5 did not, Supplementary Figure 3). Interestingly, 8 out of 11 units with significant place selectivity also had significant peaks in their stEODr averages (Supplementary Figures 2 and 3). Four of these units (2 fish) further showed a clear peak in SmpD before spike time. There was no clear correlation between lack of place specificity and absence of a peak in stEODr (5 out of the 10 units that were not place specific, also showed a significant peak in their stEODr average, and the other 5 did not, Supplementary Figure 3). Interestingly, changes in landmark configurations often resulted in changes in the firing patterns of the units and gain or loss of significance in conveyed place information. Relation between spiking activity, EOD rate, swim speed and sampling density Sampling density: We next used units that showed significant peaks in both stEODr and stSpeed averages (10 units) and calculated the spike-triggered sampling density as the ratio of stEODr to stSpeed (stSmpD, exemplar unit is shown in Figure 4E, see Supp. Fig 2 for all units). The sampling density has units of EOD pulses emitted per unit length (pulses/cm), it is a measure of electrosensory spatial acuity, and has been previously proposed to be an indicator of spatial attention (Jun et al 2016). Mirroring stSpeed, we found that for most units the stSmpD average showed a peak before spike time and a dip after it (Figure 4F orange and blue arrows, respectively). Figure 4F shows the probability distribution for the peaks (orange bars) and dips in stSmpD (blue bars) for all units and all fish (median stSmpD peak time =-0.56 s, median stSmpD dip time = 0.24 s). The timing of the peak and dip in stSmpD were not significantly different from the timing of dip and peak in stSpeed, and occurred significantly before and after spike time, respectively (for peaks: p= 2.68 e-24, for dips: 1.1 e-10, non-parametric sign test). This pattern was not present in the timing of dips and peaks calculated for stSmpD of randomly time-shifted spikes (grey bars show the peak times, similar pattern for the dips, data not shown). In summary, despite a large degree of variability in the dynamics of EODr and speed around individual spikes as indicated by the width of the timing histograms, we found that DDi spikes were most likely to occur after a dip in swim speed. EODr often increased prior to a spike and peaked around the time of the spike. The combination of the pre-spike decreasing swim speed and increasing EODr resulted in a strong increase in SmpD before the spike. Similarly, the post-spike increasing swim speed and decreasing EODr resulted in a strong SmpD dip post-spike. The changes in swim speed were clearly the dominant factor contributing to this change in SmpD. This is completely consistent 7 7 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint . Spiking occurred near boundaries and landmarks Interestingly, changes in landmark configurations often resulted in changes in the firing patterns of the units and gain or loss of significance in conveyed place information. Figure 6A shows examples of the effect of changes in landmark configuration on spiking activity of three units. Figure 6B shows a summary plot of the effect of landmark removal on the firing rate of the units in a 10 cm radius around the location of the landmark (16 trials and 10 units, 12 out of 16 trials showed a reduction). For example, removing landmarks often resulted in a reduction in firing rate of the unit around the location of the Figure 6A shows examples of the effect of changes in landmark configuration on spiking activity of three units. Figure 6B shows a summary plot of the effect of landmark removal on the firing rate of the units in a 10 cm radius around the location of the landmark (16 trials and 10 units, 12 out of 16 trials showed a reduction). For example, removing landmarks often resulted in a reduction in firing rate of the unit around the location of the 8 8 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: oRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint missing landmark. Remarkably, in one case where the landmark was moved to a new location, the unit shifted its preferred firing rate to the new location of that same landmark (Figure 6A-iii, small triangle). Spiking occurred near boundaries and landmarks Further, comparison of the firing rates in the two conditions normalized to the largest rate showed that the rate was significantly reduced with landmark removal (Figure 6B, inset). Therefore, we conclude that a subset of DDi units fire in a location- specific manner, and that their spatial specificity is tightly linked to the presence of landmarks. Swim direction and relative landmark location preference Gymnotus can swim in both forward and backward directions. Backward swims are often observed in the context of prey capture and spatial navigation and serve to bring prey or landmarks that the fish has swam past, back near the fish’s head and rostral trunk (Pedraja et al 2018, Jun et al 2016, Nelson and Maciver 1999), the regions containing the highest density of electroreceptors (Carr et al 1982 and Castello et al 2000). Interestingly, we found that most units in DDi showed preference for spiking during backward swims. Figure 7A shows the distribution of the swim direction preference indices (see Methods) for all units in all fish. The distribution showed a significant bias towards negative values that correspond to backward swims (mean (SD) =-0.196 (0.33), p=0.0072, non-parametric sign test). We next asked, do units in DDi have a preferred landmark location, i.e. are they more likely to fire when a landmark has a specific location relative to the body of the animal? To answer this question, we calculated a probability map for landmark locations when a given unit was active. To do this we divided spike-triggered landmark locations by position-triggered landmark locations (see Methods). Figure 7B shows normalized landmark probability at the time of spiking of four exemplar units. For each unit, we then calculated an anterior-posterior and a left/right preference index (see Methods). Figure 7C shows preference vectors plotted using these preference indices for all units in all fish. Vectors corresponding to exemplar units in Figure 7B are indicated within this plot. We found that for most units the probability of landmark presence was higher in the region 9 9 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: ioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint posterior to the body of the fish (Figure 7D, 14 out of 21 units). Swim direction and relative landmark location preference This bias was not present for left- versus right location for the landmarks (Figure 7E, 10 out of 21 units preferred spiking to contralateral objects). Because in all fish the tetrode was implanted in the left lobe of the pallium, these results indicate that activity in DDi is not unilateral. In summary, most DDi units we recorded spiked in response to objects located in the region posterior to the body of the fish as fish was swimming backwards. These units therefore appear to correlate to a scanning movement that would lead to relocating these objects from the trunk to the head and more anterior parts of the body, which contain the highest density of electroreceptors. 10 10 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint Discussion CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: oRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint neurons to looming/receding and longitudinal object motion. We note that forward object motion is equivalent to the backward swimming component of B-scans. Similarly, backward object motion is equivalent to the forward swim direction. We found that DDi units only fired when the fish was moving and the EODr was high (Figure 3, Up state: Jun et al. 2014b). Discharge typically occurred at multiple locations distributed within the experimental tank and often near tank boundaries and landmarks. Spikes from about half of the units we recorded conveyed significant spatial information (Figure 5). The spatial pattern of spiking and its spatial specificity changed when the landmark configuration was altered (Figure 6A). We found that in the majority of units, spikes were linked to the presence of landmarks and that removing landmarks resulted in a reduction in the firing rate in the location of the missing landmark(Figure 6B). These findings demonstrate that cells in DDi encode the presence of landmarks or boundaries, albeit in a stochastic manner, as spikes were not fired at every instance the animal visited a given location. Individual spikes of many DDi cells are precisely linked to the types of active sensing (EODr and sampling density increases and B-scans) that occur when the fish is learning the spatial layout of the open maze environment (Jun et al 2016). We found that over the whole recording session, for many units on average, the spike-triggered EODr average started increasing prior to spike time and continued till slightly after the spike (13 out of 21 units, Figure 4 A, B). In many cells (14 out of 21 units), the spike-triggered speed showed a dip prior to a spike and dramatically increased post-spike (Fig. 4C, D). This combination resulted in an increased sampling density before and during the spike followed by its reduction post-spike. Discussion In this study, we wirelessly recorded and characterized neural activity within the dorsal pallium (DDi) of freely swimming pulse-type weakly electric fish Gymnotus sp. This is the first study to report on location and movement related response properties of cells in a region of a teleost fish’s pallium that has similar connectivity, and may be homologous to, the CA3 area of mammalian hippocampus (Elliot et al 2017). A previous study (Elliott et al., 2015), using a related (immobilized) gymnotiform fish (Apteronotus leptorhynchus), found that, in the absence of sensory input, DDi cells were completely silent. In response to electrocommuncation and acoustic signals, DDi cells discharged very sparsely, at long latencies and with a small number of spikes; these results are comparable to our data. Further work will be required to determine whether different DDi cells respond to electrolocation versus electrocommunication signals. Gymnotiform fish utilize two active sensing behaviors. Pulse type fish (e.g Gymnotus sp.) will increase their EODr and sampling density near landmarks and food (Jun et al 2016); Apteronotus is a ‘wave type’ fish and maintains a constant EODr when foraging. Both Gymnotus (this paper; Jun et al., 2016; Pedraja et al 2018) and Apteronotus (Nelson and Maciver 1999) will use back-and-forth scanning movements (B-scans) in proximity to salient landmarks. In the discussion below we first summarize our most important results and then interpret them with respect to neuroanatomy (effectively identical in Apteronotus and Gymnotus, Giassi et al. 2012a-c) and the results presented in Jun et al (2016). Critical to our interpretation is that our experiments were carried out in naïve fish, i.e., animals that had not learned the spatial layout of the tank (open maze) environment. We therefore compare our data to the ‘early learning’ results of Jun et al, where the fish also first encountered landmarks in the same open maze environment and, by using active sensing, learned to identify landmarks and their spatial relation to food. We also incorporate the recent results of Wallach et al. (2018), who examined the response of Apteronotus PG 11 11 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint . Discussion Wallach et al (2018) report that many PG cells fire as an object approaches the fish (Apteronotus). We hypothesize that, in Gymnotus, the increased EODr and sampling density will also strongly drive PG spiking and therefore DL spiking activity. The only sensory input to DDi is from DL which leads us to conclude that DDi spikes will likely feedback (via DDmg) to DL while DL is activated by PG input. We further discuss this point below. 12 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: ioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint In the context of active sensing these fish exhibit stereotyped forward/backward swimming motions (B-scans) that are thought to be important for learning about landmarks in the context of spatial navigation (Pedraja et al 2018, Jun et al 2016). Likewise, during prey capture, the fish tend to swim backwards once they have passed a prey item to align the prey with the head region where the highest density of electroreceptors is found (Nelson and Maciver 1999). Jun et al (2016) demonstrated that EODr and sampling density are highest during the backward phase of B-scans suggesting that PG will be very strongly driven by this phase of B-scans. Remarkably, Wallach et al (2018) described PG units that responded strongly and specifically throughout the forward movement of an object, i.e., the same relative motion that would occur during backward swimming. Most appropriately, we found that most units we recorded (17 out of 21) showed a preference for spiking during the backward swim phase of B-scans (Figure 7A). Moreover, we found that most units (12 out of 17) with preference for spiking during backward swims spiked most when objects were initially located near the trunk region (Figure 7C, D). Discussion In other words, these units spiked during a movement that would result in the object ending up near the head – the region with the highest density of electroreceptors. We hypothesize that this PG activity drives DDi cells (via DL) during back-swimming. The DDi spikes will feedback (via DDmg) to DL while it still being activated by its strong ongoing PG input. Under these conditions there will be three temporally overlapping sources of excitatory synaptic input to DL cells: (1) PG spiking driven by the backward scanning of the landmark, (2) DL spiking driven by its local recurrent connectivity (Trinh et al 2016), and (3) feedback input from DDi (via DDmg). As we have previously noted, DL is likely the site for storage of spatial memory. We therefore further hypothesize that the spatial learning described by Jun et al is driven by synaptic plasticity at one or more of these synaptic inputs to DL neurons. Finally, we pose the question: are the spatially-specific units we find in this hippocampus-like region of the fish brain functionally similar to the location/landmark responsive cells found in the mammalian hippocampus? There are five clear aspects of DDi cell discharge that suggest similarity to these cells. 1. Hippocampal place and/or grid cells exhibit sparse firing (Diamantakis et al 2016; Hainmuller et al 2018; Rolls 2015) 13 13 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: ioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint and only during movement (Chen et al 2013; Winter et al 2015; Song et al 2005; Harvey et al 2018). 2. Like place cells, the spatially specific DDi cells discharge is closely tied to the presence of a landmark (Muller and Kubie 1993) and, like place cells, they will discharge upon an early encounter with a landmark (Alme et al 2014, Wilson and McNaughton 1993). 3. Discussion In a ‘large environment’, place cells will exhibit multiple place fields (Fenton et al 2008, Park et al 2011) much like the DDi cell discharge near multiple landmarks. 4. Place cell discharge is very variable across different visits to their preferred place field (Fenton and Muller 1998) much like the variability we observed in DDi cell discharge across visits to the same landmark. 5. Lastly, and most interestingly, discharge of these DDi units near landmarks is associated with active sensing, i.e., increased EODr and sampling density and scanning behavior (B-scans). Rodents use head-scans as ‘a spatially directed investigative behavior’ (Poulter et al 2018); there is a clear functional analogy between head-scans and B-scans. An important recent study showed that head- scans drive formation or strengthening of place field discharge of hippocampal cells (Monaco et al 2014). We hypothesize that, during B-scans, DDi feedback to DL will also drive the formation of place/object- associated discharge of DL cells, and that this is a key element of spatial learning in gymnotiform fish. In blind rats, place cells can have place fields far from any landmark (Save et al 1998). Unfortunately, the fish hardly ever swam far from the tank boundary and landmarks and so we cannot determine whether this is also true for DDi cells. Further experiments with fish that have been extensively trained in the experimental tank and willing to traverse long empty spaces will be required to investigate this critical issue. We propose that place and/or landmark-associated cells are not unique to the mammalian hippocampus but, instead, have evolutionary precursors stretching back 450 million years to the last common ancestor of mammals and teleosts fish (Jun et al 2014b). A critical objective of future experiments will be to improve our ability to record neural activity across distinct pallial regions (e.g., DD, DC and DL, see Giassi et al 2012b) in freely swimming fish and over many days of exploration and spatial learning. This will allow us to gain a better understanding of the core neural mechanisms underlying spatial learning 14 14 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint . Discussion CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: oRxiv preprint by revealing commonalities and differences between the mechanisms responsible for memory guided navigation in the teleost versus mammalian brain. 15 15 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint Wireless transmitter and tetrode fabrication A wireless transmitter/receiver system (Figure 1A, TBSI-W16, Triangular Biosystems Intl, Durham, NC) was used for transmitting and receiving neural recordings in freely swimming fish. Tetrodes were constructed using 12-micron stablohm wires (stablohm 650, California Fine Wire Company, Grover Beach, CA) and wound using a Neuralynx Tetrode Spinner (Neuralynx, Bozeman, MT). Each tetrode was made 15-cm long and passed through slightly shorter flexible polyethylene tubing (PE, PE10, 0.61 mm OD x 0.28 ID, Warner Instruments Corp, Hamden, CT) such that either end of the tetrode was sticking outside the tube (Figure 1B). The one end of the tetrode to be implanted was further passed through a 0.5 cm long 180-micron diameter polyimide tubing (PI, Microlumen, 068-I, Lot#24331) and fixed in place using a mixture of Krazy glue and dental cement (Jet denture repair powder, Lang Dental Mfg. Co Inc., Wheeling, IL) for additional rigidity (Figure 1B). The PE tubing containing the tetrode was attached to a glass capillary, which could be solidly inserted inside an electrode holder attached to a micro- manipulator (Figure 1C). Melted Polyethylene glycol (PEG) was used to attach the tubing to the glass capillary. At room temperature PEG solidifies and acts as a water-soluble glue. Once the electrode was implanted, the tubing could be separated from the glass capillary by gently pouring water on the solidified PEG. The other end of the tetrode protruding from the PE tubing was also stabilized inside the PE tubing using another piece of PI tubing and glue. Individual tetrode wires were then separated at that end and attached to the input ports of the electrode interface board (EIB) which could then be plugged in to the transmitter. One of the 4 electrodes was used as reference. At this point the electrodes were first electroplated using Neuralynx gold plating solution and then with a solution of Ethylene Dioxythiopene monomer (EDOT) and Polystyrene sulfonate (PSS) using nanoZ plating protocols and software (MultiChannel Sytems MCS GmbH, Germany). Individual tetrode impedances varied between 100-200 kOhms. The EIB was then attached to the transmitter-battery ensemble and they were then put inside a cut-open Ping-Pong ball, which was used as a float for the transmitter system. A few pieces of vibration absorbing gel (Z8006, Kyosho, Lake Forest, CA) were added inside the ball and around the transmitter system to prevent movement and vibrations that could result in transmission noise. . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint Wireless transmitter and tetrode fabrication CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made avail The copyright holder for this preprint (which this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint Pong ball was then closed back (Fig 1C) and water proofed using mouldable glue (Sugru, London, UK). Wireless transmitter and tetrode fabrication The Ping- A wireless transmitter/receiver system (Figure 1A, TBSI-W16, Triangular Biosystems Intl, Durham, NC) was used for transmitting and receiving neural recordings in freely swimming fish. Tetrodes were constructed using 12-micron stablohm wires (stablohm 650, California Fine Wire Company, Grover Beach, CA) and wound using a Neuralynx Tetrode Spinner (Neuralynx, Bozeman, MT). Each tetrode was made 15-cm long and passed through slightly shorter flexible polyethylene tubing (PE, PE10, 0.61 mm OD x 0.28 ID, Warner Instruments Corp, Hamden, CT) such that either end of the tetrode was sticking outside the tube (Figure 1B). The one end of the tetrode to be implanted was further passed through a 0.5 cm long 180-micron diameter polyimide tubing (PI, Microlumen, 068-I, Lot#24331) and fixed in place using a mixture of Krazy glue and dental cement (Jet denture repair powder, Lang Dental Mfg. Co Inc., Wheeling, IL) for additional rigidity (Figure 1B). The PE tubing containing the tetrode was attached to a glass capillary, which could be solidly inserted inside an electrode holder attached to a micro- manipulator (Figure 1C). Melted Polyethylene glycol (PEG) was used to attach the tubing to the glass capillary. At room temperature PEG solidifies and acts as a water-soluble glue. Once the electrode was implanted, the tubing could be separated from the glass capillary by gently pouring water on the solidified PEG. The other end of the tetrode protruding from the PE tubing was also stabilized inside the PE tubing using another piece of PI tubing and glue. Individual tetrode wires were then separated at that end and attached to the input ports of the electrode interface board (EIB) which could then be plugged in to the transmitter. One of the 4 electrodes was used as reference. At this point the electrodes were first electroplated using Neuralynx gold plating solution and then with a solution of Ethylene Dioxythiopene monomer (EDOT) and Polystyrene sulfonate (PSS) using nanoZ plating protocols and software (MultiChannel Sytems MCS GmbH, Germany). Individual tetrode impedances varied between 100-200 kOhms. The EIB was then attached to the transmitter-battery ensemble and they were then put inside a cut-open Ping-Pong ball, which was used as a float for the transmitter system. A few pieces of vibration absorbing gel (Z8006, Kyosho, Lake Forest, CA) were added inside the ball and around the transmitter system to prevent movement and vibrations that could result in transmission noise. The Ping- 16 16 . Animal preparation and electrode implantation CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: ioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint DD and at a site rostral to the very caudal DDmg. The penetration depth was adjusted to pass through DDs but remain confined to DDi (Supplementary Figure 1). Once the electrode was implanted, the opening of the skull was covered with small pieces of thin plastic sheets (0.5 mil thick FEP film made with Teflon fluoroplastic, CS Hyde Company, Lake Villa, IL). The sheets were then secured to the rest of the skull using a UV curable sealant (Aegis Pit & Fissure Sealant, Keystone Industries, Germany), which was also used to seal any other small remaining openings. After curing with UV light, a mixture of dental cement and Krazy glue was used to further seal the opening and to cover the incision areas on the fish’s skin. At this point the MS-222 solution was slowly diluted with DI water and the PE tubing containing the tetrode was released from the glass capillary by applying a gentle flow of water. Once the fish resumed breathing, it was gently taken out of the setup and transferred to the experimental tank and allowed to recover overnight (Figure 1D). Recordings were obtained the next day in four out of 5 fish and on day 4 after surgery in one fish that took longer to recover. The recordings were obtained as long as the battery lasted. This was between 5-6 hours in 3 out of 5 fish. For the fish that took a long time to recover (i.e. Animal preparation and electrode implantation to resume normal activity level and to swim consistently around the tank) we could only record for about an hour during the 4th day before the battery ended, as we had attempted recording the days before, when the fish was not swimming reliably. For the fifth fish, we have a little over 2 hours of recording after which the fish pulled out the implant and had to be sacrificed right after. At the end of a recording session, we deeply anaesthetized the fish and perfused it in a standard manner (Giassi et al 2012a). We attempted to remove the tetrode and extract the brain with minimal damage and to then section and stain (Cresyl Violet, Giassi et al 2012a) in order to identify the tetrode location. We were successful in locating the tetrode track in two fish (Supplementary Figure 1). Animal preparation and electrode implantation All animal procedures were performed in accordance with the regulations of the animal care committee of the University of Ottawa. Gymnotus sp. of either sex were used for all experiments. Before implanting, tetrodes were first cleaned by submersion in 70% ethanol for 15 minutes and then in sterile saline solution (0.9% NaCl) for another 15 minutes to rinse off the ethanol. They were then further sterilized using UV illumination. Fish were anesthetized in a small container with tricane methanesulfonate (MS-222; Aqua Life, Syndel Laboratories) in tank water solution. They were then transferred to a holder outside of water where their head and body could be stabilized in preparation for surgery and electrode implantation. The MS-222 solution in oxygenated deionized (DI) water was continuously administered in this setup through a tube that was inserted in the mouth, and the fish’s body was covered with wet sponges and Kimwipes to protect the skin. Before opening the skull, it was first completely dried, then some crazy glue was applied on the contralateral side of the planned implant to make the skull surface rough. This procedure helped with the final closure step as the dental cement mixture could better adhere to the remaining pieces of roughed bone. Additionally, a few indentations were made using a dentist drill on the surface of the contralateral skull. These indentations served as extra attachment points as they were filled with the dental cement mixture at the closing stage. The dorsal pallium was exposed by a small craniotomy and the tetrode was micro-manipulated to the DD region. DD of gymnotiform fish is divided into superficial (DDs), intermediate (DDi) and magnocellular (DDmg) sub-regions (Giassi et al 2012a). Elliott et al (2017) suggested that DDi, on the basis of its connectivity pattern, was similar and perhaps homologous to the CA3 hippocampal field. We directed our tetrodes towards DDi using the data of Giassi et al (2012a), an Atlas of the Gymnotus brain (Corrêa et al 1998) and a high-resolution lab atlas of the Gymnotus brain for guidance. This atlas has previously successfully directed tracer injections precisely into DDi (Giassi et al 2012c; Elliott et al 2017). Surface sulci delimiting DD from the more medial dorsomedial pallium and more lateral dorsolateral pallium were used to guide the electrodes to 17 17 . Experimental setup and trials Landmarks were made of acrylic and were secured to the bottom of the tank using suction cups and could be added or removed. Each trial lasted between 30min – 1hour during which the landmark configuration was stable. Landmarks were sometimes removed or displaced to test the effect on the firing properties of the units. 2104a, 2016). The length of the tetrode (15cm) was chosen such that the wireless transmitter float would not exert any force on fish’s head. Landmarks were made of acrylic and were secured to the bottom of the tank using suction cups and could be added or removed. Each trial lasted between 30min – 1hour during which the landmark configuration was stable. Landmarks were sometimes removed or displaced to test the effect on the firing properties of the units. Electric Organ Discharge (EOD) signals were captured using 4 pairs of graphite electrodes (Mars Carbon 2-mm type HB, Staedler, Germany) attached to the tank walls at equal spacing (Jun et al 2014a). All experiments were performed in the dark and the animal’s behavior was monitored under IR illumination using a camera (C910, Logitech, IR filter removed) that was mounted above the tank. The camera acquired images at 1600x1200 resolution and had a frame rate of 15 Hz. Wireless data reception and spike sorting. Analog signals received at the receiver were digitized using (CED mkII, Cambridge Electronic Design, UK) and further analyzed using CED’s Spike2 software. EOD signals sensed by four pairs of tank electrodes were also acquired simultaneously using the CED acquisition system. Neural recordings also contained spikes from the EOD. To facilitate spike sorting, EOD spikes were removed from the recording offline, by setting the neural recording trace in the time-window -2.8:2.8 ms around each EOD spike to zero. Figure 2A shows an example recording: the three blue traces are extracellular recordings from the three tetrode channels after removing EOD spikes. and the red trace shows instantaneous EOD rate calculated based on tank electrodes. Spike sorting was done using Spike2 software based on spike waveform shape. The threshold for spike detection was set high and kept constant for all trials in one fish, therefore, only units with high signal to noise ratio were kept for subsequent analysis. Initial sorting by shape was followed by fine tuning spike clusters using principle component analysis and visual inspection of individual spikes (Figure 2B). Experimental setup and trials The experimental tank was 1.5 m in diameter and fish were tested in shallow water (~10 cm) to facilitate video tracking by restricting fish’s swimming trajectory in 2 dimensions (Figure 1E). A full description of the tank construction and the landmark shapes can be found in (Jun et al 18 18 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: ioRxiv preprint 2104a, 2016). The length of the tetrode (15cm) was chosen such that the wireless transmitter float would not exert any force on fish’s head. Landmarks were made of acrylic and were secured to the bottom of the tank using suction cups and could be added or removed. Each trial lasted between 30min – 1hour during which the landmark configuration was stable. Landmarks were sometimes removed or displaced to test the effect on the firing properties of the units. Electric Organ Discharge (EOD) signals were captured using 4 pairs of graphite electrodes (Mars Carbon 2-mm type HB, Staedler, Germany) attached to the tank walls at equal spacing (Jun et al 2014a). All experiments were performed in the dark and the animal’s behavior was monitored under IR illumination using a camera (C910, Logitech, IR filter removed) that was mounted above the tank. The camera acquired images at 1600x1200 resolution and had a frame rate of 15 Hz. 2104a, 2016). The length of the tetrode (15cm) was chosen such that the wireless transmitter float would not exert any force on fish’s head. Experimental setup and trials Due to low firing rate of units, clusters were sometimes not completely separable and therefore some units may be considered as multi-unit. Analysis of EOD rate – spike relationship. The following data analysis were all performed in MATLAB (MathWorks Inc.). Spike-triggered EOD rate (stEODr) and speed (stSpeed) averages were calculated in an 8s window around the timing of each spike (+/- 4 s). Spike-triggered sampling density (stSmpD) was calculated by dividing stEODr by stSpeed for each spike. For 19 19 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: ioRxiv preprint each unit then the spike-triggered averages were calculated using all spikes fired by that unit across all trials. The same procedure was repeated 100 times for spikes circularly shifted by a random time (by at least 30 s and at most the length of the trial minus 30 s) for comparison. The average of stEODr over the entire 8 s window for each spike was compared to that calculated for the stEODr for randomly time shifted spikes using Kruskal-Wallis test. To calculate the significance of the peak in stEODr and stSpeed averages, the value of the stEODr and stSpeed for each spike at the time of the peak in the average was calculated. These values were then compared to average EODr and speed calculated over the whole window for all spikes using the Wilcoxon sign rank test. To calculate probability density functions for peak times in stEODr, stSpeed and stSmpD, the timing of the largest peak in EODr, Speed or SmpD was measured for each spike. Experimental setup and trials It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint that unit. Only bins with more than 5 visits were included in the analysis. To calculate statistical significance of spatial information, this information measure was recalculated for 1000 randomly time shifted spike trains superimposed on the same swimming trajectory. The spatial information conveyed by a unit was considered significant if it exceeded 95th percentile of the distribution of the information calculated for the randomly shifted spike trains. To examine the effect of removing a landmark on the firing rate of a given unit we calculated the average firing rate for the bins that were within a 10-cm radius of the landmark, before and after its removal. Swimming direction and landmark location preference analysis. Spike rate during forward (backward) swim was calculated as the number of spikes that occurred during forward (backward) swim divided by the total time spent swimming forward (backward). Because of the small number of spikes, we pooled forward(backward) turns with forward (backward) swims. Direction preference index was calculated as the difference of the firing rate during forward swims and backward swims divided by their sum. Negative values of the direction preference index indicated preference for spiking during backward swims. To calculate the spike-triggered landmark (STLM) matrix, first a 160x120 -element matrix corresponding to the absolute location of landmarks and tank boundary as viewed from the video recording was constructed (each matrix element corresponded to a 10x10 pixels area in the video recording). Matrix elements that contained landmarks or tank boundaries were set to 1 whereas other elements were set to zero. Next, at the time of each spike of a given unit, this matrix was shifted and rotated such that fish’s position was centred in the matrix and its head was facing north. The matrix sum was then calculated for all spikes from a given unit. Similarly, a Position- triggered Landmark (PTLM) matrix was calculated as the sum of such rotated and shifted landmark matrix calculated every 1.33 seconds (the fish’s trajectory, which had a resolution of 15 Hz, was down-sampled 20 times). PTLM matrix elements that were smaller than the lower 10 percentile of all elements were excluded from the analysis. STLM was then normalized to PTLM to calculate the probability of presence of landmarks within 10 cm around the fish. Experimental setup and trials To calculate the histogram of timings of the dip in stSpeed and stSmpD, the timing of the smallest local minimum was used. The same procedure was repeated for randomly time- shifted spike trains. Analysis of fish’s swimming trajectory and spatial firing rates. Position, and swimming direction of the fish in the experimental tank was calculated using custom software as well as those available from Ty Hedrick’s lab (Hedrick, 2008). To calculated spatial firing properties of the units, the area of the experimental tank was divided into 16x16 cm bins and the total number of spikes fired in each bin was divided by the time-spent in that bin. Bins that had less than 5 visits during the whole trial were not included in the analysis. A visit was counted when the fish’s head first arrived at a given bin. If the fish stayed within a bin for more than 1 frame (video frame rate =15 Hz), visit number was still counted as 1, and was allowed to increase only when the fish left the bin and returned to it another time. For visualization purposes color range shown in the firing rate map plots was clipped at 97 percentile of firing rate of that unit across all bins. This was done to avoid bins where the fish spent a very small amount of time to saturate the color plot. The maximum firing rate per bin is indicated above these plots (Figure 5). Spatial information in bits per spike was calculated using the firing rate maps as described previously (Skaggs et al 1993, Rubin et al 2014): 𝑝𝑝𝑟𝑟𝑙𝑙𝑙𝑟𝑟 Spatial information (bits/spike)= ∑ 𝑝𝑝𝑝𝑝ቀ 𝑟𝑟𝑟𝑟 𝑟𝑟𝑟𝑟ቁ𝑙𝑙𝑙𝑙𝑙𝑙2( 𝑟𝑟𝑟𝑟 𝑟𝑟𝑟𝑟) Where pi is the probability of the animal being in the ith bin, calculated as the ratio of time spent in bin i divided by the total trial time, ri is the firing rate in bin i and rm is the mean firing rate for 20 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: ioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Experimental setup and trials This distance was chosen as a conservative upper range for object detectability with the electric sense based on Jun et al 2016. We then divided the area around the fish into four regions: Left and right side of the fish, each of which was further divided into two regions: anterior 1/3 and posterior 2/3 of the fish’s body. The Swimming direction and landmark location preference analysis. Spike rate during forward (backward) swim was calculated as the number of spikes that occurred during forward (backward) swim divided by the total time spent swimming forward (backward). Because of the small number of spikes, we pooled forward(backward) turns with forward (backward) swims. Next, at the time of each spike of a given unit, this matrix was shifted and rotated such that fish’s position was centred in the matrix and its head was facing north. The matrix sum was then calculated for all spikes from a given unit. Similarly, a Position- triggered Landmark (PTLM) matrix was calculated as the sum of such rotated and shifted landmark matrix calculated every 1.33 seconds (the fish’s trajectory, which had a resolution of 15 Hz, was down-sampled 20 times). PTLM matrix elements that were smaller than the lower 10 percentile of all elements were excluded from the analysis. STLM was then normalized to PTLM to calculate the probability of presence of landmarks within 10 cm around the fish. This distance was chosen as a conservative upper range for object detectability with the electric sense based on Jun et al 2016. We then divided the area around the fish into four regions: Left and right side of the fish, each of which was further divided into two regions: anterior 1/3 and posterior 2/3 of the fish’s body. The 21 21 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. Experimental setup and trials It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint Experimental setup and trials ; https://doi.org/10.1101/495887 doi: ioRxiv preprint region located around the anterior third of the fish, corresponds to the regions near fish’s head and upper trunk (gill region) that contains the largest number of electroreceptors (Carr et al 1982; Castello et al 2000). We then calculated a left - right preference index as the difference between the maximum probability on the left side and the right side divided by the sum of the two. We similarly calculated an anterior-posterior preference. Positive left- right (anterior-posterior) preferences corresponded to locations to the right side (anterior) of the fish. For each cell, we then calculated a preference vector with its x and y value equal to the left- right and anterior posterior preference indices, respectively (Figure 7E). For each unit, we used the amplitude and direction of this vector as an indicator of the strength and orientation of preferred landmark location for that unit. Because in all fish the tetrode was implanted on the left hemisphere, landmarks to the left side of the fish were ipsilateral to the location of the electrode and those to the right of the fish were contralateral. region located around the anterior third of the fish, corresponds to the regions near fish’s head and upper trunk (gill region) that contains the largest number of electroreceptors (Carr et al 1982; Castello et al 2000). We then calculated a left - right preference index as the difference between the maximum probability on the left side and the right side divided by the sum of the two. We similarly calculated an anterior-posterior preference. Positive left- right (anterior-posterior) preferences corresponded to locations to the right side (anterior) of the fish. For each cell, we then calculated a preference vector with its x and y value equal to the left- right and anterior posterior preference indices, respectively (Figure 7E). For each unit, we used the amplitude and direction of this vector as an indicator of the strength and orientation of preferred landmark location for that unit. Because in all fish the tetrode was implanted on the left hemisphere, landmarks to the left side of the fish were ipsilateral to the location of the electrode and those to the right of the fish were contralateral. 22 22 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Acknowledgements We would like to thank Florian Engert for his helpful comments, suggestions and support for this manuscript. We also thank Bill Ellis for technical support and Erik Harvey-Girard, Armin Bahl, Martin Haesemeyer and Roy Harpaz for their helpful suggestions. This research was supported by NSERC grant 04336 to LM. 23 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint Competing interests Authors of this manuscript do not have any financial or non-financial competing int Authors of this manuscript do not have any financial or non-financial competing interests. 38 38 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint References Alme CB, Miao C, Jezek K, Treves A, Moser EI, Moser M-B (2014) Place cells in the hippocampus: eleven maps for eleven rooms. PNAS. 111:18428–18435. Bastian J (1982) Vision and Electroreception - Integration of sensory information in the optic tectum of the weakly electric fish Apteronotus-Albifrons. 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It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint 15 cm Fish’s home Water filter Landmarks ransmitter Battery PE tube with tetrode inside attached to glass capillary Fish + Transmitter float PI tube Transmitter sealed inside the ball B C E Fig 1. Experimental setup and example wireless recording A. References Wireless transmitter system used for recordings. B. A long tetrode was constructed and mounted on an electrode holder to be attached to a micromanipulator. C. The other end of the tetrode was connected to the transmitter, and the ensemble was placed in a ping-pong ball and sealed. D. Pictures of a fish after tetrode implantation together with the transmitter float (picture from a related species with the same size). E. Recordings were performed in a large experimental tank containing various landmarks made with clear and opaque plexiglass, as well as a water filtering system. . CC-BY 4.0 International license under a y p ) , g p y p p p p y A Wireless transmitter Battery PE tube with tetrode inside attached to glass capillary PI tube B Transmitter sealed inside the ball C B C A Transmitter sealed inside the ball Wireless transmitter Battery 15 cm Fish’s home Water filter Landmarks Fish + Transmitter float E E D Landmarks Fish’s home Water filter Fig 1. Experimental setup and example wireless recording A. Wireless transmitter system used for recordings. B. A long tetrode was constructed and mounted on an electrode holder to be attached to a micromanipulator. C. The other end of the tetrode was connected to the transmitter, and the ensemble was placed in a ping-pong ball and sealed. D. Pictures of a fish after tetrode implantation together with the transmitter float (picture from a related species with the same size). E. Recordings were performed in a large experimental tank containing various landmarks made with clear and opaque plexiglass, as well as a water filtering system. Fig 1. Experimental setup and example wireless recording A. Wireless transmitter system used for recordings. B. A long tetrode was constructed and mounted on an electrode holder to be attached to a micromanipulator. C. The other end of the tetrode was connected to the transmitter, and the ensemble was placed in a ping-pong ball and sealed. D. Pictures of a fish after tetrode implantation together with the transmitter float (picture from a related species with the same size). E. Recordings were performed in a large experimental tank containing various landmarks made with clear and opaque plexiglass, as well as a water filtering system. References Time (s) 0 400 800 1200 1600 40 60 0.5 mV ( ) 1 ms 0.5 mV Unit 1 Unit 2 Unit 3 Unit 4 0.1 PC3 0.1 0.1 0.05 -0.1 0.05 0 PC1 0 PC2 -0.05 -0.05 -0.1 -0.1 A B Fig 2. Example recordings in one fish and four isolated units. A. The blue traces show extracellular recordings on the three of the four tetrode channels after EOD spike removal (see Methods). The fourth channel was used as reference. The red trace shows instantaneous EOD rate calculated based on EOD recordings obtained by electrodes inside the experimental tank. Raster plots show spike timing corresponding to the four units. B. Average (SD) of waveforms of the isolated units from this recording and their first three principle components (PCs). . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint B . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint Time (s) 0 400 800 1200 1600 40 60 0.5 mV EOD rate (Hz) A CC-BY 4.0 Internationa under a y p ) g 1 ms 0.5 mV Unit 1 Unit 2 Unit 3 Unit 4 0.1 PC3 0.1 0.1 0.05 -0.1 0.05 0 PC1 0 PC2 -0.05 -0.05 -0.1 -0.1 B . al license A 0.1 PC3 0.1 0.1 0.05 -0.1 0.05 0 PC1 0 PC2 -0.05 -0.05 -0.1 -0.1 B . al license B EOD rate (Hz) Fig 2. Example recordings in one fish and four isolated units. A. The blue traces show extracellular recordings on the three of the four tetrode channels after EOD spike removal (see Methods). The fourth channel was used as reference. The red trace shows instantaneous EOD rate calculated based on EOD recordings obtained by electrodes inside the experimental tank. Raster plots show spike timing corresponding to the four units. B. Average (SD) of waveforms of the isolated units from this recording and their first three principle components (PCs). Fig 2. References Example recordings in one fish and four isolated units. A. The blue traces show extracellular recordings on the three of the four tetrode channels after EOD spike removal (see Methods). The fourth channel was used as reference. The red trace shows instantaneous EOD rate calculated based on EOD recordings obtained by electrodes inside the experimental tank. Raster plots show spike timing corresponding to the four units. B. Average (SD) of waveforms of the isolated units from this recording and their first three principle components (PCs). Average firing rate Hz 0 0.02 0.04 0.06 0.08 0.1 0 0.1 0.2 0.3 Probability Swim speed (cm/s) Probability density 0 4 8 12 16 20 0 0.04 0.08 0.12 0.16 All swimming speeds Swim speeds at the time of spikes EOD rate (Hz) A 50 60 70 80 0 0.04 0.08 0.12 0.16 All EOD rates EOD rates at the time of a spike B C Fig 3. DDi units spiked sparsely during periods swimming and active sensing. A. Probability histogram of average firing rates observed in all units and all trials (25 units, 23 trials, 5 fish). B. Probability density function of all observed swim speeds (purple bars), and swim speeds at the time of a spikes (green bars). Spikes were unlikely at very low swimming speeds. C. Probability density function of all observed EOD rates (purple bars) and those observed at the time of spikes (green bars). Spikes were unlikely at low EOD rates (<50 Hz) corresponding to down states. . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint Average firing rate Hz 0 0.02 0.04 0.06 0.08 0.1 0 0.1 0.2 0.3 Probability Swim speed (cm/s) Probability density 0 4 8 12 16 20 0 0.04 0.08 0.12 0.16 All swimming speeds Swim speeds at the time of spikes EOD rate (Hz) A 50 60 70 80 0 0.04 0.08 0.12 0.16 All EOD rates EOD rates at the time of a spike B C . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. References ; https://doi.org/10.1101/495887 doi: bioRxiv preprint A -4 -2 0 2 4 53.4 53.8 54.2 54.6 EODr (Hz) -4 -3 -2 -1 0 1 2 3 4 0 0.1 0.2 B Probability density B A -4 -3 -2 -1 0 1 2 3 4 0 0.1 0.2 A B C D E F -4 -3 -2 -1 0 1 2 3 4 0 0.1 0.2 -4 -3 -2 -1 0 1 2 3 4 0 0.1 0.2 -4 -2 0 2 4 53.4 53.8 54.2 54.6 -4 -2 0 2 4 4.8 5.2 5.6 6 -4 -2 0 2 4 11 12 13 14 EODr (Hz) Speed (cm/s) Sampling density (EOD pulses/cm) Time (s) Time (s) Probability density Probability density Probability density Fig 4. Examples stEODr, stSpeed and stSmpD averages. A. StEODr average for an exemplar unit (solid red curve, dotted curve: standard error). The black curve shows the stEODr average for 100 random time shifts of the same spike train. The dashed vertical line corresponds to the spike time (zero). B. Probability density of timing of stEODr peaks around individual spikes for units with significant peak in stEODr average (orange bars, 13 units in 5 fish) and for 100 random time shifts of the same spike trains (grey bars). There was a clear peak of stEODr around zero that did not exist in the random data. C. StSpeed average (red curve) for the same unit shown in A, and the corresponding average for randomly shifted spike times (black curve). Arrows point to the dip and peak in the stSpeed pre- and post- spike respectively. D. Probability density of speed dips (blue) preceding the spike and peaks (orange) after spikes for units with a significant peak in stSpeed average (1 units in 5 fish, grey: randomly time-shifted spike trains). E. stSmpD average for the same unit. stSmpD showed a clear peak before the spike and sharply decreased immediately post-spike, with the minimum occurring after spike time. F. Probability distribution of timing of the peak (orange) and dip (blue) in sampling density for individual spikes of units with significant peak in both stEODr and stSpeed shows the same pattern of a pre-spike increase followed by a post-spike decrease (10 units in 5 fish). Fits in all histograms are non-parametric fits with Gaussian kernels. References It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint C B C A Average firing rate Hz Fig 3. DDi units spiked sparsely during periods swimming and active sensing. A. Probability histogram of average firing rates observed in all units and all trials (25 units, 23 trials, 5 fish). B. Probability density function of all observed swim speeds (purple bars), and swim speeds at the time of a spikes (green bars). Spikes were unlikely at very low swimming speeds. C. Probability density function of all observed EOD rates (purple bars) and those observed at the time of spikes (green bars). Spikes were unlikely at low EOD rates (<50 Hz) corresponding to down states. Fig 3. DDi units spiked sparsely during periods swimming and active sensing. A. Probability histogram of average firing rates observed in all units and all trials (25 units, 23 trials, 5 fish). B. Probability density function of all observed swim speeds (purple bars), and swim speeds at the time of a spikes (green bars). Spikes were unlikely at very low swimming speeds. C. Probability density function of all observed EOD rates (purple bars) and those observed at the time of spikes (green bars). Spikes were unlikely at low EOD rates (<50 Hz) corresponding to down states. . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: ioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. References ; https://doi.org/10.1101/495887 doi: bioRxiv preprint 0 0.05 0.1 0.15 I_place=1.57 bits/spike p_place=0.0087 Max FR/bin =0.18 Hz 0 0.02 0.04 0.06 0 0.01 0.02 0 0.1 0.2 0 0.1 0.2 0 0.05 0.1 0 0.1 0.2 0.3 Firing rate/bin (Hz) B I_place=1.9 bits/spike p_place=0.02 Max FR/bin =0.11 HZ I_place=1.4 bits/spike p_place=0.03 Max FR/bin =0.06 Hz I_place=0.68 bits/spike p_place=0.02 Max FR/bin =0.48 Hz Firing rate/bin (Hz) Firing rate/bin (Hz) Firing rate/bin (Hz) I_place=1.41 bits/spike p_place=0.1 Max FR/bin =0.84 Hz Firing rate/bin (Hz) I_place=1.46 bits/spike p_place=0.58 Max FR/bin =0.43 Hz Firing rate/bin (Hz) I_place=0.66 bits/spike p_place=0.55 Max FR/bin =0.2 Hz I_place=0.41 bits/spike p_place=0.89 Max FR/bin =0.41 Hz Firing rate/bin (Hz) Firing rate/bin (Hz) 0 0.05 0.1 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available py g p p ( p , ; p g p p A 0 0.05 0.1 0.15 I_place=1.57 bits/spike p_place=0.0087 Max FR/bin =0.18 Hz 0 0.02 0.04 0.06 0 0.01 0.02 0 0.1 0.2 0 0.1 0.2 0 0.05 0.1 0 0.1 0.2 0.3 Firing rate/bin (Hz) A B I_place=1.9 bits/spike p_place=0.02 Max FR/bin =0.11 HZ I_place=1.4 bits/spike p_place=0.03 Max FR/bin =0.06 Hz I_place=0.68 bits/spike p_place=0.02 Max FR/bin =0.48 Hz Firing rate/bin (Hz) Firing rate/bin (Hz) Firing rate/bin (Hz) I_place=1.41 bits/spike p_place=0.1 Max FR/bin =0.84 Hz Firing rate/bin (Hz) I_place=1.46 bits/spike p_place=0.58 Max FR/bin =0.43 Hz Firing rate/bin (Hz) I_place=0.66 bits/spike p_place=0.55 Max FR/bin =0.2 Hz I_place=0.41 bits/spike p_place=0.89 Max FR/bin =0.41 Hz Firing rate/bin (Hz) Firing rate/bin (Hz) 0 0.05 0.1 . References CC-BY 4.0 International license under a y p ) , g p y p p p p y 0 0.1 0.2 0 0.05 0.1 I_place=1.41 bits/spike p_place=0.1 Max FR/bin =0.84 Hz Firing rate/bin (Hz) I_place=1.46 bits/spike p_place=0.58 Max FR/bin =0.43 Hz Firing rate/bin (Hz) I_place=0.66 bits/spike p place=0.55 0 0.05 0.1 0.15 I_place=1.57 bits/spike p_place=0.0087 Max FR/bin =0.18 Hz 0 0.02 0.04 0.06 Firing rate/bin (Hz) I_place=1.9 bits/spike p_place=0.02 Max FR/bin =0.11 HZ Firing rate/bin (Hz) 0.1 0.2 Firing rate/bin (Hz) Firing rate/bin (Hz) 0 0.05 0.1 0 0.02 0.04 0.06 0 0.01 0.02 0 0.1 0.2 0 0.1 0.2 0 0.05 0.1 0 0.1 0.2 0.3 ng rate/bin (Hz) I_place=1.9 bits/spike p_place=0.02 Max FR/bin =0.11 HZ I_place=1.4 bits/spike p_place=0.03 Max FR/bin =0.06 Hz I_place=0.68 bits/spike p_place=0.02 Max FR/bin =0.48 Hz Firing rate/bin (Hz) Firing rate/bin (Hz) Firing rate/bin (Hz) ng rate/bin (Hz) I_place=1.46 bits/spike p_place=0.58 Max FR/bin =0.43 Hz Firing rate/bin (Hz) I_place=0.66 bits/spike p_place=0.55 Max FR/bin =0.2 Hz I_place=0.41 bits/spike p_place=0.89 Max FR/bin =0.41 Hz Firing rate/bin (Hz) Firing rate/bin (Hz) 0 0.05 0.1 Fig 5. Spatial properties of DDi units. A. Left columns: Examples of spiking patterns of four units in four fish that conveyed significant place information. Grey curves show fish’s swimming trajectory, and black dots represent spikes. Right column: Firing rate maps of the same units shown to the left. Firing rate per bin is calculated by dividing the total number of spikes by the time spent in that bin. Only bins where the fish visited more than 5 times are used for the plot. The range of the color plot was clipped to the 97th percentile of the data for visual representation purposes (see Methods). The value of the maximum firing rate/ bin (Max FR/bin) is indicated above the plot. The place information for the unit and its level of significance compared to randomly time shifted spike trains are indicated as I_place and p_place, respectively. B. Same as A but for units that did not show statistically significant place specificity. The green rectangle corresponds to fish’s home, the black trapezoid in the bottom three plots show the location of a water filter. Other shapes represent various landmarks placed in the tank. Note that the fish could go inside the home area, but not other landmarks. Small cyan squares denote bins excluded from analysis due to visit counts less than 5 (see Methods). References D -4 -3 -2 -1 0 1 2 3 4 0 0.1 0.2 Probability density C E -4 -2 0 2 4 4.8 5.2 5.6 6 Speed (cm/s) D C E -4 -2 0 2 4 -4 -2 0 2 4 11 12 13 14 Sampling density (EOD pulses/cm) Time (s) F -4 -3 -2 -1 0 1 2 3 4 0 0.1 0.2 Time (s) Probability density E F F Fig 4. Examples stEODr, stSpeed and stSmpD averages. A. StEODr average for an exemplar unit (solid red curve, dotted curve: standard error). The black curve shows the stEODr average for 100 random time shifts of the same spike train. The dashed vertical line corresponds to the spike time (zero). B. Probability density of timing of stEODr peaks around individual spikes for units with significant peak in stEODr average (orange bars, 13 units in 5 fish) and for 100 random time shifts of the same spike trains (grey bars). There was a clear peak of stEODr around zero that did not exist in the random data. C. StSpeed average (red curve) for the same unit shown in A, and the corresponding average for randomly shifted spike times (black curve). Arrows point to the dip and peak in the stSpeed pre- and post- spike respectively. D. Probability density of speed dips (blue) preceding the spike and peaks (orange) after spikes for units with a significant peak in stSpeed average (14 units in 5 fish, grey: randomly time-shifted spike trains). E. stSmpD average for the same unit. stSmpD showed a clear peak before the spike and sharply decreased immediately post-spike, with the minimum occurring after spike time. F. Probability distribution of timing of the peak (orange) and dip (blue) in sampling density for individual spikes of units with significant peaks in both stEODr and stSpeed shows the same pattern of a pre-spike increase followed by a post-spike decrease (10 units in 5 fish). Fits in all histograms are non-parametric fits with Gaussian kernels. B . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. References Firing rate was significantly lower in after landmarks were removed. Kruskal-Wallis p value is indicated on the plot. I_place=0.52 bits/spike p_place=0.0007 I_place=0.34 bits/spike p_place=0.0045 ii iii I_place=1.3 bits/spike p_place=0.99 I_place=0.34 bits/spike p_place=0.0045 0 0.02 0.04 0.06 0.08 Firing rate (Hz) LM present LM removed B 0 0.2 0.4 0.6 0.8 1 LM present LM removed p=0.0027 B Fig 6. Removing landmarks often resulted in reduction of firing rate near the missing landmark A E l f th ff t f i di l i l d k i th it i th fi h I i Fig 6. Removing landmarks often resulted in reduction of firing rate near the missing landmark. A. Examples of the effect of removing or displacing a landmark in three units in three fish. In i and ii the home was removed and in iii the small triangle was moved to a new location. Note that spikes now occur near the triangle in its new location. Place information and its significance level compared to randomly time-shifted spikes are shown above each panel. Arrows point to the location of the moved landmark. B. Summary plot of the effect of landmark removal on the firing rate of 10 units in 3 fish, 16 trials. In 12 out of 16 trials the firing rate decreased near the removed landmark. Inset: comparison of the normalized firing rates between the two conditions for the same data set. Firing rate was significantly lower in after landmarks were removed. Kruskal-Wallis p value is indicated on the plot. Fig 6. Removing landmarks often resulted in reduction of firing rate near the missing landmark. A. Examples of the effect of removing or displacing a landmark in three units in three fish. In i and ii the home was removed and in iii the small triangle was moved to a new location. Note that spikes now occur near the triangle in its new location. Place information and its significance level compared to randomly time-shifted spikes are shown above each panel. Arrows point to the location of the moved landmark. B. Summary plot of the effect of landmark removal on the firing rate of 10 units in 3 fish, 16 trials. In 12 out of 16 trials the firing rate decreased near the removed landmark. Inset: comparison of the normalized firing rates between the two conditions for the same data set. References ; https://doi.org/10.1101/495887 doi: bioRxiv preprint 0 0.02 0.04 0.06 0.08 Firing rate (Hz) LM present LM removed A B I_place=0.52 bits/spike p_place=0.0007 I_place=0.55 bits/spike p_place=0.4 I_place=0.34 bits/spike p_place=0.0045 I_place=1.3 bits/spike p_place=0.99 I_place=1.22 bits/spike p_place=0.65 I_place=1.7 bits/spike p_place=0.86 i ii iii 0 0.2 0.4 0.6 0.8 1 LM present LM removed p=0.0027 CC-BY 4.0 Intern under a not certified by peer review) is the author/funder, who has granted bioRxiv this version posted Dec ; https://doi.org/10.1101/495887 doi: bioRxiv preprint A B I_place=0.52 bits/spike p_place=0.0007 I_place=0.55 bits/s p_place=0.4 I_place=0.34 bits/spike p_place=0.0045 I_place=1.3 bits/sp p_place=0.99 I_place=1.22 bits/spike p_place=0.65 I_place=1.7 bits/spi p_place=0.86 i ii iii CC-BY under a not certified by peer review) is the author/funder, who has grant this version ; https://doi.org/10.1101/495887 doi: bioRxiv preprint A I_place=0.52 bits/spike p_place=0.0007 I_place=0.34 bits/spike p_place=0.0045 I_place=1.22 bits/spike p_place=0.65 i ii iii not certified by peer review) is the auth https://doi.org/10.11 doi: bioRxiv preprint 0 0.02 0.04 0.06 0.08 Firing rate (Hz) LM present LM removed A B I_place=0.52 bits/spike p_place=0.0007 I_place=0.55 bits/spike p_place=0.4 I_place=0.34 bits/spike p_place=0.0045 I_place=1.3 bits/spike p_place=0.99 I_place=1.22 bits/spike p_place=0.65 I_place=1.7 bits/spike p_place=0.86 i ii iii 0 0.2 0.4 0.6 0.8 1 LM present LM removed p=0.0027 CC-BY 4.0 Inter under a not certified by peer review) is the author/funder, who has granted bioRxiv this version posted De ; https://doi.org/10.1101/495887 doi: bioRxiv preprint A bio 0 0.02 0.04 0.06 0.08 Firing rate (Hz) LM present LM removed B I_place=0.52 bits/spike p_place=0.0007 I_place=0.55 bits/spike p_place=0.4 I_place=0.34 bits/spike p_place=0.0045 I_place=1.3 bits/spike p_place=0.99 i ii iii 0 0.2 0.4 0.6 0.8 1 LM present LM removed p=0.0027 Fig 6. Removing landmarks often resulted in reduction of firing rate near the missing landmark. A. Examples of the effect of removing or displacing a landmark in three units in three fish. In i and ii the home was removed and in iii the small triangle was moved to a new location. Note that spikes now occur near the triangle in its new location. Place information and its significance level compared to randomly time-shifted spikes are shown above each panel. Arrows point to the location of the moved landmark. B. Summary plot of the effect of landmark removal on the firing rate of 10 units in 3 fish, 16 trials. In 12 out of 16 trials the firing rate decreased near the removed landmark. Inset: comparison of the normalized firing rates between the two conditions for the same data set. References Firing rate/bin (Hz) Firing rate/bin (Hz) 0 0.01 0.02 0 0.1 0.2 I_place=1.4 bits/spike p_place=0.03 Max FR/bin =0.06 Hz I_place=0.68 bits/spike p_place=0.02 Max FR/bin =0.48 Hz Firing rate/bin (Hz) Firing rate/bin (Hz) Firing rate/bin (Hz) Firing rate/bin (Hz) Firing rate/bin (Hz) Firing rate/bin (Hz) Fig 5. Spatial properties of DDi units. A. Left columns: Examples of spiking patterns of four units in four fish that conveyed significant place information. Grey curves show fish’s swimming trajectory, and black dots represent spikes. Right column: Firing rate maps of the same units shown to the left. Firing rate per bin is calculated by dividing the total number of spikes by the time spent in that bin. Only bins where the fish visited more than 5 times are used for the plot. The range of the color plot was clipped to the 97th percentile of the data for visual representation purposes (see Methods). The value of the maximum firing rate/ bin (Max FR/bin) is indicated above the plot. The place information for the unit and its level of significance compared to randomly time shifted spike trains are indicated as I_place and p_place, respectively. B. Same as A but for units that did not show statistically significant place specificity. The green rectangle corresponds to fish’s home, the black trapezoid in the bottom three plots show the location of a water filter. Other shapes represent various landmarks placed in the tank. Note that the fish could go inside the home area, but not other landmarks. Small cyan squares denote bins excluded from analysis due to visit counts less than 5 (see Methods). 0 0.02 0.04 0.06 0.08 Firing rate (Hz) LM present LM removed A B I_place=0.52 bits/spike p_place=0.0007 I_place=0.55 bits/spike p_place=0.4 I_place=0.34 bits/spike p_place=0.0045 I_place=1.3 bits/spike p_place=0.99 I_place=1.22 bits/spike p_place=0.65 I_place=1.7 bits/spike p_place=0.86 i ii iii 0 0.2 0.4 0.6 0.8 1 LM present LM removed p=0.0027 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made availa The copyright holder for this preprint (which this version posted December 13, 2018. References Firing rate was significantly lower in after landmarks were removed. Kruskal-Wallis p value is indicated on the plot. Probability density Swim direction preference index A Forward swims Backward swims -0.8 -0.4 0 0.4 0.8 0 0.5 1 1.5 1 Probability density Swim direction preference index A Forward swims Backward swims -0.8 -0.4 0 0.4 0.8 0 0.5 1 1.5 L-R preference index A-P preference index LM Ant LM Post Location preference index Probability density LM right LM left 0 0.2 0.4 0.6 0.8 1 10 cm d Location preference index -0.4 -0.2 0 0.2 0.4 -0.4 -0.2 0 0.2 0.4 a b c d B C D E -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0 1 2 3 4 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0 1 2 3 4 5 a b c d Fig 7. Relationship between swimming direction and preferred landmark location. A. Probability distribution of direction preference index (21 units, 5 fish). Negative values correspond to preference for spiking during backward swims, with the maxima +/-1 corresponding to spiking only during forward or backward swims, respectively. The red curve shows a non-parametric fit with Gaussian kernels. There was a significant bias for spiking for negative swim direction preference indices (p = 0.0072, non- parametric sign test). B. Examples of normalized landmark location probability. Units b and c are from the same fish and other units are from three other fish. C. Vector plot of the direction preference indices for anterior-posterior (AP) and left- right (LR) preference indices. Small letters next to the arrows correspond to exemplar units shown in panel B. D. Most units showed preference index for landmarks that were located on the posterior 2/3 of the body (blue shaded area). E. Left- right preference indices were equally likely to be positive or negative. . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made av The copyright holder for this preprint (wh this version posted December 13, 2018. References ; https://doi.org/10.1101/495887 doi: bioRxiv preprint 0 0.2 0.4 0.6 0.8 1 0 cm d a b c d B 10 cm a b c B B B A 0 0.2 0.4 0.6 0.8 1 d d d Swim direction preference index LM right LM left Location preference index E -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 ay the preprint in perpetuity. It is made av The copyright holder for this preprint (wh . L-R preference index A-P preference index LM Ant LM Post Location preference index Probability density -0.4 -0.2 0 0.2 0.4 -0.4 -0.2 0 0.2 0.4 C D 0 1 2 3 4 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0 1 2 3 4 5 a b c d . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to displa this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint E D C Location preference index L-R preference index Fig 7. Relationship between swimming direction and preferred landmark location. A. Probability distribution of direction preference index (21 units, 5 fish). Negative values correspond to preference for spiking during backward swims, with the maxima +/-1 corresponding to spiking only during forward or backward swims, respectively. The red curve shows a non-parametric fit with Gaussian kernels. There was a significant bias for spiking for negative swim direction preference indices (p = 0.0072, non- parametric sign test). B. Examples of normalized landmark location probability. Units b and c are from the same fish and other units are from three other fish. C. Vector plot of the direction preference indices for anterior-posterior (AP) and left- right (LR) preference indices. Small letters next to the arrows correspond to exemplar units shown in panel B. D. Most units showed preference index for landmarks that were located on the posterior 2/3 of the body (blue shaded area). E. Left- right preference indices were equally likely to be positive or negative. tetrode location Fig S1. Cresyl violet stained section through the pallium of an implanted fish illustrating the location of the tetrode. Small arrows (right side) indicate the sulci used as an aid in placing the tetrode. Note that the tetrode track passed through the superficial DD (DDs) and ended within DDi. The much lower cell density in DDi compared to DL is evident in this section. . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint References DC: central division of dorsal telencephalon DDi: intermediate division of the dorsal portion of dorsal telencephalon (DD, pallium) DDs: superficial division of the dorsal portion of DD DL: dorsolateral pallium DM: dorsomedial pallium tetrode location tetrode location Fig S1. Cresyl violet stained section through the pallium of an implanted fish illustrating the location of the tetrode. Small arrows (right side) indicate the sulci used as an aid in placing the tetrode. Note that the tetrode track passed through the superficial DD (DDs) and ended within DDi. The much lower cell density in DDi compared to DL is evident in this section. DC: central division of dorsal telencephalon DDi: intermediate division of the dorsal portion of dorsal telencephalon (DD, pallium) DDs: superficial division of the dorsal portion of DD DL: dorsolateral pallium DM: dorsomedial pallium Fig S1. Cresyl violet stained section through the pallium of an implanted fish illustrating the location of the tetrode. Small arrows (right side) indicate the sulci used as an aid in placing the tetrode. Note that the tetrode track passed through the superficial DD (DDs) and ended within DDi. The much lower cell density in DDi compared to DL is evident in this section. DC: central division of dorsal telencephalon DDi: intermediate division of the dorsal portion of dorsal telencephalon (DD, pallium) DDs: superficial division of the dorsal portion of DD DL: dorsolateral pallium DM: dorsomedial pallium Fish 1 Fish 1 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. References ; https://doi.org/10.1101/495887 doi: bioRxiv preprint 56.2 56.6 57 57.4 4 4.4 4.8 5.2 5.6 14 16 18 20 56.5 57.5 58.5 4 4.4 4.8 5.2 5.6 14 16 18 56.4 56.8 57.2 57.6 3.8 4 4.4 4.8 5.2 5.6 15 17 19 21 56 57 58 3.5 4 4.5 5 5.5 14 18 22 26 -4 -2 0 2 4 56 56.4 56.8 57.2 57.6 -4 -2 0 2 4 3 4 5 -4 -2 0 2 4 15 17 19 21 23 S S S NS NS EODr (Hz) Speed (cm/s) Sampling density (spiks/cm) Time (s) Time (s) Time (s) P P P S S S NS NS 56.2 56.6 57 57.4 4 4.4 4.8 5.2 5.6 14 16 18 20 56.4 56.8 57.2 57.6 3.8 4 4.4 4.8 5.2 5.6 15 17 19 21 S S EODr (Hz) Speed (cm/s) Sampling density (spiks/cm) P P S S 0 Sampling density (spiks/cm) 0 Sampling density (spiks/cm) 19 56.5 57.5 58.5 S P 4 4.4 4.8 5.2 5.6 S 4 4.4 4.8 5.2 5.6 14 16 18 S 56 57 58 NS 3.5 4 4.5 5 5.5 14 18 22 26 NS -4 -2 0 2 4 3 4 5 -4 -2 0 2 4 15 17 19 21 23 Time (s) Time (s) NS -4 -2 0 2 4 56 56.4 56.8 57.2 57.6 NS Time (s) -4 -2 0 2 4 3 4 5 Time (s) NS Time (s) Fish 2 Sampling density . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint Sampling density . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. References ; https://doi.org/10.1101/495887 doi: bioRxiv preprint 56.6 56.8 57 57.2 57.4 4.2 4.4 4.6 4.8 15.5 16 16.5 17 17.5 S 56.5 56.7 56.9 57.1 57.3 4.2 4.6 5 5.4 14.5 15.5 16.5 17.5 S 55.8 56 56.2 56.4 56.6 4 4.4 4.8 5.2 14 16 18 S 57.4 57.8 58.2 58.6 4 4.4 4.8 5.2 5.6 14 16 18 20 NS 56.1 56.3 56.5 56.7 3.6 4 4.4 4.8 5.2 14 16 18 20 NS -4 -2 0 2 4 56.4 56.8 57.2 57.6 -4 -2 0 2 4 3.5 4.5 5.5 -4 -2 0 2 4 12 16 20 24 NS EODr (Hz) Speed (cm/s) Sampling density (spiks/cm) Time (s) Time (s) Time (s) P P P S NS NS NS S S . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpet py g p , ; p g p p 4.2 4.4 4.6 4.8 15.5 16 16.5 17 17.5 Speed (cm/s) Sampling density (spiks/cm) S . CC-BY 4.0 International license under a Sampling density (spiks/cm) . cense 56.6 56.8 57 57.2 57.4 S EODr (Hz) P ampling density (spiks/cm) 4.2 4.6 5 5.4 14.5 15.5 16.5 17.5 NS 56.5 56.7 56.9 57.1 57.3 S P 4 4.4 4.8 5.2 14 16 18 NS 55.8 56 56.2 56.4 56.6 S 4 4.4 4.8 5.2 NS 57.4 57.8 58.2 58.6 4 4.4 4.8 5.2 5.6 14 16 18 20 NS 56.1 56.3 56.5 56.7 3.6 4 4.4 4.8 5.2 14 16 18 20 NS -4 -2 0 2 4 56.4 56.8 57.2 57.6 -4 -2 0 2 4 3.5 4.5 5.5 -4 -2 0 2 4 12 16 20 24 NS Time (s) Time (s) Time (s) P NS S S 57.4 57.8 58.2 58.6 4 4.4 4.8 5.2 5.6 NS P NS 4 4.4 4.8 5.2 5.6 NS 3.6 4 4.4 4.8 5.2 S -4 -2 0 2 4 3.5 4.5 5.5 -4 -2 0 2 4 12 16 20 24 Time (s) Time (s) S Figure S2 Figure S2 Fish 3 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. References ; https://doi.org/10.1101/495887 doi: bioRxiv preprint Fish 3 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint -4 -2 0 2 4 62 63 64 65 -4 -2 0 2 4 6 7 8 9 -4 -2 0 2 4 12 13 14 15 16 Fish 3 EODr (Hz) Speed (cm/s) Sampling density (spiks/cm) Time (s) Time (s) Time (s) S S . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint -4 -2 0 2 4 6 7 8 9 -4 -2 0 2 4 12 13 14 15 16 Speed (cm/s) Sampling density (spiks/cm) Time (s) Time (s) S -4 -2 0 2 4 12 13 14 15 16 Sampling density (spiks/cm) Time (s) Sampling density (spiks/cm) -4 -2 0 2 4 6 7 8 9 Speed (cm/s) Time (s) S -4 -2 0 2 4 62 63 64 65 EODr (Hz) Time (s) S Time (s) Figure S2 Fish 4 53.6 54 54.4 54.8 4.5 5.5 6.5 11 13 15 17 53.4 53.8 54.2 54.6 4.8 5.2 5.6 6 11 12 13 14 15 53.6 53.8 54 54.2 54.4 4.6 5 5.4 13 14 15 16 53.6 54 54.2 54.6 4.8 5.2 5.6 12 13 14 15 16 53.5 54.5 55.5 4.4 4.8 5.2 5.6 6 12 14 16 S S S S S 53.5 54.5 55.5 3.5 4.5 5.5 6.5 10 14 18 22 26 54.5 55 6.5 7.5 18 22 NS EODr (Hz) Speed (cm/s) Sampling density (spiks/cm) Fish 4 NS S S S S S P P P . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. References ; https://doi.org/10.1101/495887 doi: xiv preprint 15 53.6 54 54.2 54.6 4.8 5.2 5.6 12 13 14 15 16 S S 53.6 54 54.2 54.6 S 4.6 5 5.4 13 14 15 16 S 53.6 53.8 54 54.2 54.4 S P 53.4 53.8 54.2 54.6 S P 53.5 54.5 55.5 4.4 4.8 5.2 5.6 6 12 14 16 S NS 53.5 54.5 55.5 S 3.5 4.5 5.5 6.5 10 14 18 22 26 S 53.5 54.5 55.5 NS 4 -4 -2 0 2 4 3.5 4.5 5.5 6.5 7.5 -4 -2 0 2 4 10 14 18 22 Time (s) Time (s) S -4 -2 0 2 4 53.5 54 54.5 55 -4 -2 0 2 4 3.5 4.5 5.5 6.5 7.5 -4 -2 0 2 4 10 14 18 22 NS Time (s) Time (s) Time (s) S P Time (s) Figure S2 Figure S2 Fish 5 62 64 66 68 4 6 8 10 10 14 18 22 S EODr (Hz) Speed (cm/s) Sampling density (spiks/cm) -4 -2 0 2 4 62 64 66 68 -4 -2 0 2 4 4 5 6 7 8 -4 -2 0 2 4 12 16 20 NS Time (s) Time (s) Time (s) P S NS . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint 62 64 66 68 4 6 8 10 10 14 18 22 S EODr (Hz) Speed (cm/s) Sampling density (spiks/cm) -4 -2 0 2 4 62 64 66 68 -4 -2 0 2 4 4 5 6 7 8 -4 -2 0 2 4 12 16 20 NS Time (s) Time (s) Time (s) P S NS Fig S2. stEOD, stSpeed and stSmpD averages for all units and all fish (red curves). Black curves show the average for 100 random time- shifts of the same spike train. Dashed curves are standard errors. Units with significant (non-significant) peak in their stEOD average are labeled with S (NS). Units that further showed significant place specificity are denoted with a ‘P’. . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. References ; https://doi.org/10.1101/495887 doi: oRxiv preprint 53.6 54 54.4 54.8 4.5 5.5 6.5 11 13 15 17 S EODr (Hz) Speed (cm/s) Sampling density (spiks/cm) Fish 4 S . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint 53.6 54 54.4 54.8 4.5 5.5 6.5 11 13 15 17 53.4 53.8 54.2 54.6 4.8 5.2 5.6 6 11 12 13 14 15 53.6 53.8 54 54.2 54.4 4.6 5 5.4 13 14 15 16 53.6 54 54.2 54.6 4.8 5.2 5.6 12 13 14 15 16 53.5 54.5 55.5 4.4 4.8 5.2 5.6 6 12 14 16 S S S S S 53.5 54.5 55.5 3.5 4.5 5.5 6.5 10 14 18 22 26 -4 -2 0 2 4 53.5 54 54.5 55 -4 -2 0 2 4 3.5 4.5 5.5 6.5 7.5 -4 -2 0 2 4 10 14 18 22 NS NS EODr (Hz) Speed (cm/s) Sampling density (spiks/cm) Time (s) Time (s) Time (s) NS S S S S S S P P P . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in p The copyright hol this version posted December 13, 2018. References It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: ioRxiv preprint -4 -2 0 2 4 4 5 6 7 8 -4 -2 0 2 4 12 16 20 Time (s) Time (s) NS Fig S2. stEOD, stSpeed and stSmpD averages for all units and all fish (red curves). Black curves show the average for 100 random time- shifts of the same spike train. Dashed curves are standard errors. Units with significant (non-significant) peak in their stEOD average are labeled with S (NS). Units that further showed significant place specificity are denoted with a ‘P’. Fig S2. stEOD, stSpeed and stSmpD averages for all units and all fish (red curves). Black curves show the average for 100 random time- shifts of the same spike train. Dashed curves are standard errors. Units with significant (non-significant) peak in their stEOD average are labeled with S (NS). Units that further showed significant place specificity are denoted with a ‘P’. . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: ioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted December 13, 2018. ; https://doi.org/10.1101/495887 doi: bioRxiv preprint 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1-P place 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1-P stEODr average Fig S3. Significance levels for place information and the peak amplitude for the stEODr average for each of the 21 units recorded in 5 fish (1- p values). 11 out of 21 units conveyed significant place information (1-p> 0.95, red and purple circles), 8 of which also had a significant peak in their stEODr average (red circles). Out of the 10 units which did not show place specificity, 5 had significant peaks in their stEODr average (green circles). 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1-P place 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1-P stEODr average Fig S3. References Significance levels for place information and the peak amplitude for the stEODr average for each of the 21 units recorded in 5 fish (1- p values). 11 out of 21 units conveyed significant place information (1-p> 0.95, red and purple circles), 8 of which also had a significant peak in their stEODr average (red circles). Out of the 10 units which did not show place specificity, 5 had significant peaks in their stEODr average (green circles).
https://openalex.org/W2081257571	https://europepmc.org/articles/pmc4310241?pdf=render	Latin	null	Coronary Arteries Segmentation Based on the 3D Discrete Wavelet Transform and 3D Neutrosophic Transform	BioMed research international	2,015	cc-by	5,436	Shuo-Tsung Chen,1,2,3 Tzung-Dau Wang,4 Wen-Jeng Lee,5 Tsai-Wei Huang,6 Pei-Kai Hung,1 Cheng-Yu Wei,7,8 Chung-Ming Chen,1 and Woon-Man Kung7,9 Shuo-Tsung Chen,1,2,3 Tzung-Dau Wang,4 Wen-Jeng Lee,5 Tsai-Wei Huang,6 Pei-Kai Hung,1 Cheng-Yu Wei,7,8 Chung-Ming Chen,1 and Woon-Man Kung7,9 1Institute of Biomedical Engineering, National Taiwan University, Taipei 10617, Taiwan 2Department of Applied Mathematics, Tunghai University, Taichung 40704, Taiwan 3Sustainability Research Center, Tunghai University, Taichung 40704, Taiwan 4Cardiovascular Center and Division of Cardiology, Department of Internal Medicine, National Taiwan University Hospital, Taipei 10048, Taiwan 5Department of Medical Imaging, National Taiwan University Hospital, Taipei 10048, Taiwan 6Department of Nursing, College of Medicine and Nursing, Hungkuang University, Taichung 43302, Taiwan 7Department of Exercise and Health Promotion, College of Education, Chinese Culture University, Taipei 11114, Taiwan 8Department of Neurology, Chang Bing Show Chwan Memorial Hospital, Changhua 50544, Taiwan 9Department of Neurosurgery, Lo-Hsu Foundation, Lotung Poh-Ai Hospital, Luodong, Yilan 26546, Taiwan 1Institute of Biomedical Engineering, National Taiwan University, Taipei 10617, Taiwan 2Department of Applied Mathematics, Tunghai University, Taichung 40704, Taiwan 1Institute of Biomedical Engineering, National Taiwan University, Taipei 10617, Taiwan 2D f A li d M h i T h i U i i T i h 0 0 T i Department of Exercise and Health Promotion, College of Education, Chinese Culture University, Taipei 11114, Taiw Department of Neurology, Chang Bing Show Chwan Memorial Hospital, Changhua 50544, Taiwan Department of Neurosurgery, Lo-Hsu Foundation, Lotung Poh-Ai Hospital, Luodong, Yilan 26546, Taiwan orrespondence should be addressed to Chung-Ming Chen; chung@ntu.edu.tw and Woon-Man Kung; nskungwm Correspondence should be addressed to Chung-Ming Chen; chung@ntu.edu.tw and Woon-Man Kung; nskungw Received 19 July 2014; Accepted 11 October 2014 Received 19 July 2014; Accepted 11 October 2014 Received 19 July 2014; Accepted 11 October 2014 Academic Editor: Kuo-Sheng Hung Academic Editor: Kuo-Sheng Hung Copyright © 2015 Shuo-Tsung Chen et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Purpose. Most applications in the field of medical image processing require precise estimation. To improve the accuracy of segmentation, this study aimed to propose a novel segmentation method for coronary arteries to allow for the automatic and accurate detection of coronary pathologies. Methods. The proposed segmentation method included 2 parts. First, 3D region growing was applied to give the initial segmentation of coronary arteries. Next, the location of vessel information, HHH subband coefficients of the 3D DWT, was detected by the proposed vessel-texture discrimination algorithm. Hindawi Publishing Corporation BioMed Research International Volume 2015, Article ID 798303, 9 pages http://dx.doi.org/10.1155/2015/798303 Hindawi Publishing Corporation BioMed Research International Volume 2015, Article ID 798303, 9 pages http://dx.doi.org/10.1155/2015/798303 Hindawi Publishing Corporation BioMed Research International Volume 2015, Article ID 798303, 9 pages http://dx.doi.org/10.1155/2015/798303 Shuo-Tsung Chen,1,2,3 Tzung-Dau Wang,4 Wen-Jeng Lee,5 Tsai-Wei Huang,6 Pei-Kai Hung,1 Cheng-Yu Wei,7,8 Chung-Ming Chen,1 and Woon-Man Kung7,9 Based on the initial segmentation, 3D DWT integrated with the 3D neutrosophic transformation could accurately detect the coronary arteries. Results. Each subbranch of the segmented coronary arteries was segmented correctly by the proposed method. The obtained results are compared with those ground truth values obtained from the commercial software from GE Healthcare and the level-set method proposed by Yang et al., 2007. Results indicate that the proposed method is better in terms of efficiency analyzed. Conclusion. Based on the initial segmentation of coronary arteries obtained from 3D region growing, one-level 3D DWT and 3D neutrosophic transformation can be applied to detect coronary pathologies accurately. Research Article Coronary Arteries Segmentation Based on the 3D Discrete Wavelet Transform and 3D Neutrosophic Transform Shuo-Tsung Chen,1,2,3 Tzung-Dau Wang,4 Wen-Jeng Lee,5 Tsai-Wei Huang,6 Pei-Kai Hung,1 Cheng-Yu Wei,7,8 Chung-Ming Chen,1 and Woon-Man Kung7,9 1. Introduction they usually obtain a seriously narrowed segmentation in the neighborhood of a branch point in the vessel. Level-set approaches [6–13] are computationally expensive. They also suffer from leakage at places where the intensity gradients of the edges are relatively weak and are very sensitive to the placement of the initial contour of the propagating front. Metz et al. [14] used the minimum cost path of the specified start and end points in vessel to detect the coronary arteries centerline. This is not an automatic method; detecting branches is difficult. Friman [15] proposed multiple hypothesis template tracking, which follows the direction of Efficient and automatic image segmentation methods are useful for the isolation and visualization of vessels in com- puted tomographic angiography (CTA). There are many proposed methods for the segmentation of vessels [1–14]. A vessel filter [1] can be used to enhance tubular structure; however, it cannot address the problem of the image force and veins, which can lead to a narrowed or broken seg- mentation of vessels. Parametric shape models [2–5] do not directly allow for the detection of topological changes, and 2 2 BioMed Research International centerline obtained in advance. However, it is difficult to detect small branches and vessels by using this method. where 𝑖and 𝑛are the dilation and translation parameters. Orthogonal wavelet basis functions not only provide a simple method to calculate coefficient expansion but also span 𝐿2(R) in signal processing. As a result, signal 𝑆(𝑡) ∈𝐿2(R) can be expressed as a series expansion of orthogonal scaling functions and wavelets. More specifically, y g In this study, we propose a new method for automatically and correctly segmenting coronary arteries from CTA data sets. In image preprocessing, we detected the aorta auto- matically by using methods proposed in the literature [5, 16]. The proposed coronary arteries segmentation method is summarized as follows. First of all, we automatically obtained the seed point of a 3D region growing by the difference between the two adjacent slices due to the small changes of the aorta between two adjacent slices. Next, 3D region growing was applied to initially search for the probable location of coronary arteries, which was then dilated by 3 voxels. Based on the dilation of the probable location, we detected the coronary arteries accurately by applying the 3D discrete wavelet transformation (DWT) and 3D neutrosophic transformation to the CTA volume. 1. Introduction The location of vessel information, in HHH subband coefficients, was detected by the proposed vessel-texture discrimination algorithm. Accordingly, HHH subband coefficients were used, which were characterized and classified by 𝛼-means operation and 𝐾-means clustering. Finally, the proposed method was tested on several CTA data sets, and the experimental results indicated that the proposed method had a good performance.h 𝑆(𝑡) = ∑ ℓ 𝑐𝑗0 (ℓ) 𝜑𝑗0,𝑘(𝑡) + ∑ 𝑘 ∞ ∑ 𝑗=𝑗0 𝑑𝑗(𝑘) 𝜓𝑗,𝑘(𝑡) , (2) (2) where 𝑐𝑗(ℓ) = ∫R 𝑆(𝑡)𝜑𝑗,ℓ(𝑡)𝑑𝑡and 𝑑𝑗(𝑘) = ∫R 𝑆(𝑡)𝜓𝑗,𝑘(𝑡)𝑑𝑡 are the low-pass and high-pass coefficients, respectively; 𝑗0 is an integer to define an interval on which 𝑆(𝑡) is a piecewise constant. The two-scale equations for scaling and wavelet basis function are given as follows: 𝜑(𝑡) = √2 ∑ 𝑚∈Z ℎ𝑚𝜑(2𝑡−𝑚) , (3) 𝜓(𝑡) = √2 ∑ 𝑚∈Z 𝑔𝑚𝜑(2𝑡−𝑚) , (4) (3) (4) where 𝑔𝑚= (−1)𝑚ℎ1−𝑚. The coefficient ℎ𝑚in (3) has to meet several conditions for the set of the wavelet basis function to be unique and orthonormal and have a certain degree of regularity.hfi The rest of this study is organized as follows. Section 2 reviews some preliminaries, and Section 3 uses 3D region growing and 3D DWT to propose a new method for segment- ing coronary arteries. Section 4 contains the experiments and discussion, and the conclusions are drawn in Section 5. The coefficients ℎ𝑚and 𝑔𝑚play a very crucial role in a given DWT. Performing the wavelet transformation does not require the explicit forms of 𝜑(𝑡) and 𝜓(𝑡) but only depends on ℎ𝑚and 𝑔𝑚. The final output of the wavelet decomposition includes a set of 𝑗-level wavelet coefficients. One method to implement DWT is to use a filter bank that provides perfect reconstruction. DWT involves local analysis of frequency in space and time domains, and it provides multiscale image details step by step. If the scale becomes smaller, every part becomes more accurate, and ultimately all imaging details can be focalized accurately. If DWT is applied to a volume, it will produce the highest-frequency, middle-frequency, and lowest-frequency parts. Figure 1 shows the results of applying 3D DWT to a volume, which includes eight parts: LLL, LLH, LHL, LHH, HLL, HLH, HHL, and HHH. The lowest- frequency and highest-frequency parts are LLL and HHH, respectively [16, 17]. 3. The Proposed Segmentation Method growing. We chose a 26-connected neighborhood for our adjacent pixel relationship, and then the 3D region growing method was applied with a set of prespecified seed voxel(s) and grown from these seeds by merging neighboring voxels whose properties were most similar to the premerged region.hif growing. We chose a 26-connected neighborhood for our adjacent pixel relationship, and then the 3D region growing method was applied with a set of prespecified seed voxel(s) and grown from these seeds by merging neighboring voxels whose properties were most similar to the premerged region. The homogeneity criterion was defined as the difference between the intensity of the candidate voxel and the average intensity of the premerged region. The selection of the seed point was initially intended to check which slice began the information of the coronary arteries. Next, the homogeneity criterion was applied to group voxels with the same or similar intensities into one region. If the homogeneity criterion was satisfied, the candidate voxel was merged with the premerged region. The process was repeated until no more voxels were assigned to the region, and then the number of all merged voxels was calculated. In order to avoid leakage, the total number of merged voxels was limited to 12000; otherwise, 3D region growing was restarted by automatically using an improved homogeneity criterion. Finally, the initial segmentation of the coronary arteries in a volume was completed. In order to segment coronary arteries accurately from CTA data sets, 3D region growing was initially applied to search for the probable location of the coronary arteries. Next, we used the 3D DWT and 3D neutrosophic transformation to accurately detect the coronary arteries. 3.1. Initial Segmentation of Coronary Arteries. This section discusses the initial segmentation of coronary arteries using 3D region growing. In image preprocessing, we found the aorta automatically by using methods proposed in the liter- ature [5, 16]. The selection of the seed point of 3D region growing was initially made to check which slice began the information of the coronary arteries. Due to the small changes in aorta area between the two adjacent slices, we automatically obtained the seed points by the difference between the two adjacent slices.f As shown in Figure 2, we use the difference between the two adjacent slices (a) and (b) to automatically obtain the seed points bounded by the blue line in (c) which indicates the boundary of coronary arteries. 2. Preliminaries In this section, we will briefly introduce the concepts of DWT and provide an overview of some fundamental mathematical concepts that are used in this study. 2.1. Region Growing. Region growing is a simple, well- developed, region-based image segmentation technique [17]. It postulates that neighboring voxels within the same region have similar intensity values and is also classified as a voxel- based image segmentation method since it involves the selection of initial seed points. In other words, this method of segmentation examines neighboring voxels of initial seed points and determines whether neighboring voxels should be added to the region. Consequentially, the general concept of region growing is to group voxels with the same or similar intensities to one region according to the given seed points and a homogeneity criterion. 2.3. 𝐾-Means Clustering. 𝐾-means clustering is a method of cluster analysis which aims to partition 𝑛observations into 𝑘 clusters in which each observation belongs to the cluster with the nearest mean. Given a set of observations (𝑥1, 𝑥2, . . . , 𝑥𝑛), where each observation is a 𝑑-dimensional real vector, 𝑘- means clustering aims to partition the 𝑛observations into 𝑘(𝑘≤𝑛) sets {𝑠1, 𝑠2, . . . , 𝑠𝑘} so as to minimize the within- cluster sum of squares: 2.2. Discrete Wavelet Transform. Wavelet transform is obtained by a single prototype function 𝜓(𝑥) which is regulated with scaling and shift parameters. To construct 𝜓(𝑥), a scaling function 𝜑(𝑥) is determined. The discrete normalized scaling and wavelet basis functions are defined arg min 𝑘 ∑ 𝑖=1 ∑ 𝑥𝑗∈𝑠𝑖 󵄩󵄩󵄩󵄩󵄩𝑥𝑗−𝜇𝑖 󵄩󵄩󵄩󵄩󵄩 2 , (5) as (5) 𝜑𝑖,𝑛(𝑡) = 2𝑖/2𝜑(2𝑖𝑡−𝑛) , 𝜓𝑖,𝑛(𝑡) = 2𝑖/2𝜓(2𝑖𝑡−𝑛) , 𝜑𝑖,𝑛(𝑡) = 2𝑖/2𝜑(2𝑖𝑡−𝑛) , 𝜓𝑖,𝑛(𝑡) = 2𝑖/2𝜓(2𝑖𝑡−𝑛) , (1) (1) where 𝜇𝑖is the mean of points in 𝑠𝑖. where 𝜇𝑖is the mean of points in 𝑠𝑖. BioMed Research International 3 3 Rows Columns Slices Original volume LxLyLz LxLyHz LxHyLz LxHyHz HxLyLz HxLyHz HxHyLz HxHyHz LA ↓ LA ↓ LA ↓ LA ↓ LA ↓ LA ↓ LA ↓ HA ↓ HA ↓ HA ↓ HA ↓ HA ↓ HA ↓ HA ↓ Figure 1: The structure of applying 3D DWT to a volume. HA ↓ Figure 1: The structure of applying 3D DWT to a volume. 3. The Proposed Segmentation Method The boundary of coronary arteries denotes the high-frequency subband 𝑑𝑗(𝑘) in (2) when comparing vessel lumen and background. 3.2. Vessel-Texture Discrimination. According to Parseval’s theorem, the energy in a signal 𝑆(𝑡) is given as follows [16, 17]: Since coronary arteries do not exhibit abrupt intensity changes along their centerline [4], a rough tubular mask of coronary arteries can be easily constructed by 3D region ∫\|𝑆(𝑡)\|2 𝑑𝑡= ∞ ∑ 𝑙=−∞ \|𝑐(𝑙)\|2 + ∞ ∑ 𝑗=0 ∞ ∑ 𝑘=−∞ 󵄨󵄨󵄨󵄨󵄨𝑑𝑗(𝑘)󵄨󵄨󵄨󵄨󵄨 2 . (6) (6) BioMed Research International BioMed Research International 4 BioMed Research International This equation implies that the energy of a signal is the sum- mation of low-frequency and high-frequency coefficients. DWT is a good analytic tool for image texture analysis or line-based patterns [18–21]. Since a vessel is a type of 3D line- based pattern in CT volume, we used 𝑙2-norm of these wavelet coefficients to find the energy of line-based patterns. 𝑙2-norm was defined as Next, the subbands HHH were characterized by 3 member- ship sets 𝑇, 𝐹, and 𝑈. Consider This equation implies that the energy of a signal is the sum- mation of low-frequency and high-frequency coefficients. This equation implies that the energy of a signal is the sum- mation of low-frequency and high-frequency coefficients. DWT is a good analytic tool for image texture analysis or line-based patterns [18–21]. Since a vessel is a type of 3D line- based pattern in CT volume, we used 𝑙2-norm of these wavelet coefficients to find the energy of line-based patterns. 𝑙2-norm was defined as Next, the subbands HHH were characterized by 3 member- ship sets 𝑇, 𝐹, and 𝑈. Consider This equation implies that the energy of a signal is the sum- mation of low-frequency and high-frequency coefficients. q y g q yfi DWT is a good analytic tool for image texture analysis or line-based patterns [18–21]. Since a vessel is a type of 3D line- based pattern in CT volume, we used 𝑙2-norm of these wavelet coefficients to find the energy of line-based patterns. 3. The Proposed Segmentation Method 𝑙2-norm was defined as 𝑇HHH (𝑖, 𝑗, 𝑘) = HHH (𝑖, 𝑗, 𝑘) −HHHmin HHHmax −HHHmin , 𝐹HHH (𝑖, 𝑗, 𝑘) = 1 −𝑇HHH (𝑖, 𝑗, 𝑘) , 𝑈HHH (𝑖, 𝑗, 𝑘) = 𝛿(𝑖, 𝑗, 𝑘) −𝛿min 𝛿max −𝛿min , (10) 𝑇HHH (𝑖, 𝑗, 𝑘) = HHH (𝑖, 𝑗, 𝑘) −HHHmin HHHmax −HHHmin , (10) 𝐸(𝐶) = ‖𝐶‖2 = 𝑝 ∑ 𝑖=1 󵄨󵄨󵄨󵄨𝑐𝑖󵄨󵄨󵄨󵄨 2 , (7) (7) where the vector 𝐶= [𝑐𝑖]1×𝑝was the wavelet coefficients of a frequency channel. The searching algorithm is summarized as follows. where HHHmin = min {HHH (𝑖, 𝑗, 𝑘)} , HHHmax = max {HHH (𝑖, 𝑗, 𝑘)} , Algorithm 1. (1) Transform a given vessel volume into fre- quency channels by a specified number of decomposition levels. We usually set the number to one in the first search. 𝛿(𝑖, 𝑗, 𝑘) = 󵄨󵄨󵄨󵄨󵄨HHH (𝑖, 𝑗, 𝑘) −HHH (𝑖, 𝑗, 𝑘)󵄨󵄨󵄨󵄨󵄨, (11) 𝛿(𝑖, 𝑗, 𝑘) = 󵄨󵄨󵄨󵄨󵄨HHH (𝑖, 𝑗, 𝑘) −HHH (𝑖, 𝑗, 𝑘)󵄨󵄨󵄨󵄨󵄨, 𝛿min = min {𝛿(𝑖, 𝑗, 𝑘)} , 𝛿max = max {𝛿(𝑖, 𝑗, 𝑘)} . (11) (11) yi (2) Use (7) to calculate the average 𝑙2-norm of each channel and maximum of 𝐸(𝐶) for the vessel volume.i yi (2) Use (7) to calculate the average 𝑙2-norm of each channel and maximum of 𝐸(𝐶) for the vessel volume.i 𝛿min = min {𝛿(𝑖, 𝑗, 𝑘)} , 𝛿max = max {𝛿(𝑖, 𝑗, 𝑘)} . (3) If the maximum of 𝐸(𝐶) was significantly greater than another channel’s 𝐸(𝐶), the search was stopped. Otherwise, the number of decomposition levels was increased followed by a repeat of step 1. That is, a pixel could be represented as a neutrosophic domain 𝑃(𝑡, 𝑓, 𝑢) which means the pixel is 𝑡% true, 𝑓% false, and 𝑢% uncertain, where 𝑡varies in 𝑇, 𝑓varies in 𝐹, and 𝑢varies in 𝑈. In order to reduce the uncertainty 𝑢%, 𝛼-means operation was employed as follows: By using the above algorithm, we observed that the most significant information of the vessel texture often appeared in the high frequency channels. Thus, we used the subband HHH to detect vessels in this study. 𝑇(𝛼) = {𝑇, if 𝑈< 𝛼, 𝑇𝛼, if 𝑈≥𝛼, 𝑇(𝛼) = {𝑇, if 𝑈< 𝛼, 𝑇𝛼, if 𝑈≥𝛼, 3.3. Accurate Detection of Coronary Arteries. The initial segmentation of coronary arteries in a volume was completed by 3D region growing as described in Section 3.1. 3. The Proposed Segmentation Method Since region growing is a simple region-based image segmentation method, it was only used to search for the initial location of the coronary arteries. We then accurately detected the coronary arteries by applying DWT to each slice in the volume, as described in this subsection. 𝐹(𝛼) = {𝐹, if 𝑈< 𝛼, 𝐹𝛼, if 𝑈≥𝛼, (12) (12) 𝑈𝛼(𝑖, 𝑗, 𝑘) = 𝛿𝑇(𝑖, 𝑗, 𝑘) −𝛿𝑇min 𝛿𝑇max −𝛿𝑇min , First, the initial location of the coronary arteries obtained from 3D region growing was dilated by 3 voxels. Next, we used the Haar wavelet bases in (1) to transform the host images into the orthogonal DWT domain by one-level decomposition. Only HHH subbands were employed for further processes, because most of the information on the coronary arteries and boundaries were in the HHH subbands. We calculated the mean energy using coefficients of HHH subbands in a local window 𝑤as follows: where the parameter 𝛼is a positive number and where the parameter 𝛼is a positive number and 𝑇𝛼(𝑖, 𝑗, 𝑘) = 1 𝑤× 𝑤× 𝑤 𝑟 ∑ 𝑙=𝑖 𝑠 ∑ 𝑚=𝑗 𝑡 ∑ 𝑛=𝑘 𝑇(𝑙, 𝑚, 𝑛) , 𝐹𝛼(𝑖, 𝑗, 𝑘) = 1 𝑤× 𝑤× 𝑤 𝑟 ∑ 𝑙=𝑖 𝑠 ∑ 𝑚=𝑗 𝑡 ∑ 𝑛=𝑘 𝐹(𝑙, 𝑚, 𝑛) , 𝛿𝑇(𝑖, 𝑗, 𝑘) = 󵄨󵄨󵄨󵄨󵄨󵄨󵄨𝑇(𝑖, 𝑗, 𝑘) −𝑇(𝑖, 𝑗, 𝑘) 󵄨󵄨󵄨󵄨󵄨󵄨󵄨, 𝑇(𝑖, 𝑗, 𝑘) = 1 𝑤× 𝑤× 𝑤 𝑟 ∑ 𝑙=𝑖 𝑠 ∑ 𝑚=𝑗 𝑡 ∑ 𝑛=𝑘 𝑇(𝑙, 𝑚, 𝑛) . (13 𝑇𝛼(𝑖, 𝑗, 𝑘) = 1 𝑤× 𝑤× 𝑤 𝑟 ∑ 𝑙=𝑖 𝑠 ∑ 𝑚=𝑗 𝑡 ∑ 𝑛=𝑘 𝑇(𝑙, 𝑚, 𝑛) , 𝐹𝛼(𝑖, 𝑗, 𝑘) = 1 𝑤× 𝑤× 𝑤 𝑟 ∑ 𝑙=𝑖 𝑠 ∑ 𝑚=𝑗 𝑡 ∑ 𝑛=𝑘 𝐹(𝑙, 𝑚, 𝑛) , We calculated the mean energy using coefficients of HHH subbands in a local window 𝑤as follows: (13) HHH (𝑖, 𝑗, 𝑘) = 1 𝑤× 𝑤× 𝑤 𝑟 ∑ 𝑙=𝑖 𝑠 ∑ 𝑚=𝑗 𝑡 ∑ 𝑛=𝑘 HHH (𝑙, 𝑚, 𝑛) , (8) 𝛿𝑇(𝑖, 𝑗, 𝑘) = 󵄨󵄨󵄨󵄨󵄨󵄨󵄨𝑇(𝑖, 𝑗, 𝑘) −𝑇(𝑖, 𝑗, 𝑘) 󵄨󵄨󵄨󵄨󵄨󵄨󵄨, 𝑇(𝑖, 𝑗, 𝑘) = 1 𝑤× 𝑤× 𝑤 𝑟 ∑ 𝑙=𝑖 𝑠 ∑ 𝑚=𝑗 𝑡 ∑ 𝑛=𝑘 𝑇(𝑙, 𝑚, 𝑛) . (8) where where 𝑟= round (𝑖+ 𝑤 2 ) , 𝑠= round (𝑗+ 𝑤 2 ) , 𝑡= round (𝑘+ 𝑤 2 ) . (9) The inverse DWT was then applied to obtain a new volume which possessed the true subset. 3. The Proposed Segmentation Method Finally, we applied 𝐾-means clustering (𝐾= 3) in (2) to differentiate vessel lumen, vessel boundary (true subset), and background. The true subset 𝑇 was retained, respectively. (9) BioMed Research International 5 (a) (b) (c) Figure 2: The 3D region growing seed points in (c) were automatically obtained by using the difference between the two areas bounded by the blue lines in adjacent slices (a) and (b). (b) (a) (b) (a) (c) (c) Figure 2: The 3D region growing seed points in (c) were automatically obtained by using the difference between the two areas bounded by the blue lines in adjacent slices (a) and (b). Figure 2: The 3D region growing seed points in (c) were automatically obtained by using the difference between the two areas bounded by the blue lines in adjacent slices (a) and (b). 4. Experiments and Discussion line are coronary arteries. The segmenting efficiency was compared with the manually delineated ground truth data 𝑁𝑅 in Figure 3(a) by using OM which was defined as To test the proposed method, CTA volumes obtained from a CT system were segmented for coronary arteries. The slice thickness was 0.625 mm and the volume was 512∗512∗(⋅) in different data sets. The window size 𝑤was set to 3 which was enough to capture the local texture characteristics. The parameter 𝛼was set to 0.2. We tested 20 data sets, most of which were segmented successfully except for a few small branches that were lost in 2 of the data sets due to the local failure in region growing. To evaluate the performance of our segmented coronary arteries, we compared our results with that obtained from the ground truth values obtained from the commercial software from GE Healthcare and the level-set method. We used the overlapping metric (OM) and Hausdorff distance (𝑑𝐻) to analyze the efficiency of each method. OM = 2 (𝑁𝑇∩𝑁𝑅 𝑁𝑇+ 𝑁𝑅 ) , (14) (14) where 𝑁𝑇indicates the pixels/voxels of the segmented coro- nary arteries. The OM was close to 1 when the segmenta- tion was well matched to the reference ground truth and approached zero when the results had no similarity to the reference. In the 6 slices in Figure 3(b), the segmentation results showed that the proposed method detected coronary arteries accurately. As shown in Table 1, the average OM of the proposed method was 0.96. 4.2. The Segmenting Efficiency on a 3D Volume. The first focus of the comparison was the correctness of the 4 main branches: the right coronary artery (RCA), the left anterior descending artery (LAD), the circumflex (CRX), and the first diagonal 4.1. The Segmenting Efficiency of 2D Imaging. Figure 3(b) shows the results of 6 slices in 1 CTA volume obtained using the proposed method. The areas bounded by the red 6 BioMed Research International 6 (a) Ground truth data (b) The segmented results Figure 3: Comparison of the 2D segmentation (b) with respect to the ground truth data (a). (DA). Figures 4 and 5 show the detected coronary s in the CTA volume obtained from the GE Health- nd the proposed method, and the proposed method nted these 4 main branches correctly compared to the n branches in Figure 5 Another test of performance is the correctness of the remaining branches. 4. Experiments and Discussion Due to the multiresolution of the DWT, each subbranch in Figures 3 and 5 was correctly segmented. Furthermore, the coronary arteries obtained from the pro- posed method were much better than those obtained from (a) Ground truth data (b) The segmented results Figure 3: Comparison of the 2D segmentation (b) with respect to the ground truth data (a). (a) Ground truth data (b) The segmented results (b) The segmented results (a) Ground truth data (a) Ground truth data Figure 3: Comparison of the 2D segmentation (b) with respect to the ground truth data (a). Another test of performance is the correctness of the remaining branches. Due to the multiresolution of the DWT, each subbranch in Figures 3 and 5 was correctly segmented. Furthermore, the coronary arteries obtained from the pro- posed method were much better than those obtained from artery (DA). Figures 4 and 5 show the detected coronary arteries in the CTA volume obtained from the GE Health- care and the proposed method, and the proposed method segmented these 4 main branches correctly compared to the 4 main branches in Figure 5. BioMed Research International 7 Figure 4: The 3D coronary arteries manually edited by a radiologist using an AW workstation (GE Healthcare, Wisconsin, USA). 150 150 150 200 200 250 100 100 100 50 50 50 3D coronary tree Figure 6: The 3D coronary arteries in the CTA volume obtained from the level-set approach. 150 150 150 200 200 250 100 100 100 50 50 50 3D coronary tree Figure 6: The 3D coronary arteries in the CTA volume obtained from the level-set approach. Figure 4: The 3D coronary arteries manually edited by a radiologist using an AW workstation (GE Healthcare, Wisconsin, USA). 200 150 100 100 100 50 50 50 150 150 Figure 5: The 3D coronary arteries in the CTA volume obtained from the proposed method. 150 100 100 100 50 50 50 150 150 Table 3: Comparison of the cross-sectional diameter. Table 2 lists the mean OM and mean Hausdorff distance for the proposed method and the method of Yang et al. [11]. The results show that the proposed method was much better than that of Yang et al. [11] in terms of both OM and Hausdorff distance. 4.3. Diameter Measurement. In this subsection, we computed the diameters of segmented coronary arteries using the proposed method and the method of Yang et al. 5. Conclusions [3] D. Nain, A. Yezzi, and G. Turk, “Vessel segmentation using a driven flow shape,” in Proceeding of International Conference on Medical Image Computing Computer-Assisted Intervention- MICCAI 2004, vol. 3216 of Lecture Notes in Computer Science, pp. 51–59, 2004. Accurate extraction of coronary arteries is important to assess artery lesions in clinical practice. In this study, we propose a novel method to segment coronary arteries automatically. Based on the initial segmentation obtained from 3D region growing, one-level 3D DWT and 3D neutrosophic transfor- mation were applied to detect coronary arteries accurately. The location of vessel information, in HHH subband coef- ficients of DWT, was successfully detected by the proposed vessel-texture discrimination algorithm. Accordingly, the HHH subband coefficients were used and characterized and classified by 3D neutrosophic transformation and 𝐾-means clustering. The experimental results verify the efficiency of the proposed method. [4] H. Tek, Y. Zheng, M. A. Gulsun, and G. Funka-Lea, “An automatic system for segmenting coronary arteries from CTA,” in Proceedings of the MICCAI Workshop on Computing and Visualization for Intravascular Imaging (MICCAI-CVII ’11), pp. 47–54, 2011. [5] A. Hennemuth, T. Boskamp, D. Fritz et al., “One-click coronary tree segmentation in CT angiographic images,” International Congress Series, vol. 1281, pp. 317–321, 2005. [6] Y. Yang, A. Tannenbaum, and D. Giddens, “Knowledge-based 3D segmentation and reconstruction of coronary arteries using CT images,” in Proceedings of the 26th Annual International Conference of the IEEE Engineering in Medicine and Biology Society (EMBC ’04), vol. 1, pp. 1664–1666, San Francisco, Calif, USA, September 2004. 4. Experiments and Discussion Many efficient algorithms have been proposed to extract the tube centerline. We applied the algorithm proposed by Lee et al. [23] to extract the centerline of the segmented coronary arteries. Using these extracted centerlines, we obtained the cross sections of the segmented coronary arteries, as shown in Figure 7. By computing the area 𝐴of each cross section, the diameter 𝑟was estimated as follows: Figure 5: The 3D coronary arteries in the CTA volume obtained from the proposed method. Table 1: Comparison of the 2D image segmentation results. the level-set method, which had several leakages as seen in Figure 6.f 𝑟≈√𝐴 𝜋. (16) (16) In addition to the OM, the difference between the segmented vessel surface and the manually delineated ground truth data was measured by the Hausdorff distance [22] which was defined as follows: Table 3 shows the estimated diameter of the same cross section for the proposed method and the method of Yang et al. [11]. The diameter of the proposed method was closer to the diameter in ground truth data at the same cross section than to that obtained from the method of Yang et al. 𝑑𝐻(𝑋, 𝑌) = max {sup 𝑥∈𝑋 inf 𝑦∈𝑌𝑑(𝑥, 𝑦) , sup 𝑦∈𝑌 inf 𝑥∈𝑋𝑑(𝑥, 𝑦)} , (15) where 𝑋and 𝑌are the vertices of the mesh surfaces of the arteries corresponding to the segmentation results and the ground truth and 𝑑(𝑥, 𝑦) measure the Euclidean distance between points 𝑥and 𝑦belonging to vertices 𝑋and 𝑌. 4.4. Experimental Environment and Execution Time. The proposed method was implemented in MATLAB (R2011a) on a standard specification PC with a 3.2 GHz CPU and 12 GB RAM. The average execution time was 58 seconds to 8 8 BioMed Research International (b) (a) (b) Figure 7: (a) The cross section of the segmented coronary arteries. (b) Magnified view of (a). (b) (a) Figure 7: (a) The cross section of the segmented coronary arteries. (b) Magnified view of (a). [2] P. J. Yim, J. J. Cebral, R. Mullick, H. B. Marcos, and P. L. Choyke, “Vessel surface reconstruction with a tubular deformable model,” IEEE Transactions on Medical Imaging, vol. 20, no. 12, pp. 1411–1421, 2001. extract the entire coronary tree, compared to approximately 47 seconds for the method by Yang et al. for the same process. Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper. [7] H. Shikata, G. McLennan, E. A. Hoffman, and M. Sonka, “Segmentation of pulmonary vascular trees from thoracic 3D CT images,” International Journal of Biomedical Imaging, vol. 2009, Article ID 636240, 11 pages, 2009. Acknowledgments [8] T. Brox and J. Weickert, “Level set segmentation with multiple regions,” IEEE Transactions on Image Processing, vol. 15, no. 10, pp. 3213–3218, 2006. This study was supported by the National Taiwan University Hospital and the National Science Council, Taiwan, under the NSC Grant: NSC 98-2221-E-002-098-MY3. [9] W. K. Pratt, Digital Image Processing, John Wiley & Sons, Los Altos, Calif, USA, 4th edition, 2007. [10] L. Wang, L. He, A. Mishra, and C. Li, “Active contours driven by local Gaussian distribution fitting energy,” Signal Processing, vol. 89, no. 12, pp. 2435–2447, 2009. References [1] A. F. Frangi, W. J. Niessen, K. L. Vincken, and M. A. Viergever, “Multiscale vessel enhancement filtering,” in Medical Image Computing and Computer-Assisted Interventation—MICCAI’98, vol. 1496 of Lecture Notes in Computer Science, pp. 130–137, Springer, Berlin, Germany, 1998. [11] Y. Yang, A. Tannenbaum, D. Giddens, and A. Stillman, “Auto- matic segmentation of coronary arteries using bayesian driven implicit surfaces,” in Proceedings of the 4th IEEE International Symposium on Biomedical Imaging: From Nano to Macro (ISBI ’07), pp. 189–192, April 2007. BioMed Research International BioMed Research International 9 [12] Y. Wang and P. Liatsis, An Automatic Method for Segmentation of Coronary Arteries X-Ray in Coronary CT Imaging, IEEE Computer Society Developments in E-Systems Engineering, 2010. [13] Y. Wang and P. Liatsis, “Automatic segmentation of coronary arteries in CT imaging in the presence of kissing vessel artifacts,” IEEE Transactions on Information Technology in Biomedicine, vol. 16, no. 4, pp. 782–788, 2012. [14] C. T. Metz, M. Schaap, A. C. Weustink, N. R. Mollet, T. Van Walsum, and W. J. Niessen, “Coronary centerline extraction from CT coronary angiography images using a minimum cost path approach,” Medical Physics, vol. 36, no. 12, pp. 5568–5579, 2009. [15] O. Friman, M. Hindennach, C. K¨uhnel, and H.-O. Peitgen, “Multiple hypothesis template tracking of small 3D vessel structures,” Medical Image Analysis, vol. 2010, no. 14, pp. 160– 171, 2010. [16] S. C. Saur, C. K¨uhnel, T. Boskamp, G. Szekely, and P. C. Cattin, “Automatic ascending aorta detection in CTA datasets,” in Bildverarbeitung f¨ur die Medizin 2008, pp. 323–327, Springer, Berlin, Germany, 2008. [17] T. Pavlidis and Y.-T. Liow, “Integrating region growing and edge detection,” IEEE Transactions on Pattern Analysis and Machine Intelligence, vol. 12, no. 3, pp. 225–233, 1990. [18] M. Unser, “Local linear transforms for texture measurements,” Signal Processing, vol. 11, no. 1, pp. 61–79, 1986. [19] T. Chang and C. C. J. Kuo, “Texture analysis and classification with tree-structured wavelet transform,” IEEE Transactions on Image Processing, vol. 2, no. 4, pp. 429–441, 1993. [20] M. Unser, “Texture classification and segmentation using wavelet frames,” IEEE Transactions on Image Processing, vol. 4, no. 11, pp. 1549–1560, 1995. [21] Z.-Z. Wang and J.-H. Yong, “Texture analysis and classification with linear regression model based on wavelet transform,” IEEE Transactions on Image Processing, vol. 17, no. 8, pp. 1421–1430, 2008. [22] R. T. Rockefellar and R. J.-B. Wets, Variational Analysis, vol. 317, Springer, New York, NY, USA, 1988. [23] T. C. Lee, R. L. Kashyap, and C. N. Chu, “Building skeleton models via 3-D medial surface axis thinning algorithms,” Graphical Models and Image Processing, vol. 56, no. 6, pp. 462– 478, 1994.
https://openalex.org/W3008411277	https://figshare.com/articles/journal_contribution/Moral_and_contextual_dimensions_of_inappropriate_antibiotic_prescribing_in_secondary_care_A_three-country_interview_study_/11815176/2/files/22543424.pdf	English	null	Moral and Contextual Dimensions of “Inappropriate” Antibiotic Prescribing in Secondary Care: A Three-Country Interview Study	Frontiers in sociology	2,020	cc-by	8,894	ORIGINAL RESEARCH published: 20 February 2020 doi: 10.3389/fsoc.2020.00007 Moral and Contextual Dimensions of “Inappropriate” Antibiotic Prescribing in Secondary Care: A Three-Country Interview Study Carolyn Tarrant 1, Eva M. Krockow 2, W. M. I. Dilini Nakkawita 3, Michele Bolscher 4, Andrew M. Colman 2, Edmund Chattoe-Brown 5, Nelun Perera 6, Shaheen Mehtar 4 and David R. Jenkins 6 Carolyn Tarrant 1, Eva M. Krockow 2, W. M. I. Dilini Nakkawita 3, Michele Bolscher 4, Andrew M. Colman 2, Edmund Chattoe-Brown 5, Nelun Perera 6, Shaheen Mehtar 4 and David R. Jenkins 6 1 Department of Health Sciences, University of Leicester, Leicester, United Kingdom, 2 Department of Neuroscience, Psychology and Behaviour, University of Leicester, Leicester, United Kingdom, 3 Faculty of Medicine, General Sir John Kotelawala Defence University, Colombo, Sri Lanka, 4 Tygerberg Academic Hospital and Faculty of Health Sciences, Stellenbosch University, Cape Town, South Africa, 5 School of Media, Communication and Sociology, University of Leicester, Leicester, United Kingdom, 6 Department of Clinical Microbiology, University Hospitals of Leicester NHS Trust, Leicester, United Kingdom Keywords: antibiotic prescribing, antimicrobial resistance, hospital, qualitative investigation, international Edited by: Nicola Kay Gale, University of Birmingham, United Kingdom Overuse of broad-spectrum antibiotics in secondary care is a key contributor to the emergence and spread of antimicrobial resistance (AMR); efforts are focused on minimizing antibiotic overuse as a crucial step toward containing the global threat of AMR. The concept of overtreatment has, however, been difﬁcult to deﬁne. Efforts to address the overuse of medicine need to be informed by an understanding of how prescribers themselves understand the problem. We report ﬁndings from a qualitative interview study of 46 acute care hospital prescribers differing in seniority from three countries: United Kingdom, Sri Lanka and South Africa. Prescribers were asked about their understanding of inappropriate use of antibiotics. Prescriber deﬁnitions of inappropriate use included relatively clear-cut and unambiguous cases of antibiotics being used “incorrectly” (e.g., in the case of viral infections). In many cases, however, antibiotic prescribing decisions were seen as involving uncertainty, with prescribers having to make decisions about the threshold for appropriate use. Decisions about thresholds were commonly framed in moral terms. Some prescribers drew on arguments about their duty to protect public health through having a high threshold for prescribing, while others made strong arguments for prioritizing risk avoidance for the patients in front of them, even at a cost of increased resistance. Notions of whether prescribing was inappropriate were also contextually dependent: high levels of antibiotic prescribing could be seen as a rational response when prescribers were working in challenging contexts, and could be justiﬁed in relation to ﬁnancial and social considerations. Inappropriate antibiotic use is framed by prescribers not just in clinical, but also in moral and contextual terms; this has implications for the design and implementation of antibiotic stewardship interventions aiming to reduce inappropriate use of antibiotics globally. Reviewed by: Brian Godman, Karolinska Institutet (KI), Sweden Laurie Denyer Willis, University of Cambridge, United Kingdom *Correspondence: Carolyn Tarrant ccp3@le.ac.uk Specialty section: This article was submitted to Medical Sociology, a section of the journal Frontiers in Sociology Specialty section: This article was submitted to Medical Sociology, a section of the journal Frontiers in Sociology Received: 31 October 2019 Accepted: 04 February 2020 Published: 20 February 2020 Received: 31 October 2019 Accepted: 04 February 2020 Published: 20 February 2020 Design g This study used a qualitative interview design, involving interviews with prescribers in secondary care in Sri Lanka, South Africa and the United Kingdom. Semi-structured interviews were conducted between 2016 and 2017. Interviews were conducted in each country by local researchers. We used a detailed shared topic guide (see Appendix A), containing 17 questions about antibiotic use. The guide included questions exploring a range of aspects of antibiotic use, with several questions focusing speciﬁcally on identifying the participant’s understanding of inappropriate prescribing and asking for examples. In developing the guide we drew on previous research into the determinants of prescribing in hospitals (Krockow et al., 2019), as well as theoretical literature on social dilemmas as this was our overarching theoretical perspective for the study (Tarrant et al., 2019). We piloted and revised the topic guide based on interviews with two junior doctors. We conducted in-depth training and practice interviews for researchers, and held regular telephone meetings to discuss emerging ﬁndings through the course of data collection. The interviews were audio recorded and ranged in length between 20 and 80 min. Written consent was obtained from participants for recording of interviews and use of anonymised quotes in reports and publications. All data were anonymised prior to analysis, and participating institutions were oﬀered debriefs about the research ﬁndings. Ethical approval was obtained separately in Sri Lanka, South Africa and the United Kingdom. One challenge for stewardship is that it may be diﬃcult to pinpoint what inappropriate or excessive antibiotic use means in practice, although eﬀorts have been undertaken to try to develop consensus deﬁnitions and quality indicators for antibiotic prescribing (Spivak et al., 2016). Deﬁning and measuring inappropriate or suboptimal use is complicated by the tensions that exist between the aim of reducing antibiotic prescribing in order to tackle the growing systemic problem of AMR, and the risks of failing to administer medication to individual patients when there is a potential risk of mortality and morbidity (Fitzpatrick et al., 2019). While prescribing antibiotics in the absence of bacterial infection is clearly inappropriate, clinicians have to base the majority of initial prescribing decisions on clinical judgement–prescribing empirically based on indicative signs and symptoms as opposed to a deﬁnitive diagnosis. This is particularly the case for acute medical patients presenting with a spectrum of symptoms that could possibly be indicative of infection. Citation: Tarrant C, Krockow EM, Nakkawita WMID, Bolscher M, Colman AM, Chattoe-Brown E, Perera N, Mehtar S and Jenkins DR (2020) Moral and Contextual Dimensions of “Inappropriate” Antibiotic Prescribing in Secondary Care: A Three-Country Interview Study. Front. Sociol. 5:7. doi: 10.3389/fsoc.2020.00007 February 2020 \| Volume 5 \| Article 7 Frontiers in Sociology \| www.frontiersin.org 1 “Inappropriate” Antibiotic Prescribing Tarrant et al. Design This initial decision could be supported by guidelines and anti-biograms (Liang et al., 2016) where available, and subsequently be reﬁned based on microbiological results or review as part of a hospital’s stewardship programme. For individual physicians, however, making these initial treatment decisions under conditions of uncertainty often involves balancing risks; their views about what constitutes the “correct” or most appropriate course of action may diﬀer. INTRODUCTION not to prescribe an antibiotic, and whether to use a broad- spectrum antibiotic as the primary treatment. This variety of conﬂicting opinions may be grounded in diﬀerent contextual inﬂuences of their medical training, past clinical experiences and current work situation, as well as their orientation toward the uncertainties and risks involved in managing patients with potentially serious conditions. Antimicrobial resistance (AMR) is a health threat with potentially devastating global consequences (O’Neill, 2016). A key contributor to resistance is the overuse of antibiotics in healthcare; there are a range of drivers including unregulated access to antibiotics in the community in some lower- income settings, and unnecessary and excessive prescribing in community and hospital settings. Previous research indicates that more than one third of antibiotic prescriptions for hospital patients globally may be inappropriate (Zarb et al., 2010). Drawing on qualitative interview data involving prescribers from a range of diﬀerent international hospital contexts, this article aims to provide insights into the opinions held by prescribers about what counts as inappropriate prescribing, and the factors that mediate their judgements. Appropriate prescribing choices are typically deﬁned as the right drug, administered at the right time, using the right dose, for the right duration (Dryden et al., 2011). Antimicrobial stewardship interventions in hospitals focus on reducing the excessive use of antibiotics, and avoiding the use of inappropriate types of antibiotic, broad-spectrum antibiotics in particular (Hood et al., 2019). Broad-spectrum antibiotics are eﬀective against a wider range of pathogens than narrow-spectrum antibiotics. While they are typically necessary in situations where information is lacking about the cause of an infection, broad- spectrum antibiotics come at the cost of being stronger drivers of AMR (Karam et al., 2016), and ideally their use should be limited to emergency cases (e.g., severe sepsis of unknown origin). Stewardship programmes have been implemented in hospitals worldwide, although with more diﬃculty in some contexts (Cox et al., 2017; Charani et al., 2019), resulting in positive but variable impact (Hulscher and Prins, 2017; Nathwani et al., 2019). Frontiers in Sociology \| www.frontiersin.org Participants dose, and duration existed, but the prescriber failed to prescribe in accordance with these guidelines without justiﬁcation– resulting in the patient receiving an inappropriate antibiotic or the incorrect dose or duration of treatment. We interviewed a total of 46 participants: 18 participants in Sri Lanka, 13 participants in South Africa, and 15 participants in the United Kingdom. The majority of participants were doctors and ranged in seniority from junior doctors to consultants. In the United Kingdom, two advanced nurse prescribers were also included in the sample. If there’s a clear clinical scenario of infection that we know this is hospital-acquired pneumonia, and you know what kind of antibiotic is that, and you start prescribing a very broad spectrum, then you are not following guidelines, then you are just harming the patient. (SA 009) RESULTS Participants also pointed to situations in which the diagnosis was unambiguous, where clear guidelines about antibiotic choice, Participants also pointed to situations in which the diagnosis was unambiguous, where clear guidelines about antibiotic choice, dose, and duration existed, but the prescriber failed to prescribe in accordance with these guidelines without justiﬁcation– resulting in the patient receiving an inappropriate antibiotic or the incorrect dose or duration of treatment. Data Analysis A related type of inappropriate prescribing described by participants was the use of antibiotics in the absence of any symptoms pointing to a bacterial infection. For example, participants reported cases where the mere acuity of a patient triggered a prescription of antibiotics despite the absence of any infection-speciﬁc symptoms. All interview recordings were transcribed verbatim and anonymised data were analyzed by the United Kingdom-based research team using the constant comparative method (Charmaz, 2014). Starting with open, descriptive coding of a selection of transcripts, an initial coding framework was created using NVivo Software. This was followed by an iterative process of coding and evolution of the coding framework, with reference to existing literature and theoretical concepts (Tarrant et al., 2019). Drawing on this coded data, we focused on codes speciﬁcally pertaining to participants’ understanding of inappropriate antibiotic prescribing. We generated data summaries for key themes. Visual methods were used to display data extracts and clusters of codes, and to map themes. They just come into the emergency unit, and [they are...] getting antibiotics, even though they have a multitude of other reasons for their admission. (SA 012) So the inappropriate use will be you don’t have any evidence that the patient’s having bacterial infection. The patient might be unwell due to other reasons, for example they might have asthma exacerbation with very little evidence of infection. (UK 007) They just come into the emergency unit, and [they are...] getting antibiotics, even though they have a multitude of other reasons for their admission. (SA 012) So the inappropriate use will be you don’t have any evidence that the patient’s having bacterial infection. The patient might be unwell due to other reasons, for example they might have asthma exacerbation with very little evidence of infection. (UK 007) “Incorrect” Use of Antibiotics “Incorrect” Use of Antibiotics Prescriber deﬁnitions of inappropriate use included examples of relatively clear-cut and unambiguous cases of antibiotics being used “incorrectly.” These deﬁnitions included situations where antibiotics were prescribed but where infection was unlikely to be the cause of symptoms, for example, in cases in which symptoms or patient presentation indicated a diﬀerent root cause such as a viral infection. Indeed, the vast majority of study participants across all countries and hospitals discussed detailed examples of patients being treated with antibiotics for viral illnesses such as the ﬂu. This was seen as problem for patients in primary care settings, but also in hospitals, particularly in ambulatory emergency care. Recruitment of participants was conducted using a snowball sampling approach: researchers were introduced to potential participants via email or personal introduction by the local contact in each hospital, or by previous interviewees. We aimed to purposively sample participants to include prescribers with diﬀerent roles and levels of seniority. We aimed for a minimum sample size of 12 participants per country (total of 36 participants) as our previous experience indicted that this would be a reasonable number to enable us to fully explore the issues. We continued to recruit participants to interviews in each country until the team agreed we had reached a point of data saturation (Aldiabat and Le Navenec, 2018). I think the most common scenario, too common personally in my experience, where [...] antibiotics in general are prescribed inappropriately, are viral illnesses. [...] especially in, in the ambulatory [emergency] care setting. (UK 001) Some patients clearly having viral infections but they are on antibiotics. (SL 013) Participants Our study participants included prescribers from three diﬀerent countries (Sri Lanka, South Africa and the United Kingdom), recruited from a total of seven diﬀerent hospitals across the three countries. These countries and participating hospitals were selected based on existing collaborations between the research team, and included high and lower resource settings, with diverse challenges in terms of resourcing and patient population. In Sri Lanka and South Africa a signiﬁcant proportion of medical care happens in the private sector (in Sri Lanka around 50% of outpatient and 10% of inpatient care is in the private sector (The Economist, 2014), and doctors commonly work across both sectors; around 20% of the South African population are seen in the private sector; Meyer et al., 2017) In both these countries we included public and private hospital settings to One of the underlying challenges to antimicrobial stewardship is a lack of agreement amongst physicians on what constitutes a “right” choice when making decisions about whether or February 2020 \| Volume 5 \| Article 7 Frontiers in Sociology \| www.frontiersin.org 2 “Inappropriate” Antibiotic Prescribing Tarrant et al. explore how these diﬀerent contexts shaped prescribing. Public health care makes up the majority of care the United Kingdom (Klein, 2005) (and is often used combination with public healthcare) hence both hospitals chosen for the study were public (National Health Service) hospitals. In Sri Lanka we included one private hospital and one public hospital located in a major city, and a publicly funded hospital located in a rural area. In South Africa we included two diﬀerent hospitals located in a major city. One hospital was publicly funded while the other belonged to a chain of private hospitals. The two hospitals in the UK included a large city teaching hospital and a smaller hospital in an urban area. what they judged to be a “correct” decision. Excessive antibiotic use, and high levels of reliance on broad spectrum antibiotics could be justiﬁed based on arguments about the duties of a doctor/healthcare professional to their individual patients, and as being appropriate given the local context. Ambiguities of Inappropriate Use: Uncertainty and Moral Framing of Antibiotic Prescribing Decisions Ambiguities of Inappropriate Use: Uncertainty and Moral Framing of Antibiotic Prescribing Decisions Beyond these shared deﬁnitions of incorrect antibiotic use, participants described gray areas of inappropriate use, where the appropriateness of prescribing decisions was less objectively clear. Participants recognized that many antibiotic prescribing decisions involved decision-making under uncertainty, where clinicians were using their clinical judgement to assess the likelihood of infection, the likely source of infection and infective agent, and therefore the best course of action. Prescribers had to make decisions about the threshold at which they would prescribe antibiotic treatment, and their certainty over whether they could use a targeted narrow-spectrum antibiotic as opposed to a broad spectrum antibiotic. This threshold might vary from patient to patient, depending on their vulnerability and level of risk (e.g., young children, frail older people), but also individual prescribers were seen to vary in their approach. It’s kind of a public health like obligation, isn’t it, to make sure that you’re giving decent antibiotics correctly, to reduce resistant strains. (UK 009) So we have to balance that risk constantly. And I would say obviously people can argue that your individual patient takes priority, but then other considerations would be society as a whole, or the broader community has to be taken as the priority. [...] You have to be cognizant of the fact that these treatment decisions you make on this patient has an impact on the next one and society as a whole. (SA 010) It’s kind of a public health like obligation, isn’t it, to make sure that you’re giving decent antibiotics correctly, to reduce resistant strains. (UK 009) So we have to balance that risk constantly. And I would say obviously people can argue that your individual patient takes priority, but then other considerations would be society as a whole, or the broader community has to be taken as the priority. [...] You have to be cognizant of the fact that these treatment decisions you make on this patient has an impact on the next one and society as a whole. (SA 010) They made critical judgements about other clinicians who were quick to prescribe antibiotics or relied too heavily on broad spectrum antibiotics. They made critical judgements about other clinicians who were quick to prescribe antibiotics or relied too heavily on broad spectrum antibiotics. [There] are generally two camps that you get with dealing with uncertainty. I think I try to do what is good for the patient and that is the only thing [...] The only agenda I have is that. (SL 007) As a doctor, the most important is the patient’s interests, you know, so you try and do the right thing for that speciﬁc patient, and then, the other interests are probably less important. (SA 006) I have that sense at a societal level [of the problem of AMR], but my job as a doctor is to treat the person in front of me. [...] so I don’t balance... [...I’m] just doing what I can to make the patient better. (UK 012) Ambiguities of Inappropriate Use: Uncertainty and Moral Framing of Antibiotic Prescribing Decisions So you get the one which is very prone to jump in and do something, and that might be prescribing antibiotics [...], which may or may not be appropriate. And then you get the other, which is more likely to just, to try to investigate and work out what’s happening before giving an antibiotic. (UK 004) For the vast majority of cases, the use of broad-spectrum antibiotics is [...] a consequence of lackadaisical or poorly worked up clinical decision-making. [...] You’ve got a better chance of getting [the patient] better quicker, because you’re covering all possible ills. But it’s not good medicine. (UK 002) Under conditions of uncertainty, where judgements had to be made about where to set a threshold for prescribing, antibiotic use was less easy to classify as appropriate or inappropriate in objective terms. Participants recognized that setting a low threshold–i.e., erring on the side of caution and prescribing antibiotics to acutely ill patients “when in doubt,” was an easy and low risk approach to avoiding the risks of deterioration and death for their patients. They also recognized, however, that overuse of antibiotics had negative consequences for society by contributing to the problem of AMR. The tension between the interests of diﬀerent stakeholders was well-understood by most. Where prescribers were prepared to set the threshold was seen as reﬂecting, to some extent, their experience and conﬁdence in assessing risk and tolerating uncertainty. While this reasoning sits in line with broader goals of antibiotic stewardship, conversely, other participants made counterarguments to this position, also grounded in moral terms. Although recognizing the tensions in balancing the interests of individuals and society, some participants framed their duty, and correspondingly their understanding, of what it meant to be a good doctor or healthcare professional, in terms of prioritizing the wellbeing of the individual patients in front of them. Although they recognized the clinical importance of treating patients appropriately, they minimized the risk of AMR and their responsibility for the problem, in comparison with the risks and their responsibilities for sick patients in front of them. When I started working in this setting, I would be very over-careful of missing something. I think, as I got more conﬁdent, I start the conversation with “I don’t like prescribing antibiotics. If I feel your child needs an antibiotic, I will give it. Findings: Deﬁnitions of Inappropriate Antibiotic Use Our ﬁndings highlighted diverse deﬁnitions of inappropriate use. There was consensus that some cases of antibiotic use could be seen to be objectively “incorrect” based on the patient’s condition or symptoms, but participants’ accounts demonstrated that there was often signiﬁcant ambiguity and lack of consensus about Overall, participants from all hospitals and countries shared similar opinions about what constituted an unambiguously clinically “incorrect” decision about antibiotic prescribing, or suboptimal antibiotic use. These types of incorrect or suboptimal February 2020 \| Volume 5 \| Article 7 Frontiers in Sociology \| www.frontiersin.org 3 “Inappropriate” Antibiotic Prescribing Tarrant et al. uses of antibiotics were commonly seen as reﬂecting unjustiﬁed individual preferences and habits, a lack of appropriate knowledge, or, for more junior doctors a lack of experience or senior supervision. Organizational systems and processes were also seen as playing into this, for example, a lack of access to guidelines, workload and demand on practitioners, or ineﬃcient systems for monitoring and regulating antibiotic use. Participants shared the view that these types of incorrect uses could and should be tackled to reduce antibiotic overuse. participants on drew on moral arguments in relation to balancing the interests of the diﬀerent stakeholders. These arguments reﬂected participants’ underpinning beliefs about what it meant, for them, to be a good doctor or good healthcare professional, and resulted in nuanced, and sometimes contradictory, accounts, of what was inappropriate and why. Some participants made moral arguments about the importance of considering their duty to broader society (and protecting public health) when making decisions about antibiotic use under uncertainty. For some, appropriate antibiotic use was seen as being grounded in a consideration of the risks to society of excessive antibiotic use, balanced against their duty to their individual patients. Ambiguities of Inappropriate Use: Uncertainty and Moral Framing of Antibiotic Prescribing Decisions But I would prefer to rather wait and see” (SA 007) In considering how they judged whether their own, and others’, levels of prescribing were inappropriately high or low, February 2020 \| Volume 5 \| Article 7 Frontiers in Sociology \| www.frontiersin.org 4 “Inappropriate” Antibiotic Prescribing Tarrant et al. We are seeing a lot of [...] infections in our wards because we [don’t] have the facilities, I mean like the beds are very close and they are not in separate parts, cubicles. (SL 015) We are seeing a lot of [...] infections in our wards because we [don’t] have the facilities, I mean like the beds are very close and they are not in separate parts, cubicles. (SL 015) These participants drew on such arguments as justiﬁcation for using antibiotics, and particularly broad spectrum antibiotics, when in doubt, even if this was at a cost of increased resistance. We are seeing a lot of [...] infections in our wards because we [don’t] have the facilities, I mean like the beds are very close and they are not in separate parts, cubicles. (SL 015) As a doctor we need to save patients, [...] even if we think that this is broad-spectrum antibiotics, and [we should be concerned about] resistance with the [hospital] trust, but at that time I think the most important thing is to treat your patient well. (UK 005) In these cases, it was not the antibiotic use that was seen as wrong or inappropriate, but the precipitating conditions. Participants felt they were able to respond to these conditions only in the best way that they could. While these problems–access to hospitals and use of antibiotics in the community, and insanitary hospital environments–remained as they were, participants were able to justify high levels of antibiotic use and saw little opportunity for reducing their use. As such, judgements about the appropriateness of antibiotic prescribing decisions could not always be pinned down in objective clinical terms. Instead, how participants justiﬁed their approach to antibiotic prescribing under conditions of uncertainty reﬂected their orientation to risk, and their position about what it meant to be a good doctor in terms of moral responsibilities. Participants took diﬀerent standpoints in relation to where their duties as a doctor or healthcare professional lay, and therefore, what was and was not appropriate practice. Ambiguities of Inappropriate Use: Uncertainty and Moral Framing of Antibiotic Prescribing Decisions g Other contextual factors that shaped views about whether antibiotic prescribing was the “right” thing to do, even at a cost of increased resistance, related to ﬁnancial and social considerations. Although some participants ﬂagged the problem of costs of excessive use of antibiotics for their organizations, others argued that ﬁnancial and social considerations for patients could make antibiotic use the appropriate choice. In private hospitals, some argued that using broad-spectrum antibiotics could help reduce costs for patients arising from length of stay. In lower income settings, participants recognized that a hospital stay could be ﬁnancially devastating for wage earners in families and had an impact on the economy: treating patients aggressively to get them well and out of hospital quickly was seen in some cases as a priority. Ambiguities of Inappropriate Use: Inappropriate Prescribing as Contextually Dependent Notions of whether levels of antibiotic prescribing were considered to be inappropriate were also contextually dependent: what could potentially be seen as over-use of antibiotics, or excessive reliance on broad-spectrum antibiotics, was re-framed in some cases by participants as a rational and appropriate response to the conditions in which they worked. Although they recognized such antibiotic use as excessive, they did not always see it as inappropriate in the context of the demands they faced and the resources available to them. This was a particularly common response from participants in low resource settings. Most people with private medical insurance, they have to [...] get out of the hospital as soon as possible. They work for themselves, a lot of the people, they cannot aﬀord to stay long in the hospital. So if you give [...] a good broad-spectrum antibiotic to start oﬀwith [...] it’s a win-win situation all the way. (SA 004) Most people with private medical insurance, they have to [...] get out of the hospital as soon as possible. They work for themselves, a lot of the people, they cannot aﬀord to stay long in the hospital. So if you give [...] a good broad-spectrum antibiotic to start oﬀwith [...] it’s a win-win situation all the way. (SA 004) Broad-spectrum beneﬁt was, [...] start medications, there will be improvement, so these people are working, I mean, going back to working. (SL 005) Such challenging circumstances in low resource settings included conditions of high patient throughput–including high numbers of patients presenting at a late stage when they were acutely ill. Problems also arose when patients who had already taken (often unspeciﬁed) antibiotics in the community prior to coming into hospital–including antibiotics that had been prescribed without any microbiological testing, or had been purchased. This constrained the choices about how these patients could be treated once they arrived in hospital. It also meant that that waiting for microbiological tests prior to prescribing was commonly seen as futile, and this futility was exacerbated in some contexts by the lack of rapid and high quality testing services. DISCUSSION AND CONCLUSIONS The interview ﬁndings from doctors and nurse prescribers across three countries and diﬀerent hospital sites suggest ambiguities in opinions about what counts as inappropriate antibiotic prescribing and antibiotic over-use in hospital settings. Our focus in this paper was on how prescribers made judgements about the appropriateness of antibiotic use, and how they justiﬁed their own and others’ use of antibiotics. In terms of prescribers’ own understanding of appropriateness, there was consensus that antibiotic use under certain circumstances could be judged to be clinically “incorrect” (e.g., “incorrect” the use of antibiotics for viral illnesses). Not all decisions about antibiotic use could, however be judged as objectively appropriate/inappropriate in clinical terms. There was signiﬁcant ambiguity about judgements of appropriateness of antibiotic use in case of diagnostic uncertainty. Such judgements were mediated by personal perceptions of working within the frames of risk and uncertainty, and participants’ comfort in tolerating risk. As identiﬁed in previous research, this could vary dependent on individual training, experience, and seniority, but also on When the patients come very late [i.e., present at hospital with infections at an advanced stage] by that time they will have at least more than one system aﬀected. [...] so we will again be using the broad spectrum even without [waiting] for the cultures and things like that (SL 003) Unfortunately, because our diagnostic tests are not that great, and turnaround times are poor, and sensitivities etc. are not that good, you might have to go [with] broad [spectrum antibiotics] (SA 012) When the patients come very late [i.e., present at hospital with infections at an advanced stage] by that time they will have at least more than one system aﬀected. [...] so we will again be using the broad spectrum even without [waiting] for the cultures and things like that (SL 003) Unfortunately, because our diagnostic tests are not that great, and turnaround times are poor, and sensitivities etc. are not that good, you might have to go [with] broad [spectrum antibiotics] (SA 012) Unsanitary and overcrowded environments were seen as vastly increasing the risk of hospital-associated infections, increasing the need to rely on antibiotics. February 2020 \| Volume 5 \| Article 7 Frontiers in Sociology \| www.frontiersin.org 5 “Inappropriate” Antibiotic Prescribing Tarrant et al. the extent of concern about the impact of negative patient outcomes, and the risk of personal and reputational damage (Krockow et al., 2019). DISCUSSION AND CONCLUSIONS tending to the needs of the population as a whole has been recognized as a central ethical problem in diverse areas of medicine, particularly preventative medicine (Rosenberg, 1998; Griﬃths et al., 2006). Our study highlights how this tension underpinned moral judgements about antibiotic use: what one prescriber judged to be excessive antibiotic use, based on their perceptions of duty to consider public health, could be seen by another as an appropriate response based on their sense of responsibility to minimize risk to the individual patient in front of them. These ﬁndings raise questions about what good practice can mean within existing health care systems: with attendant regulatory and structural drivers that prioritize immediate patient outcomes; and formalized ethical principles for professional practice (General Medical Council, 2019; Sri Lanka Medical Association, 2019)1 that deﬁne being a “good doctor” in terms of making the care of the individual patient their primary concern, and protecting the life of their patients. J d t b t th i t l ﬂ t d th Our study has limitations. We included participants from three countries, including high and lower resource settings, hence the generalizability of our ﬁndings to other international settings is necessarily limited. We conducted interviews with a small number of participants in each hospital, although we included prescribers with diﬀerent roles and diﬀerent levels of seniority and experience. Our study design did not allow us to explore how practitioners actually behaved in practice in relation to decision-making about antibiotic use. Also, our analysis focused speciﬁcally on antibiotic prescribing decisions; we did not explore other dimensions of antibiotic use such as medication review, stopping or switching antibiotics. Reviewing antibiotic prescribing is an important focus for stewardship, providing a way of updating or correcting initial prescribing decisions particularly in the light of new information that can provide more certainty about the best clinical course of action. Activities around reviewing, stopping and switching antibiotics present a range of diﬀerent challenges (Schouten et al., 2007) which were not the focus of our study. y A strength of our study is the inclusion of a range of diﬀerent organizations across three international contexts, including high and lower-income settings, and public and private hospitals. We did not include a private hospital in the United Kingdom, because the majority of acute healthcare provision is through publicly-funded NHS providers. DISCUSSION AND CONCLUSIONS et al., 2010,?; Pearson et al., 2018; Wilkinson et al., 2019): our study shows how these factors also played into prescribers’ reasoning about appropriateness of antibiotic use. “Excessive” antibiotic use could be recognized as such by prescribers but nonetheless be seen as representing a reasonable response to local conditions. In this sense, although levels of prescribing were seen as excessive, they were not seen as inappropriate. As such, judgements about the appropriateness of antibiotic use did not solely reﬂect a ﬁxed individual moral position, but were situated in context of the local systems and structures of care, and the temporality of the patient’s presentation. It is apparent that the way doctors and other prescribers make judgements about appropriateness are grounded in individual moral reasoning, and are highly contextualized: they cannot be reduced to purely technical criteria. Judgements about the appropriateness of antibiotic use also drew on moral reasoning about what it meant to be a good doctor or healthcare professional. This reﬂects what has been referred to as relational ethical reasoning: reasoning aimed at working out “what is the right thing to do” based on an individual’s role, and relationship with and responsibility for others (Austin et al., 2003; Pollard, 2015). In the context of medicine, relational ethical reasoning is directed at answering such questions as: What makes a good doctor or health professional? Am I a good doctor or healthcare professional (Lindseth, 1992)? This reasoning reﬂects not only individual skills and experience, but also how an individual sees themselves as positioned, and where their responsibilities lie, in relation to their patients and other stakeholders (Sørlie et al., 2001). Prescribers see themselves as “acting wisely in the face of inevitable uncertainty” (Tanenbaum, 1993), but make diﬀerent interpretations of what it means to do so. Individual participants varied in their views of their responsibilities in relation to public health and for considering wider society in their decision making; some felt the individual patient was their only concern. This ﬁnding builds on other research demonstrating that individual prescriber decisions about antibiotic use are underpinned by diﬀerent perceptions: the extent to which they are oriented toward AMR and infectious diseases (Björkman et al., 2010) as opposed to having a dominating focus on the care of the patient. This tension between attending to the needs of individual patients vs. 1Africa HPCoS. Ethical guidelines for good practice in the health care professions [Available from: https://www.hpcsa.co.za/Uploads/Professional_Practice/ Ethics_Booklet.pdf]. DISCUSSION AND CONCLUSIONS A further strength is the conduct of the interviews in each locality by local researchers, who were familiar with local health systems and could build rapport eﬀectively with participants. Although contextual factors, patient characteristics, and stewardship activities varied signiﬁcantly between countries and hospitals, it is notable that we found strong concordance across the settings in terms of deﬁnitions of “incorrect” use, and of uncertainty and moral aspects of decision- making. Findings relating to contextual inﬂuences mainly came from the interviews in lower-income settings, although NHS staﬀin the United Kingdom did reﬂect on some of these considerations including cost to the healthcare system. Judgements about the appropriateness also reﬂected the context within which prescribers were working: high levels of antibiotic use could be seen as a rational and morally justiﬁable response to challenging conditions such as patient acuity and poor environments in hospitals for hygiene and infection control. The importance of cultural and contextual factors in shaping antibiotic use is well-recognized (Hulscher Our ﬁndings have implications for antimicrobial stewardship. As highlighted earlier in this paper, lack of consensus among prescribers about what constitutes inappropriate use presents a challenge for stewardship eﬀorts. Our ﬁndings suggest that this lack of consensus is unlikely to have a technical solution–for example, through drawing up more speciﬁc deﬁnitions–because February 2020 \| Volume 5 \| Article 7 Frontiers in Sociology \| www.frontiersin.org 6 “Inappropriate” Antibiotic Prescribing Tarrant et al. judgements about appropriateness are morally and contextually framed. Stewardship interventions that directly target behavior change using techniques such as education, restrictions and controls on prescribing, and audit and feedback (Davey et al., 2017) may have value where there is consensus that prescribing is wrong or suboptimal. These types of interventions may, however, be less eﬀective at addressing the underpinnings of moral reasoning about antibiotic use, or the structural and contextual factors, that from the point of view of prescribers can make antibiotic overuse a rational and justiﬁable action. Aiming to tackle inappropriate prescribing may be problematic where consensus is lacking about what in fact constitutes “inappropriate” prescribing: where this phenomenon is morally contestable and contextually-embedded. The terminology of “inappropriate” or “suboptimal” prescribing itself may be unhelpful, given the implicit assumption that this can always be judged objectively based on the facts of the matter. Chandler, 2019), particularly in resource limited settings. ETHICS STATEMENT The studies involving human participants were reviewed and approved by University of Leicester Research Ethics Committee, Sri Lanka Medical Association Ethics Review Committee, and University of Stellenbosch Health Research Ethics Committee 1. The patients/participants provided their written informed consent to participate in this study. DATA AVAILABILITY STATEMENT The datasets generated for this study are available on request to the corresponding author. One implication of our ﬁndings is that, rather than assuming that inappropriate prescribing can be objectively speciﬁed and therefore reduced through simple interventions, there may be a need to look at how to provide more support for prescribers in managing uncertainty. Stewardship approaches that aim to support empirical decision making, improve documentation of rationale for antibiotic use, and focus on reviews of antibiotic prescriptions (based on updated information providing more certainty, such as microbiology results) are clearly important. There is also a need, however, to address the moral aspects of prescribing decisions. This might involve including vignette-based debates in stewardship training, and providing opportunities for collective input to diﬃcult decisions. We may also need more explicit societal debate, and the establishment of collective agreements around, the duty of prescribers to consider the interests of society in making antibiotic prescribing decisions (Tarrant et al., 2019). Consensus guidelines and decision-support tools have been identiﬁed as approaches to managing moral dilemmas in antibiotic prescribing (Leibovici et al., 2012). Another implication is the need to recognize that eﬀorts to reduce inappropriate antibiotic use by targeting prescribing behavior (for example, through education, or auditing) may be futile if they fail to conceptualize antibiotic overuse as a rational response to local cultural and contextual conditions. Even antibiotic use that can be objectively deﬁned as “clinically incorrect” could reﬂect the accepted practice of using antibiotics as a “quick ﬁx” to complex problems such as poorly integrated health systems (Denyer Willis and DISCUSSION AND CONCLUSIONS This points to the need for a more holistic approach (McLeod et al., 2019) that considers the broader drivers of antibiotic use in secondary care settings globally, including issues such as sanitation, community healthcare, and the ﬁnancial implications for patients of hospitalization. Our study suggests that inappropriate antibiotic use is framed by prescribers not just in clinical, but also in moral and contextual terms; this has implications for the design and implementation of antibiotic stewardship interventions aiming to reduce inappropriate use of antibiotics globally. ACKNOWLEDGMENTS We thank those who participated in interviews, and Elizabeth Shaw for help with data coding and producing data summaries. FUNDING This research was funded by the Global Challenges Research Fund—Grant No. ES/P004784/1 awarded by the Economic and Social Research Council (ESRC) on behalf of the Research Councils United Kingdom (RCUK). AUTHOR CONTRIBUTIONS CT, AC, EC-B, DJ, NP, SM, and EK planned and designed the study. EK, CT, WN, and MB conducted interviews. CT and EK conducted the data analysis and drafted the ﬁrst manuscript version. All authors helped to review and reﬁne the ﬁnal version. Cox, J. A., Vlieghe, E., Mendelson, M., Wertheim, H., Ndegwa, L., Villegas, M. V., et al. 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Identiﬁcation of targets for quality improvement in antimicrobial prescribing: the web-based ESAC Point Prevalence Survey 2009. J. Antimicrobial. Chemother. 66, 443–449. doi: 10.1093/jac/dkq430 Liang, B., Wheeler, J. S., and Blanchette, L. M. (2016). Impact of combination antibiogram and related education on inpatient ﬂuoroquinolone prescribing patterns for patients with health care–associated pneumonia. Ann. Pharmacother. 50, 172–179. doi: 10.1177/1060028015625658 Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Lindseth, A. (1992). “The role of caring in nursing ethics,” in Quality Development in Nursing Care: From Practice to Science, ed G, Udén (Linköping: World Health Organization in Collaboration with Linköping Centre), 99–100. McLeod, M., Ahmad, R., Shebl, N. A., Micallef, C., Sim, F., and Holmes, A. (2019). A whole-health–economy approach to antimicrobial stewardship: analysis of current models and future direction. PLoS Med. 16:e1002774. doi: 10.1371/journal.pmed.1002774 Copyright © 2020 Tarrant, Krockow, Nakkawita, Bolscher, Colman, Chattoe-Brown, Perera, Mehtar and Jenkins. 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. Frontiers in Sociology \| www.frontiersin.org REFERENCES No use, distribution or reproduction is permitted which does not comply with these terms. Copyright © 2020 Tarrant, Krockow, Nakkawita, Bolscher, Colman, Chattoe-Brown, Perera, Mehtar and Jenkins. 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. Meyer, J. C., Schellack, N., Stokes, J., Lancaster, R., Zeeman, H., Defty, D., et al. (2017). Ongoing initiatives to improve the quality and eﬃciency of medicine use within the public healthcare system in south africa; a preliminary study. Front. Pharmacol. 8:751. doi: 10.3389/fphar.2017.00751 February 2020 \| Volume 5 \| Article 7 Frontiers in Sociology \| www.frontiersin.org 8 “Inappropriate” Antibiotic Prescribing Tarrant et al. APPENDIX A: TOPIC GUIDE FOR INTERVIEWS 6. What would you see as the risks of prescribing a BSA, as opposed to a narrow spectrum antibiotic? 6. What would you see as the risks of prescribing a BSA, as opposed to a narrow spectrum antibiotic? 6. What would you see as the risks of prescribing a BSA, as opposed to a narrow spectrum antibiotic? 7. Do diﬀerent stakeholders have diﬀerent interests? [patient / doctor / hospital / society] To what extent do you consider these in your day to day prescribing, and how do you balance these interests? Prescribing Decisions I would be interested to hear your thoughts on choosing between a broad vs. a narrow spectrum antibiotic. Broad spectrum antibiotics being an antibiotic with activity against a wide range of pathogens. A narrow spectrum antibiotic is one that is targeted at a speciﬁc organism. I would be interested to hear your thoughts on choosing between a broad vs. a narrow spectrum antibiotic. Broad spectrum antibiotics being an antibiotic with activity against a wide range of pathogens. A narrow spectrum antibiotic is one that is targeted at a speciﬁc organism. 14. Do you get information about overall levels of antibiotic resistance in this hospital? 15. Do you think that reducing the use of BSAs in hospitals would make an important diﬀerence to addressing the overall AMR problem? Why yes or no? 4. How easy do you ﬁnd this decision? What do you see as the uncertainties and how do you deal with them? What sort of inﬂuences are there on your decision? Questions About Their Role Can you tell me brieﬂy about your job role? What is your involvement in the antibiotic prescribing for acute medical patients? 8. If you prescribe a BSA, how likely is it that the patient would be switched to a narrow spectrum antibiotic at a later point? Why? What are the barriers to this? What helps make it easier? What education or training have you had speciﬁcally on antibiotic prescribing? 9. How do you know whether you are making good decisions about antibiotic prescribing? Do you get any feedback about your antibiotic prescribing approach? Prescribing Decisions I’d like you to consider antibiotic prescribing for an acute medical patient with a suspected infection, that is, when it is not conﬁrmed that the patient has an infection, or what the infective organism might be (also known as empirical prescribing). 10. Do you ever feel patients are prescribed BSAs inappropriately? Could you start by saying what you see as inappropriate use? Are there common situations where this happens? Why do you think this happens? 11. What steps could be taken to stimulate appropriate use of BSAs? 1. How do you go about making the decision whether or not to prescribe an antibiotic? 1. How do you go about making the decision whether or not to prescribe an antibiotic? • Main barriers to improving the way BSAs are used in this hospital? e.g.: local culture / lack of lab facilities / organizational policies / external incentives or pressure • Are there any ‘rules of thumb’ that you use? What inﬂuences this decision? • Are there any ‘rules of thumb’ that you use? What inﬂuences this decision? 2. Can you tell me about how you decide which antibiotic to use, for an acute medical patient with a suspected infection? I’d like to focus now on antibiotic resistance, that is, the ability of a bacteria to stop an antibiotic from working against it, meaning that some antibiotic treatments become ineﬀective, infections persist and can spread to others. This can mean having reduced or no antibiotic treatment options. I’d like to focus now on antibiotic resistance, that is, the ability of a bacteria to stop an antibiotic from working against it, meaning that some antibiotic treatments become ineﬀective, infections persist and can spread to others. This can mean having reduced or no antibiotic treatment options. • Local or national guidelines on antibiotic prescribing? • Any limitations/restrictions on the antibiotics you can use? • Do you ever get advice on your prescribing decisions? Who from & why? Do you worry about the problem of antimicrobial resistance in your day to day practice? Why? 3. How important do you feel it is to collect microbiology specimens, in making antibiotic prescribing decisions? Why? y y y y 12. Do you ever see examples of resistance? How often does this happen in your experience? 13. How much does the problem of antibiotic resistance inﬂuence your decision-making about prescribing antibiotics? Frontiers in Sociology \| www.frontiersin.org ENDING 5. What would you see as the beneﬁts of prescribing a broad spectrum antibiotic (BSA), as opposed to a narrow spectrum antibiotic? Is there anything else you’d like to add about the use of BSAs, or the problem of AMR? Thanks for participating! February 2020 \| Volume 5 \| Article 7 Frontiers in Sociology \| www.frontiersin.org 9
https://openalex.org/W2946403183	https://alustath.uobaghdad.edu.iq/index.php/UJIRCO/article/download/840/706	English	null	The Problem – Solution Pattern In Hemingway’s Cat in the Rain	Al-ustād̲	2,019	cc-by	6,669	The Problem – Solution Pattern In Hemingway’s Cat in the Rain Asst. Prof. Sarab Khalil Hameed, Ph.D. University of Baghdad College of Arts sarab_khalil@yahoo.com Received:2/11/2019 Accepted:18/1/2019 Abstract The ‘problem – Solution’ model of discourse structure, developed by Winter (1969, 1976, 1977) and extended by Hoey (1979, 1982, 1983) is one of the few models which combine surface linguistic study and local levels of text analysis with the overall structure of text. This model has been used in the analysis of non-narrative texts. It is hypothesized that: It is hypothesized that: 1. The problem -Solution pattern is used in narrative texts. 2. The lexical items can signal the rhetorical pattern in the text. 2. The lexical items can signal the rhetorical pattern in the text. The study aims at investigating the validity of the above hypotheses by using Hoey’s model of analysis on Hemingway’s Cat in the Rain. The study aims at investigating the validity of the above hypotheses by using Hoey’s model of analysis on Hemingway’s Cat in the Rain. The analysis of the short story proved the validity of the proposed hypotheses and revealed the following: The analysis of the short story proved the validity of the proposed hypotheses and revealed the following:  The writer followed a conventional order in presenting the information i.e. situation, problem, solution and evaluation.  He communicated the rhetorical pattern to the reader through his clever use of the lexical items.  The solution is always preceded by a pre-solution.  The solution is always preceded by a pre-solution.  The positive solution signals the end of the discourse and the negative solution signals another problem which confirms the reader’s expectations. Key Words: problem-solution , Hemingway , Cat in the Rain نمط (المشكلة– الحل) في قصة قطة تحت المطر للك اتة هيمنغواي ا.م.د ســراب خليل حميذ جامعة تغذاد كلية االداب Vol.(58) No.(1)(March-2019 AD, 1440 AH) Vol.(58) No.(1)(March-2019 AD, 1440 AH) AL-USTATH الملخص : اٌ ًَط ْٛكهٛح انخطاب "انًشكهح– ( انحم" ، انرٙ ٔضعٓا َٔرش9191 ، 9199 ، 9199 ْٕ٘ ) ٔانرٙ طٕسْا ( 9191،9191،9191 ) ْٕ ٔاحذ يٍ انًُارج انقهٛهح انرٙ ذجًع تٍٛ انذساسح انهغٕٚح انسطحٛح ٔانًسرٕٚاخ انًحهٛح ٔ .نرحهٛم انُض يع انٓٛكم انعاو نهُض ن.قذ اسرخذو ْزا انًُٕرج فٙ ذحهٛم انُظٕص غٛش انسشدٚح ٌٚفرشع ْزا انثحث تأ: 9 . ًَط( انًشكهح- انحم )ٚسرخذو فٙ انُظٕص انسشدٚح. 1. ًٚكٍ نهًفشداخ انًعجًٛح اٌ ذؤشش انًُط االسهٕتٙ فٙ انُض. 121 Vol.(58) No.(1)(March-2019 AD, 1440 AH) AL-USTATH ٚٓذف انثحث إنٗ انرح قق يٍ طحح انفشضٛح أعالِ تاسرخذاو ًَٕرج ْٕ٘ نهرحهٛم عهٗ قظح انكاذة ًُْغٕا٘ " قطح "ذحد انًطش ٔ أثثد ذحهٛم انقظح انقظٛشج طحح انفشضٛاخ انًقرشحح ذٕطم انٗ يٙا ٚه:  ٚرثع انكاذة ذشذٛثا ذقهٛذٚا فٙ ذقذٚى انًعهٕياخ يثم انحانح ، انًشكهح، انحم ٔانرقٛٛى  نقذ أطم انًُطٙانخطات نهقاسٖء يٍ خالل اسرخذايّ ان زكٙ نهًفشداخ انًعجًٛح  ٚكٌٕ انحم انُٓائٙ دائًا يسثٕقا تًقرشح حم نه  ٙٚؤد٘ انحم االٚجات انٗ آَاء انخطاب ٔٚؤد٘ انحم انسهثٙ انٗ يشكهح اخشٖ يًا ٚؤكذ ذٕقعاخ انقاسٖء Theoretical Background 1. The Rhetorical Patterns 1. The Rhetorical Patterns Using Winter’s (1977) previous model of clause relations as a point of departure, Hoey (1979, 1983, 1986, 1993, 1994 and 2001) proposes a new model for discourse analysis in terms of rhetorical relations. He broadens the scope of Winter’s model by showing how certain lexical items can signal the rhetorical pattern of the whole texts instead of confining the role of lexical items to the signaling of clause relations alone. Whatever the pattern used in a discourse, Hoey (1979: 33) identifies a way to detect the presence of a rhetorical pattern by projecting the discourse under analysis into a dialogue involving questions and answers. To demonstrate this method, Hoey (1986: 191) offers the following made-up text: (1) Charles was a language teacher. (2) His students came to him unable to write coherently. (3) He taught them discourse analysis. (4) Now they all write novels. (1) Charles was a language teacher. (2) His students came to him unable to write coherently. (3) He taught them discourse analysis. Projecting this text into a dialogue, the result can be as follows: Projecting this text into a dialogue, the result can be as follows: Projecting this text into a dialogue, the result can be as follows: 1. D (iscourse): Charles was a language teacher. Q(uestion): What problem arose for him? D (iscourse): Charles was a language teacher p 2. D: His students came to him unable to write coherently. Q: What did he do about it? 2. D: His students came to him unable to write coherently. 2. D: His students came to him u Q: What did he do about it? Q: What did he do about it? 3. D: He taught them discourse Q: What was the result? 3. D: He taught them discourse analysis. 3. D: He taught them disco Q: What was the result? Q: What was the result? 4. D: Now they all write novels. Such interrogative method helps establish the relationships between the sentences in the text. Thus, sentence (1) can be identified as the situation, sentence (2) as the problem, sentence (3) as the Solution, and sentence (4) as the positive evaluation. Aspect of Situation Requiring a Response (i.e. problem) Aspect of Situation Requiring a Response (i.e. 1. The Rhetorical Patterns problem) Vol.(58) No.(1)(March-2019 AD, 1440 AH) AL-USTATH AL-USTATH Positive evaluation positive result positive result/evaluation Combined in a single statement Basis Positive evaluation (optional) (optional) Figure (1) The Basic Problem-Solution pattern Adapted from (Hoey 1983 ) Positive evaluation positive positive result result/evaluation Combined in a single statement Basis (optional) Positive evaluation (optional) Figure (1) The Basic Problem-Solution pattern Adapted from (Hoey 1983 ) However, in real discourse the case is more complicated, and Hoey’s (1979) model may fall short of analyzing complex texts. Therefore, Hoey (1983) modified his earlier pattern by considering evaluation as either positive or negative. If it is positive, the same pattern given in Figure (1) would emerge; if the evaluation is negative, it signals another problem that leads to an evaluation, introducing the concept of a multi-layering. A multi-layered pattern is represented in Figure (2) herein: Situation (optional) Problem Response positive evaluation Negative Evaluation And/or result and/or result (all three options in figure (1) Figure (2) the recycling effect of negative evaluation in problem-solution patterns Adapted from (Hoey 2001 ) Intending to render his model universally applicable, Hoey (1986: 196) recommends the following more elegant and abstract presentation of his pattern. Situation (optional) Response Negative Evaluation and/or result (all three options in positive evaluation And/or result Figure (2) the recycling effect of negative evaluation in problem-solution patterns Adapted from (Hoey 2001 ) Intending to render his model universally applicable, Hoey (1986: 196) recommends the following more elegant and abstract presentation of his pattern. Intending to render his model universally applicable, Hoey (1986: 196) recommends the following more elegant and abstract presentation of his pattern. The purpose of the element situation is to provide sufficient background information for the readers to enable them in comprehending the problem being 123 Vol.(58) No.(1)(March-2019 AD, 1440 AH) AL-USTATH discussed however, when the situation is known or can be inferred from the general context, there is no need to provide further details about it. discussed however, when the situation is known or can be inferred from the general context, there is no need to provide further details about it. It can also be said that any coherent piece of thought, analysis or discussion introduced in the situation element is intended to be as a basis that allows the reader to make his own judgment. In other words, it is the setting. 1. The Rhetorical Patterns The key to the understanding of the informational structure in texts is the ability to recognize the lexical signaling of the problem. The element of the problem has been employed in its wider sense to mean any form of dissatisfaction or hazard that creates a need to improve or change a situation. Thus, it can be included under the element of problem any form of unhappiness, discomfort, danger, disease, dilemma, a need to know something, an obstacle, an aim too much or not enough of something etc., in short, any adverse evaluation of a situation or a Solution can be identified as a problem. When the problem is recognized, then an attempt might be expected, proposed or an actual solution to follow. In fact, the element of Solution involves all the actions taken as an effort to introduce a kind of improvement to overcome the identified problem. If these actions succeed in solving the problem, they will be considered them as a Solution, but when they fail, they are termed as attempted Solution. Actual Solutions are most frequently preceded by a pre evaluation which informs the reader that an actual Solution will follow. The element of evaluation is generally used to indicate the effectiveness of the Solution in overcoming the identified problem. The concept of evaluation implies any further information presented not only about the Solution, but also about something that informs us how good or bad that thing is in respect to a class of attributes to which the thing belongs in comparison with similar things. Hence, good evaluation signals the end of discourse, while, negative evaluation signals another problem. For this reason, evaluation should include reliable data, non- controversial evidence, an assessment based on that evidence, or a combination of all these matters. 2. Lexical Signals and Text Organization Lexical signals were first identified by Winter (1968, 1974 and 1977). He divided lexical signals into three groups: vocabulary 1 is the first groups which combine subordinators, vocabulary 2 is the second group for grammatical connectives and the third vocabulary 3 are lexical items such as verbs, nouns and adjectives. For example problem, solution, solve, evaluate, etc. These lexical items serve as signposts in the text. The function of these items is to inform the readers about the type of information presented and how they are organized. y Winter (ibid: 18 – 19) suggested three connective roles for these items: 3. The Analysis of the Story y y The first problem Solution pattern takes a linear progression in which the basic elements of the pattern are arranged in the conventional order of situation-problem- Solution- evaluation. This means that the writer proceeds linearly in exposing the information to the reader. Therefore ,the Solution offered to the identified problem in the text will always end up with a positive evaluation which signals the end of the discourse .No reference is made to another problem This has been so powerful in confirming the reader’s expectations as to the development of discourse. The situation has been established in part (1). The position of this chunk as being first in the discourse provides expectations to the context of the subsequent sentences. The situation is established through the lexical items: hotel, room, garden , and floor signal the setting of the situation ; The participants and their attitudes to each other and to the others are signaled through the following items : two Americans, people, and artists, did not know any ; the medium of communication is a written form by the writer himself. The concept of problem is communicated through the direct lexical items crouched, dripping, compact, and the negative lexical item not dripped in. The Solution offered has been introduced by a pre- Solution which is an alternative Solution immediately preceding the final Solution. This is largely intended to assist the readers’ expectation concerning the nature of the adopted evaluation. Thus (3 and 4) provide the writer’s pre- Solution which lexically signaled by the item ‘get’. The last Solution is followed by a positive evaluation which is signaled by the items ‘don’t get wet’ which means he is convinced that his wife should get the cat. In the above type of problem Solution – pattern there is a chained progression in which each Solution or evaluation results in a different problem. Unlike the linear progression which ends with a positive evaluation, in the chained type a negative evaluation signals another problem. This accounts for the multi-layering phenomenon in discourse, or the existence of more than one problem- Solution pattern. The first problem Solution pattern takes a linear progression in which the basic elements of the pattern are arranged in the conventional order of situation-problem- Solution- evaluation. This means that the writer proceeds linearly in exposing the information to the reader. 3. The Analysis of the Story Therefore ,the Solution offered to the identified problem in the text will always end up with a positive evaluation which signals the end of the discourse .No reference is made to another problem This has been so powerful in confirming the reader’s expectations as to the development of discourse. The situation has been established in part (1). The position of this chunk as being first in the discourse provides expectations to the context of the subsequent sentences. The situation is established through the lexical items: hotel, room, garden , and floor signal the setting of the situation ; The participants and their attitudes to each other and to the others are signaled through the following items : two Americans, people, and artists, did not know any ; the medium of communication is a written form by the writer himself. The concept of problem is communicated through the direct lexical items crouched, dripping, compact, and the negative lexical item not dripped in. The Solution offered has been introduced by a pre- Solution which is an alternative Solution immediately preceding the final Solution. This is largely intended to assist the readers’ expectation concerning the nature of the adopted evaluation. Thus (3 and 4) provide the writer’s pre- Solution which lexically signaled by the item ‘get’. The last Solution is followed by a positive evaluation which is signaled by the items ‘don’t get wet’ which means he is convinced that his wife should get the cat. In the above type of problem Solution – pattern there is a chained progression in which each Solution or evaluation results in a different problem. Unlike the linear progression which ends with a positive evaluation, in the chained type a negative evaluation signals another problem. This accounts for the multi-layering phenomenon in discourse, or the existence of more than one problem- Solution pattern. PATTERN (1) The text PSP-COM There were only two Americans stopping at the hotel. They did not know any of the people they passed on the stairs on their way to and from their room. Their room was on the second floor facing the sea. It also faced the public garden and the war monument. There were big palms and green benches in the public garden. In the good weather there was always an artist with his easel. Artists liked the way the palms grew…The American wife stood at the window looking out. 1. The Rhetorical Patterns The writer uses these lexical items as explicit signals for the text organization. Winter (ibid:23) stated that what makes these items lexical is that they are selected in the same way as other lexical items, nouns, verbs and adjectives to be subjects, verbs, objects and/or a complement to the clause. Being an open-class words, they can be modified easily. Wi (ibid 18 19) d h i l f h i 2. Lexical Signals and Text Organization Lexical signals were first identified by Winter (1968, 1974 and 1977). He divided lexical signals into three groups: vocabulary 1 is the first groups which combine subordinators, vocabulary 2 is the second group for grammatical connectives and the third vocabulary 3 are lexical items such as verbs, nouns and adjectives. For example problem, solution, solve, evaluate, etc. These lexical items serve as signposts in the text. The function of these items is to inform the readers about the type of information presented and how they are organized. The writer uses these lexical items as explicit signals for the text organization. p g g Winter (ibid:23) stated that what makes these items lexical is that they are selected in the same way as other lexical items, nouns, verbs and adjectives to be subjects, verbs, objects and/or a complement to the clause. Being an open-class words, they can be modified easily. Winter (ibid:23) stated that what makes these items lexical is that they are selected in the same way as other lexical items, nouns, verbs and adjectives to be subjects, verbs, objects and/or a complement to the clause. Being an open-class words, they can be modified easily. Winter (ibid: 18 – 19) suggested three connective roles for these items: Winter (ibid: 18 – 19) suggested three connective roles for these items: 124 Vol.(58) No.(1)(March-2019 AD, 1440 AH) AL-USTATH a. Certain items like situation, problem and solution can signal the organization of the whole text. a. Certain items like situation, problem and solution can signal the organization of the whole text. a. Certain items like situation, problem and solution can signal the organization of the whole text. g b. Some items perform an attitudinal function in discourse structure. g b. Some items perform an attitudinal function in discourse structure. c. Certain items may function anaphorically i.e. talk about the preceding sentence. c. Certain items may function anaphorically i.e. talk about the preceding sentence. 3. The Analysis of the Story situation Outside right under their window a cat was crouched under one of the dripping green tables. The cat was trying to make herself so compact that she would not dripped on. problem ‘I’m going down and get that kitty,’ the American wife said. Pre- The text p pp ‘I’m going down and get that kitty,’ the American wife said. Pre- 125 Vol.(58) No.(1)(March-2019 AD, 1440 AH) AL-USTATH solution ‘I’ll do it.’ Her husband offered from the bed. Pre- solution ‘No, I’ll get it.’ The poor kitty out trying to keep dry under a table solution The husband went on reading, lying propped up with the two pillows at the foot of the bed. ‘Don’t get wet,’ he said. P- evaluation ‘I’ll do it.’ Her husband offered from the bed. The situation here is presented to symbolize the problems arising from lack of understanding between spouses. These problems may affect the marriage to the point of making it miserable. g Right from the title of the story one senses the existence of a problem. Cats hate water therefore exposure to water means an unsatisfactory situation that requires avoidance so the cat seeks shelter to avoid being "dripped on" The public garden in the opening of the story may be taken to mean the ideal state of marriage or how should an ideal marriage be, whereas the war monument refers to conflict and tragic events that may spoil the ideal state. The Paradise of the public garden is spoiled by war and thus marriage of the American Adam and Eve was spoiled by conflict which does not necessarily need to be violent. A conflict of ideas, attitudes and lack of common vision and understanding is symbolized by the rain falling on the public garden. The cat may be taken as resembling the American woman stuck in a hotel room with a careless and not very sympathizing husband puts the wife in a position similar to that of the cat which indicated that the situation is already problematic. y p An attempt to solve the problem is shown as the wife decides to rescue the "kitty". An attempt to solve the problem is shown as the wife decides to rescue the "kitty". An attempt to solve the problem is shown as the wife decides to rescue the kitty . The husband meekly offers to help but reclines as he is engrossed with reading. The husband meekly offers to help but reclines as he is engrossed with reading. The wife takes the initiative to solve the cat's problem thus unconsciously attempts to solve her own. The wife takes the initiative to solve the cat's problem thus unconsciously attempts to solve her own. The term "kitty" is childish reflecting a childish yearning in a grown up woman. The problem in Pattern one remains unsolved and the only positive remark the wife receives is that of "Don’t get wet." PATTERN (2) The text PSP-COM The wife went down stairs and the hotel owner stood up and bowed to her as she passed the office. His desk was at the far end of the office. ‘I’ll do it.’ Her husband offered from the bed. He was an old man and very tall ‘Il poive ,1’ the wife said. She liked the hotel- keeper. ‘Si.Si, Signora, brutto tempo.2 It is very bad weather’ situation He stood behind his desk in the far end of the dim room. The wife liked him. She liked the deadly serious way he received any complaints. She liked his dignity. She liked the way he wanted to serve her……… She liked his old , heavy and big hands Problem Liking him, she opened the door and looked out. Solution It was raining harder. A man in a rubber cape was N-evaluation AL-USTATH AL-USTATH crossing the empty square to the café. The cat would be around to the right Perhaps she could go along under the eaves As she stood in the doorway, an umbrella opened behind her. It was the maid who looked after their room. ‘You must not get wet,’ she smiled, speaking Italian. Of course, the hotel –keeper had sent her. 1 Problem Pre-solution Solution 1 ‘It’s raining.’ 2 2 ‘Yes, yes, Madam, awful weather’ In this part of the story the situation begins as the wife descends the stairs. The hotel owner stood up and "bowed" to the American wife. The problem arising is that of the hotel owner. He is old, very tall with heavy hands thus representing wisdom, strength and capability; all that her husband lacks. At this moment the writer presents the second problem: the wife's liking for the hotel owner. The problem is soon solved when the wife opens the door to go outside. p p g In this part readers are presented with the problem of the rain outside getting harder therefore implying subconscious comparison between husband and hotel owner on the part of the wife. Assuming the location of the cat she sets to solve the first problem. Readers are encountered with a solution for the hard rain when an umbrella is opened right behind her. The solution here is provided by the maid who must have been sent by the masculine hotel owner. With the maid holding the umbrella over her, she walked along the gravel path until she was under their window. situation With the maid holding the umbrella over her, she walked along the gravel path until she was under their window. The table was there, washed bright green in the rain, but the cat was gone. She was suddenly disappointed The maid looked up at her. problem ‘Ha perduto qualque cosa, Signora?’3 ‘Ha perduto qualque cosa, Signora?’3 ‘There was a cat,’ said the American girl. ‘A cat?’ ‘There was a cat,’ said the American girl. ‘Si , il gatto.’ ‘ A cat?’ The maid laughed. ‘A cat in the rain?’ ‘Yes, -’, she said, ‘under the table.’ Then, ‘Oh , I wanted it so much. I wanted a kitty.’ y When she talked English the maid’s face tightened. ‘Come, Signora,’ she said. ‘We must get back inside. You will be wet.’ solution ‘Come, Signora,’ she said. The text 126 Vol.(58) No.(1)(March-2019 AD, 1440 AH) AL-USTATH ‘We must get back inside. You will be wet.’ ‘I suppose so’, said the American girl. 3 3 ‘Have you lost something, Madam?’ 127 Vol.(58) No.(1)(March-2019 AD, 1440 AH) AL-USTATH In this pattern the situation presented is that of a woman sheltered from rain walking along a gravel path. In this pattern the situation presented is that of a woman sheltered from rain walking along a gravel path. Hemingway may have sought to refer to women sheltered by the umbrella of marriage while walking along the path of life or the umbrella could be taken to refer to American superiority and the inclination to be served for having superior financial status. Either way the situation leads to the problem of the missing cat. To the wife's Disappointment, the cat is no where to be seen. Knowing the creature's hatred for water the maid laughingly inquires how can a cat possibly remain under the rain? The problem persists though the wife actually admits her need to have found the cat. The solution presented is for the rain problem when the maid decides that they "must get back" and the wife reluctantly agrees ". I suppose PATTERN (4) PATTERN (4) The text PSP-COM They went back along the gravel path and passed the door. The maid stayed outside to close the umbrella. As the American girl passed the office, the padrone bowed from his desk. situation Something felt very small and tight inside the girl. The padrone made her feel very small and at the same time really important……. problem She went on up the stairs She opened the door of the room. solution George was on the bed, reading. N-evaluation ‘Did you get the cat?’ he asked, putting the book down. Pre-problem ‘It was gone.’ ‘ Wonder where it went to,’ he said ,resting his eyes from reading. She sat down on the bed. problem ‘I wanted it so much,’ she said. ‘I don’t know why I wanted it so much. I wanted that poor kitty. It is’nt any fun to be a poor kitty out in the rain.’ solution George was reading again P-evaluation PATTERN (4) The text PSP-COM They went back along the gravel path and passed the door. The maid stayed outside to close the umbrella. As the American girl passed the office, the padrone bowed from his desk. situation Something felt very small and tight inside the girl. The padrone made her feel very small and at the same time really important……. problem She went on up the stairs She opened the door of the room. solution George was on the bed, reading. N-evaluation ‘Did you get the cat?’ he asked, putting the book down. Pre-problem ‘It was gone.’ ‘ Wonder where it went to,’ he said ,resting his eyes from reading. She sat down on the bed. problem ‘I wanted it so much,’ she said. ‘I don’t know why I wanted it so much. I wanted that poor kitty. It is’nt any fun to be a poor kitty out in the rain.’ solution George was reading again P-evaluation George was on the bed, reading. ‘Did you get the cat?’ he asked, putting the book down. ‘It was gone.’ ‘It was gone.’ ‘It was gone.’ ‘ Wonder where it went to,’ he said ,resting his eyes from reading. She sat down on the bed. ‘I wanted it so much,’ she said. ‘I don’t know why I wanted it so much. I wanted that poor kitty. It is’nt any fun to be a poor kitty out in the rain.’ solution George was reading again George was reading again The situation is presented when the American woman goes inside and passes the Padron's office. The maid remains outside to take care of the umbrella. The problem arises when she feels "tight", "very small" and "important". The problem shown in Pattern 2 of "liking" is expanded by the above mentioned feelings. These feminine inclinations exist in one way or another in almost every female. Thus the American girl becomes an archetype. Women usually seek shelter in the arms of wise masculinity which provides an "umbrella" to protect her from life discomforts. This is similar the cat's case using a wooden table for shelter. For the American girl the hotel Padron is as solid as the wooden table. The solution is provided by going upstairs to her room. The husband's negativity is reflected in his passive role, though there is a slight change when he puts his book down, but the original problem remains unsolved. p y g g p The husband's negativity is reflected in his passive role, though there is a slight change when he puts his book down, but the original problem remains unsolved. 128 Vol.(58) No.(1)(March-2019 AD, 1440 AH) AL-USTATH The wife admits wanting the cat but ignores the reasons behind this want. She subconsciously finds her as an equal and expresses concern over the "Kitty's" miserable situation. All this is met by carelessness on the husband's part. subconsciously finds her as an equal and expresses concern over the "Kitty's" miserable situation. All this is met by carelessness on the husband's part. PATTERN (5) The text PSP-COM She went over and sat in front of the mirror of the dressing table looking at herself with the hand glass. She studied her profile, first one side and then the other. Then she studied the back of her head and of her neck. situation ‘Don’t you think it would be a good idea if I let my hair grow out?’ She asked, looking at her profile again. problem George looked up and saw the back of her neck, clipped close like a boy’s. ‘I like it the way it is.’ Pre-solution solution ‘I get so tired of it.’ she said, ‘I get so tired of looking like a boy.’ N-evaluation George shifted his position in the bed. He hadn’t looked away from her since she started to speak.’ ‘You look pretty darn nice,’ he said. George was reading again She laid down the mirror down on the dresser and went over to the window and looked out. It was getting dark. N-evaluation ‘I want to pull my hair back tight and smooth and make a big knot at the back that I can feel,’ she said.’ ‘I want to have a kitty to sit on my lap and purr when I stroke her.’ problem ‘Yeah?’ George said from the bed. N -evaluation And I want to eat at a table with my own silver and I want candles. And I want it to be spring and I want to brush my hair out in front of a mirror and I want a kitty and I want some clothes problem Oh, shut up and get something to read,’ George said solution He was reading again. His wife was looking out of the window. It was quiet dark now and still raining in the palm trees. N-evaluation ‘Anyway , I want a cat,’she said, I want a cat now. If cannot have long hair or any fun, I can have a cat. problem George was not listening. He was reading his book. solution His wife looked out of the window where the light had come on in the square. N-evaluation Someone Knocked at the door. problem ‘Avanti, George said. The text She went over and sat in front of the mirror of the dressing table looking at herself with the hand glass. She studied her profile, first one side and then the other. Then she studied the back of her head and of her neck. dressing table looking at herself with the hand glass. She studied her profile, first one side and then the other. Then she studied the back of her head and of her neck. ‘Don’t you think it would be a good idea if I let my hair grow out?’ She asked, looking at her profile again. George looked up and saw the back of her neck, clipped close like a boy’s. ‘I get so tired of it.’ she said, ‘I get so tired of looking like a boy.’ George shifted his position in the bed. He hadn’t looked away from her since she started to speak.’ George shifted his position in the bed. He hadn’t looked away from her since she started to speak.’ ‘You look pretty darn nice,’ he said. ‘Excuse me,' she said, ‘the padron asked me to bring this for the Signora’ solution ‘Excuse me,' she said, ‘the padron asked me to bring this for the Signora’ solution The final situation is presented when the wife sits in front of the mirror. The hand glass she uses to see her profile is like self analysis as she moves it from one side to the other. The major problem brewing inside is expressed through the simple question; "Don't you think it is a good idea if I let my hair grow out?" When the husband, now called "George" replies, a solution for the superficial problem is provided. The widely used name "George" is used here to resemble the majority of American men and the husband becomes an archetype. yp George is "Adam" who ignores "Eve". George is "Adam" who ignores "Eve". The wife's concise evaluation of her problem is fully expressed when she admits t "getting tired" of it meaning tired of such life. She manages to draw her husband's attention for a short while but his evaluation is once more negative. "George" does not realize the depth of the problem he is only interested in the apparent meaning of his wife's complaint not realizing the problem brimming inside. Readers realize the existence of a major problem when the wife cites the minor problems one after the other. She wants to wear her hair "long" to pull it back in a "tight" knot. This hairdo resembles the sheltered calm female of nobility often portrayed with a cat on her lap. This is supported by her desire to have silverware and candles. The American woman is tired of practicality and equality with men represented by "short hair". She longs for being taken care of and seeks some romance through wanting it to be "spring". Thus her apparently childish needs carry a much deeper meaning. All her efforts are curbed when George tells her to "shut up" and "get something to read" thus emphasizing the idea of equality, yet killing all her aspirations. His negative evaluation for the point she tries to make sends her back to the window. The unsolved problem is more persistent as it gets dark and the rain continues. ‘You look pretty darn nice,’ he said. ‘You look pretty darn nice,’ he said. She laid down the mirror down on the dresser and went over to the window and looked out. It was getting dark. ‘I want to pull my hair back tight and smooth and make a big knot at the back that I can feel,’ she said.’ ‘I want to have a kitty to sit on my lap and purr when I stroke her.’ ‘Yeah?’ George said from the bed. g And I want to eat at a table with my own silver and I want candles. And I want it to be spring and I want to brush my hair out in front of a mirror and I want a kitty and I want some clothes Oh, shut up and get something to read,’ George said He was reading again. His wife was looking out of the window. It was quiet dark now and still raining in the palm trees. ‘Anyway , I want a cat,’she said, I want a cat now. If cannot have long hair or any fun, I can have a cat. I want a cat now. If cannot have long hair or any fun, I can have a cat. George was not listening. He was reading his book. George was not listening. He was reading his book. His wife looked out of the window where the light had come on in the square. Someone Knocked at the door. ‘Avanti, George said. 129 Vol.(58) No.(1)(March-2019 AD, 1440 AH) AL-USTATH He looked up from his book. She held a big tortoise-shell cat pressed tight against her and swung down against her body ‘Excuse me,' she said, ‘the padron asked me to bring this for the Signora’ solution The wife ceases to follow reason "Anyway I need a cat", "I want a cat now", "If I cannot have long hair or "any fun", I can have a cat." y The husband solves the problem by ignoring it and going back to his book. The husband solves the problem by ignoring it and going back to his book. The lights come on outside foretelling a solution. This is provided through a knock on the door and the appearance of the maid holding a big cat. The lights come on outside foretelling a solution. This is provided through a knock on the door and the appearance of the maid holding a big cat. Though the cat may not have been the same and probably a substitute for the original, the wife had seen from her window, but it solves the problem. A substitute solution is provided by the "Padron" assuming George's protective role. 130 Vol.(58) No.(1)(March-2019 AD, 1440 AH) AL-USTATH Conclusions sions lysis of the story has shown the following results: The analysis of the story has shown the following results: The analysis of the story has shown the following results: 1. Hemingway followed a conventional order in presenting the information i.e. situation, problem, solution and evaluation. This proves the first hypothesis that the problem-solution pattern works with literary texts. 2. He communicated the rhetorical pattern to the reader through his clever use of the lexical items. This proves the second hypothesis in which the lexical items marked in bold signal the structure of the text. g 3. The solution is always preceded by a pre-solution. 3. The solution is always preceded by a pre-solution. 4. The positive solution signals the end of the discourse and the negative solution signals another problem which confirms the expectations of the reader. 4. The positive solution signals the end of the discourse and the negative solution signals another problem which confirms the expectations of the reader. 131 Vol.(58) No.(1)(March-2019 AD, 1440 AH) AL-USTATH References Hemingway, Ernest. (1987). The Complete Short Stories of Ernest Hemingway. Simon & Schuster inc. New York. Hoey, Michael. (1983). On the Surface of Discourse. Allen and Unwin, London. -----------------. (1983). ‘some text properties of certain nouns’, in Tony McEnery and Simon Botley (eds.), New Approaches to Discourse Anaphora: Proceddings of the second Colloquium on Discourse Anaphor Resolution DAARC2, University of Lancaster. -----------------. (1991). Patterns of Lexis in Text. Oxford University Press. -----------------. (1993). Data, description, Discourse: Papers on the English Language in Honour of John McH Sinclair. Harper Collins, London. ----------------. (2001). Textual Interaction: An Introduction to Written Discourse Analysis. Routledge, London. Winter, Eugene. (1977). ‘A clause-relational approach to English texts’. Instructional science (special edn.), 6,1-92. -----------------. (1982). Towards a Contextual Grammar of English: The Clause and Its Place in the Definition of Sentence. Allen and Unwin, London. ----------------. (1992). ‘the notion of Unspecific Versus Specific as One Way of Analysing The Information of a Fund-Raising Letter’, in William C. Mann and Sandra A. Thompson (eds.), Discourse Description: Diverse Linguistic Analyses of a Fund- Raising Text, Amesterdam: John Benjamins, 131-70 Hemingway, Ernest. (1987). The Complete Short Stories of Ernest Hemingway. Simon & Schuster inc. New York. Hoey, Michael. (1983). On the Surface of Discourse. Allen and Unwin, London. -----------------. (1983). ‘some text properties of certain nouns’, in Tony McEnery and Simon Botley (eds.), New Approaches to Discourse Anaphora: Proceddings of the second Colloquium on Discourse Anaphor Resolution DAARC2, University of Lancaster. -----------------. (1991). Patterns of Lexis in Text. Oxford University Press. ----. (1991). Patterns of Lexis in Text. Oxford University Press. ---. (1993). Data, description, Discourse: Papers on the English Language in Honour of John McH Sinclair. Harper Collins, London. --. (2001). Textual Interaction: An Introduction to Written Discourse Analysis. Routledge, London. Winter, Eugene. (1977). ‘A clause-relational approach to English texts’. Instructional science (special edn.), 6,1-92. ---. (1982). Towards a Contextual Grammar of English: The Clause and Its Place in the Definition of Sentence. Allen and Unwin, London. ----------------. (1992). ‘the notion of Unspecific Versus Specific as One Way of Analysing The Information of a Fund-Raising Letter’, in William C. Mann and Sandra A. Thompson (eds.), Discourse Description: Diverse Linguistic Analyses of a Fund- Raising Text, Amesterdam: John Benjamins, 131-70 132 132
https://openalex.org/W4285576992	https://zenodo.org/records/6644246/files/Gul%20KJET-2.1-2020-0129072021124300.pdf	English	null	Steel Structures Usage and Steel Production: A Case Study in Afghanistan	Kardan journal of engineering and technology	2,020	cc-by	5,682	Mr. Gul Rahman Abdulrahimzai, Teaching Assistant, Dept. of Civil Engineering, Kardan University, Kabul Afghanistan. <agulrahman@gmail.com > Mr. Haroon Rasheed Safi and Mr. Asil Ahmad Halimyar, are Graduated Student, Dept. of Civil Engineering, Kardan University, Kabul Afghanistan. Steel Structures Usage and Steel Production: A Case Study in Afghanistan Gul Rahman Abdulrahimzai Haroon Rasheed Safi Asil Ahmad Halimyar Gul Rahman Abdulrahimzai Haroon Rasheed Safi Asil Ahmad Halimyar Keywords: Steel Structure’s Usage, Steel Production Industry and Market, SPSS, Steel Imports, Investment. Article Article Kardan Journal of Engineering and Technology 2 (1) 1–19 ©2020 Kardan University Kardan Publications Kabul, Afghanistan DOI: 10.31841/KJET.2021.9 https://kardan.edu.af/Research/Kardan_jour nal_of_engineering_and_technology.aspx# Kardan Journal of Engineering and Technology 2 (1) 1–19 ©2020 Kardan University Kardan Publications Kabul, Afghanistan DOI: 10.31841/KJET.2021.9 https://kardan.edu.af/Research/Kardan_jour nal_of_engineering_and_technology.aspx# Steel Structures Usage and Steel Production: A Case Study in Afghanistan Abstract Concrete structures dominant in the construction sector and the lack of steel structure usage is a propagating problem in Afghanistan. This problem not only affects the steel production industry and market but also the construction Industry. To determine factors contributing to this problem and their extent, a series of surveys and interviews were conducted with steel traders, wholesalers, steel production companies, construction companies, and engineers. The collected data was analyzed by SPSS (Software package for the Social Sciences) program. After a thorough investigation of the results, it was concluded that, the low investment in steel sector has created a shortage in steel products’ market. Therefore, traders import steel products legally and illegally, which in turn weakens the domestic production industry on basis of unfair competition. Moreover, a serious shortage of skilled labor, design professionals, and quality materials has shifted more attention towards the concrete structure. To address these concerns, this paper has attempted to suggest solutions that can help improve the market condition and shift attention towards building steel structures. The outcome suggests that training professionals in the steel sector, importing steel production and erection technologies, increasing tariffs on steel imports, controlling illegal imports, exploring mines and iron reserves for raw materials, and making productive investments in the steel sector are likely to contribute to the reduction of the present problems. Keywords: Steel Structure’s Usage, Steel Production Industry and Market, SPSS, Steel Imports, Investment. 1 Steel Structures Usage and Steel Production: A Case Study in Afghanistan Introduction Steel’s use as a construction material roughly follows the same timeline as steel innovation in general as railroads became a popular mode of transport [1]. In the 1800s, there were three forms of “ferrous metals” in use: wrought iron, cast iron, and steel. Wrought iron was very familiar to blacksmiths who had been working with the material for years. While we now think of it as mostly decorative, in the Victorian era, it was also commonly used as a building material. Cast iron, strong but brittle were more widely used for cooking and farming but was not very appropriate for the building. Steel was expensive to produce and was used for higher-end items like watches, swords, and scythes. [1]. Steel has become one of the most significant materials in construction industry. It is often seen as an extremely important component of modern buildings and housing [2]. With more than 1,869.9 million tons produced in 2019, It’s become viable for any kind of project and offers several benefits, which many building plans rely on for structural safety [3]. Due to factors such as weight, availability, strength, and sustainability [4]. However, unlike other countries, construction in Afghanistan heavily relies on concrete structures and steel is seldom used for construction. Concrete dominance over market can be a grave problem since it creates a monopoly on the steel industry and creates hurdles in way of steel resources exploitation and new company commencements. Moreover, construction industry is changing and new trends are emerging [5]; since Steel plays an important role in these trends, Its applications will increase substantially in future. Therefore, Afghanistan needs to address these problems and adapt with the fast-evolving industry. Consequently, this paper aims to investigate steel usage and the problems in its way in construction sector in Afghanistan. Furthermore, it will attempt to suggest potential solutions to the existing problems. 1.3. Objectives The purpose of this research paper is to investigate the barriers and challenges in way of steel usage in constructional sector in Afghanistan. It will suggest possible solutions to some of the most persistent problems causing the lack of interest and investment in building steel structures in Afghanistan. • Challenges in way of steel production. • Challenges in way of steel production. • Barriers in way of steel utilization in construction industry. • Market problems. • Market problems. • Market problems. • Solutions to abovementioned challenges. 1.2. Problem Statement Although steel is an important component in developing infrastructures and construction industry, it is barely utilized in construction sector in Afghanistan. Currently the market is dominated by concrete structures because of the following problems: first, there is low level of investment in steel sector and companies are unable to fulfill market demands. Therefore, imported products from other countries dominate the market. Secondly, even though Afghanistan has a very high potential for steel production, none of its mines has been explored and producers only use scrap metals. Furthermore, there is a serious shortage of professionals and skilled labor involved in this sector. overall costs increase substantially and projects face hurdles when steel structures are intended to be used. These problems will be discussed separately here. 1.1. An Overview of Steel Construction in Afghanistan Afghanistan is a country blessed with natural resources. It has 3 trillion mineral reserves from which iron consists of 2.3 million tons [6],[7]. However, despite being so rich in resources, mines are still untapped. The steel industry uses metal scraps, which are mostly the remaining parts of tanks and other weaponries from the decades-long war. During this long unrest, Afghanistan has completely lost its infrastructure. There were very few opportunities for investments and many sectors completely shut down. Many Industries including steel industry had to start all over again after the establishment of new government in 2001. Despite vigorous damages to the steel industry, there is a strong political will and numerous efforts have been made to revive it. Large investments have been done in this sector. For 2 Abdulrahimzai, Safi & Halimyar (2020) instance, Maihan and Khan steel companies started their operations in 20 million US dollars with an investment of about 100 and 10 million dollars respectively. Whereas there were no domestic products a few years ago, 65% of the market is now occupied by domestic products. This shows that Afghanistan is now able to somewhat compete with the regional product and is going towards self-sufficiency in steel sector. 1.4. Scopes and Limitations This study is limited to the study of causes that creates hurdles in way of steel structures construction in Afghanistan. The study consists of the market condition, steel production, availability, imports, skills and expertise. Primary data is collected from Kabul, Mazar-e- Sharif, Jalal Abad and Khoust. A survey is conducted from various parties including steel product traders, steel production companies, construction companies, and engineers. This survey is only conducted in Kabul province. 3 3 Steel Structures Usage and Steel Production: A Case Study in Afghanistan 2.2. Skill and Expertise Due to the 4 decades long war, Afghanistan has lost a high number of its intellects and skilled labor. With the establishment of new government, considerable efforts have been made in education sector. A number of highly qualified experts returned to the country. However, there is still a considerable shortage in the involvement of professional engineers and skilled labor in Afghanistan [8]. Moreover, the absence of skilled labor in country appears to be having negative effect on business, industries and it is affecting investment in the country. [9] [10]Therefore, to cut expenses construction most often prefer concrete rather than steel for construction. 2. Literature Review Currently investment in the Afghan construction market is dominated by concrete for there is an overall shortage of steel products. If a steel structure is intended to be built, the company is ought to order all the required products from foreign countries which increases the costs notably. Moreover, there is a dearth of domestic products availability in market. steel products are not preferred by dealer and end users. Moreover, due to lack of government concentration and open borders regional monopoly players are trying to fail the industry. This makes it very challenging for new companies to enter market. 2.1. Raw Materials Although Afghanistan is very rich in natural resources and has an estimated 2.3 billion tons of iron reserves, none of its mines are extracted yet. Steel production companies use metal scraps as raw material. A portion of these scraps are imported from other countries which has its effect on product’s prices. No Province Location Reserve/ton 1 Badakhshan Saijar 45,000,000 2 Badakhshan Farmara 35,000,000 3 Baghlan Zirak 20,000,000 4 Bamyan Khysh 117,000,000 5 Bamyan Haji Gak 2,070,000,000 Source: Ministry of Mines report Table 1: List of Afghanistan Iron Reserves 2.2. Skill and Expertise Table 1: List of Afghanistan Iron Reserves 3. Methodology This research is based on three types of questioners for primary, secondary and tertiary data collection. Different questioners were sent and interviews were conducted with steel product dealers, steel production companies, construction companies and engineers. Quantitative research method was used to collect the data about production, capacity, and investment. Qualitative research method was used to collect the data about customers, strategies, available products, customer choices and behavior and barriers. The collected data was then analyzed using SPSS (Software package for the Social Sciences) software. SPSS statistics is a statistical software platform which delivers a specific set of feature which lets users extract insights from data [11-16]. • Analyze and understand your data, and solve complex research problems [17-18]. • Understand large and complex data sets with statistical procedures [19-20]. 3.1. Primary Data Primary data on market is collected from steel wholesale dealers of Kabul and different provinces of Afghanistan. 2.3. Technology Another contributing factor to the preference of concrete structure instead of steel structures is the lack technology and machines availability in Afghanistan. Many steel sections cannot be produced inside the country and have to be ordered from abroad. This limits the implementation of many 4 Abdulrahimzai, Safi & Halimyar (2020) steel structure designs and it is a burden on engineers and construction companies in terms of project cost and desired materials availability. Chart 1: Available Steel Products in Afghanistan Market 3. Methodology Chart 1: Available Steel Products in Afghanistan Market 3.2. Secondary Data Secondary data is collected from Afghanistan’s steel industries. Secondary data is collected from Afghanistan’s steel industries. Further data is collected from construction companies, and engineers working in the construction sector in Afghanistan. The government and businesses should invest in steel production companies to increase their production capacity. This will eliminate the need 5 5 Steel Structures Usage and Steel Production: A Case Study in Afghanistan for importing steel products and lessen the competition with regional monopoly players. 4.1. Primary Data on Market The first target of the survey is the construction steel market to determine which products are available. According to conducted survey, Afghanistan steel market is composed of domestic and imported products. During the survey it was determined that the market is dominated by imported steel products. While there is no major difference in the quality of domestic and imported products, a considerable portion of customers prefer imported products because of the unawareness about domestic products. Furthermore, weak marketing strategy of domestic producers and illegal tax, tariff free imports have created an unfair competition in the market. Chart 2: Steel Products Imported from Different Countries Shown in Percentages Chart 2: Steel Products Imported from Different Countries Shown in Percentages 4. Results and Discussion Concrete dominance over construction market is a grave problem for it creates a monopoly on the industry. There are various reasons to this problem. From materials availability to the required technologies, skills, expertise, labor, and raw materials there are a number of factor that has created this problem. Thus, many engineers and construction companies prefer concrete structures over steel structures in Afghanistan. To investigate the extent of these issues this paper results of a survey conducted in different parties involved in the construction sector in Afghanistan. 4.1.2. Available Products in the Market The conducted survey mainly focused on construction steel products in market. It was found that the following products were available in market. Chart 3: Available Imported Steel Products in Afghanistan Chart 3: Available Imported Steel Products in Afghanistan 4.1.1. Investments Afghanistan’s steel industry is not able to fulfill the market demand due to lack of sufficient productive investment in this sector. Afghan government needs to take serious steps to medicate risk to the investment. For example, other illegal imports and the illegal imports of steel goods is an alarming 6 Abdulrahimzai, Safi & Halimyar (2020) danger for domestic steel industry. An increase of tariff on imports of steel and controlling illegal imports can Support the domestic production. 4.1.3. Domestic Products in the Market The conducted survey found out the following steel products were produced and available in the market. Chart 4: Domestic Steel Products Availability in the Market Chart 4: Domestic Steel Products Availability in the Market It could be seen that a number of products are not produced in Afghanistan and must be imported from foreign countries. This creates an idea that domestic products are of lesser quality. Additionally, an engineer must order required product from other countries if he intends to design and build a steel structure. This will increment the overall costs substantially. Chart 5: Categories that Make the Primary Customers of Steel Products 4.1.4. Primary Customers Most customers that buy steel products private buyers and end users. From the chart below it can be determined that steel products are mostly bought for individual uses and are not preferred by construction companies and the government as the concrete is dominant. 7 7 Steel Structures Usage and Steel Production: A Case Study in Afghanistan 4.1.5. Product Preference As shown in the 52% of customers prefer imported products while 28% prefer domestic steel products. The high extent of imported products preference negatively affects steel industry progress in Afghanistan. The remaining 20% of the customers prefer both products whichever is available and economical. Chart 6: Product Preference 4.1.6. Domestic Product Availability Over 60% of traders and whole sellers have domestic products. However, 40% do not have domestic products. Steel production companies, therefore, must adopt new strategies to cover the remaining 40% of the market. product availability in market will undoubtedly make it easier for buyers and companies to access domestic products and in turn support domestic companies back. Chart 6: Product Preference 4.1.6. Domestic Product Availability Chart 6: Product Preference Chart 6: Product Preference Steel Structures Usage and Steel Production: A Case Study in Afghanistan Chart 5: Categories that Make the Primary Customers of Steel Products 4.2. Production Companies Afghanistan has deep roots in steel production. However, the steel industry faced a grave decline during wars. Since 2003 this sector began to emerge and made a notable progress despite the strategies of regional countries to flap it. If the government take serious steps steel industry will undoubtedly become self-sufficient. This research has collected data from 7 steel production companies on their initial investments, the source of their raw materials, used technology, production rate, cost affecting factors, mission, market competition, and the barriers they face. 4.1.6. Domestic Product Availability Over 60% of traders and whole sellers have domestic products. However, 40% do not have domestic products. Steel production companies, therefore, must adopt new strategies to cover the remaining 40% of the market. product availability in market will undoubtedly make it easier for buyers and companies to access domestic products and in turn support domestic companies back. 8 8 Abdulrahimzai, Safi & Halimyar (2020) Chart 7: Domestic Products Availability Percentage by Wholesalers 4 2 Production Companies Chart 7: Domestic Products Availability Percentage by Wholesalers 4.2.2. Raw Materials Most of raw materials used by companies are iron scrapes which are collected, melted and reformed and converted into various products. These scrapes are found inside the country however some of them is imported from Pakistan. Chart 9: Raw Materials taken as Primary Products in Afghanistan by Steel Industry Chart 9: Raw Materials taken as Primary Products in Afghanistan by Steel Industry Nonetheless, Afghanistan has yet not explored its rich mines and all the iron reserves remain untapped. Though iron scrapes are a good source of raw materials for companies, eventually they will run out. Therefore, iron mines should be studied and extracted. 4.2.1. Investments Some steel production companies have entered the market with an initial investment as low as 0.1 million dollars. However recently 2 companies have invested 30 million and 500 million dollars. This significant change that can lead Afghanistan towards self-sufficiency in this sector. Chart 8: Initial Investments Made by Steel Production Companies As charted above a number of notable investments have been made in steel sector which will change the market markedly for future. Yet the government and businesses should take steps to ensure steel production companies are adequately supported. Chart 8: Initial Investments Made by Steel Production Companies Chart 8: Initial Investments Made by Steel Production Companies As charted above a number of notable investments have been made in steel sector which will change the market markedly for future. Yet the government and businesses should take steps to ensure steel production companies are adequately supported. 9 Steel Structures Usage and Steel Production: A Case Study in Afghanistan 4.2.4. Production Capacity 4.2.4. Production Capacity It was found out that the sampled firm have a variety of production rates, from a minimum of 20 metric tons per day to a maximum of 700 tons per day. Moreover, the annual production of these firm was determined to be from 7300 to 260,000 metric tons. Chart 11: Steel Production Rate Per Day Chart 12: Annual Steel Production Rate Chart 11: Steel Production Rate Per Day Chart 11: Steel Production Rate Per Day Chart 11: Steel Production Rate Per Day Chart 12: Annual Steel Production Rate Chart 12: Annual Steel Production Rate Chart 12: Annual Steel Production Rate Currently about 80% of the annual steel needs of Afghanistan (650,000- 700000) are produced by domestic companies. This is a huge progress for steel industry in Afghanistan. Nevertheless, the main challenge is the limited variety of products that are produced. Most often steel structures and engineers are limited by this inadequate variety of steel products. Therefore, many projects are not likely to be implement using only domestic products. Steel production companies should expand their capacity by producing more varieties of steel products so that the need for importing custom ordered products is eliminated. 4.2.3. Applied Modern Technology Based on the data collected 57% of the surveyed companies uses modern technology and 43% do not have electrical machines and modern equipment. Chart 10: Modern Technology put is use by Steel Production Companies Chart 10: Modern Technology put is use by Steel Production Companies One of the reasons that domestic companies cannot produce custom ordered products is because of the lack of modern machinery. Therefore, importing modern machinery can solve this problem and shift attention towards building steel structures. 10 Abdulrahimzai, Safi & Halimyar (2020) Chart 13: Factors Caused Cost Rise of Domestic Products Since raw materials and electricity play significant role in steel product prices, exploring mines and providing low-priced electricity will bring a notable change in domestic products prices. Therefore, enabling better competition with regional countries. 4.2.5. Cost Affecting Factors Based on the answers received from the respondents, the final price is set based on the total investments plus profit. Some important factor that affects the overall prices of the steel products are raw materials, electricity, labor, transportation, and taxes. 11 11 Steel Structures Usage and Steel Production: A Case Study in Afghanistan 4.2.6. Market Competition Steel producers consider legal and illegal imports from regional countries as their biggest competitor. 86% of industries believe that regional countries are their competitors whereas 14% say they don’t have any competition. Chart 14: Market Competitors Since the emergence of steel industry in Afghanistan (,) regional competitors have used techniques like dumping and price reduction to flap the industry and keep control of it. Therefore, it is crucial for afghan industry to become self-sufficient in order to survive in the market and be able to compete. Chart 14: Market Competitors Chart 14: Market Competitors Since the emergence of steel industry in Afghanistan (,) regional competitors have used techniques like dumping and price reduction to flap the industry and keep control of it. Therefore, it is crucial for afghan industry to become self-sufficient in order to survive in the market and be able to compete. 12 12 Abdulrahimzai, Safi & Halimyar (2020) 4.2.7. Barriers The conducted survey asked industrialists about the barriers and challenges they face. They respondents pointed out factors such security, electricity, raw materials, investments and so on. The collected answers are shown below: Chart 15: Obstructions in the Steel Industry in its Production Chart 15: Obstructions in the Steel Industry in its Production It can be seen that unfair competition, electricity, and raw materials are the most persistent problems. To address these problems some serious steps must be taken. For instance, illegal trades should be controlled, electricity should be provided for companies and as mentioned before, mines should be exploited to provide quality raw materials. 5.3. Construction Companies and Engineers Afghanistan has an enormous reserves of Iron mines. Therefore, it is an ideal country for producing and utilizing steel products. However, most of construction companies and engineer prefer using concrete instead of steel. In order to discover the reason for this preference a survey was conducted in 5 construction companies and 16 engineers were interviewed, too. 5.3.1. Preference First, question was about their preference and the reasons for it. Results of the survey and interviews depicted that 81% of the of engineer and all construction companies preferred concrete over steel. Majority of the reasons are provided here: 13 Steel Structures Usage and Steel Production: A Case Study in Afghanistan Steel Structures Usage and Steel Production: A Case Study in Afghanistan Chart 16: Reasons for Preferring Concrete Rather than Steel Chart 16: Reasons for Preferring Concrete Rather than Steel To answer these concerns, more technicians and engineers should be trained in training centers and universities. As Afghan Korea vocational Training center is calling upon the government to boost capacity so that more students can benefit of such centers [21]. Furthermore, investments should be done to import specialized steel erection equipment and technologies so that high quality materials could be produced inside the country. Opportunities should be provided for students to study abroad and use their expertise in construction and steel industry to overcome the problem of professional’s shortage. Moreover, this chart demonstrates a summary of reasons that were provided by companies and engineers on the comparison of steel and concrete. Chart 17: Property Comparison of Steel and Concrete 5.3.2. Demand Chart 17: Property Comparison of Steel and Concrete Chart 17: Property Comparison of Steel and Concrete 5.3.2. Demand 5.3.2. Demand One of the reasons concretes is used by construction companies and engineers is that most of the times its customers demand. 95% of customers prefer concrete structure rather than steel structures. 14 Abdulrahimzai, Safi & Halimyar (2020) Chart 18: Demand Comparison of Steel Structure and Concrete Structure Chart 18: Demand Comparison of Steel Structure and Concrete Structure The main reason is that people are not aware of the benefits of steel structures. They believe that concrete structures is longer lasting and durable. Therefore, to solve this issue people’s awareness should be increased about steel structures. Construction companies should conduct seminars and information sessions to provide more information on steel structures and their benefits. 6. Data Obtained from SPSS To put high precession, the data we gained as a result of numerous surveys and interviews about (steel productions, imports, domestic products availability, its preference by different stakeholders “wholesalers, construction companies and steel production companies”, and barriers in steel production in Afghanistan) were then analyzed with the help of SPSS software by inputting a number of variables such as: imported products availability, domestic products availability. And the outputs of the analysis by software are tabulated as below. Product Availability Shape type Responses Percent of Cases U Channels 2 28.6% T Bar 2 28.6% Square Bar 1 14.3% Rounded Bar 1 14.3% Rebar 6 85.7% Ingots 2 28.6% Equal and Unequal Angles 4 57.1% Beam 2 28.6% Iron Strip 3 42.9% Galvanized Wire 6 85.7% Steel Sheets 5 71.4% Iron Pipe 5 71.4% 15 Steel Structures Usage and Steel Production: A Case Study in Afghanistan Table 2: Percentage of Available Steel Products that are Imported Domestic Products Availability Shape type Responses Percent of Cases U channels 1 14.3% T Bars 2 28.6% Square Bar 3 42.9% Rounded Bar 2 28.6% Rebar 5 71.4% Ingots 1 14.3% Equal and Unequal Angles 2 28.6% Beam 3 42.9% Iron Strip 5 71.4% Table 3: Percentage of Available Domestic Steel Products Nos. of companies Initial investments (Millions $) Percent 2 0.1 28.6 1 1 14.3 1 2 14.3 1 3 14.3 1 30 14.3 1 50 14.3 7 Total 100.0 Percentage of Available Steel Products that are Imported Domestic P Ingots 1 14.3% Equal and Unequal Angles 2 28.6% Beam 3 42.9% Iron Strip 5 71.4% Table 3: Percentage of Available Domestic Steel Products Nos. of companies Initial investments (Millions $) Percent 2 0.1 28.6 1 1 14.3 1 2 14.3 1 3 14.3 1 30 14.3 1 50 14.3 7 Total 100.0 Table 3: Percentage of Available Domestic Steel Products Nos. 6. Data Obtained from SPSS of companies Initial investments (Millions $) Percent 2 0.1 28.6 1 1 14.3 1 2 14.3 1 3 14.3 1 30 14.3 1 50 14.3 7 Total 100.0 Table 4: Initial Investments Made by Steel Production Companies in Afghanistan Reasons for Concrete Preference Reasons Responses Nos Percent of Cases Shortage of steel skilled labor 18 90.0% Materials availability (Concrete Structures) 16 80.0% Shortage of specialized equipment for steel construction 15 75.0% Low quality steel products 14 70.0% Shortage of steel structure design professionals 15 75.0% Table 5: List of Reasons for Concrete Preference Over Steel Barriers Barriers Responses Nos. Percent of Cases Unfair Competition 7 100.0% Brand Preference 4 57.1% Illegal Imports 5 71.4% Security 6 85.7% Raw Materials 6 85.7% Legal Imports 3 42.9% Trade Mark 3 42.9% Reasons for Concrete Preference Reasons Responses Nos Percent of Cases Shortage of steel skilled labor 18 90.0% Materials availability (Concrete Structures) 16 80.0% Shortage of specialized equipment for steel construction 15 75.0% Low quality steel products 14 70.0% Shortage of steel structure design professionals 15 75.0% Table 5: List of Reasons for Concrete Preference Over Steel Barriers Barriers Responses Nos. Percent of Cases Unfair Competition 7 100.0% Brand Preference 4 57.1% Illegal Imports 5 71.4% Security 6 85.7% Raw Materials 6 85.7% Legal Imports 3 42.9% Trade Mark 3 42.9% 16 Abdulrahimzai, Safi & Halimyar (2020) Electricity 7 100.0% Investments 5 71.4% Table 6: Barriers on way of steel production in Afghanistan Market Abdulrahimzai, Safi & Halimyar (2020) Table 6: Barriers on way of steel production in Afghanistan Market 7. Conclusion Afghanistan has had a remarkable progress in the steel sector in last two decades. However, there is a general tendency towards building concrete structures rather than steel structures. There are a number of reasons that caused this propagating problem ranging from the market conditions to domestic production, construction companies’ preferences and public demand. This paper investigates the results of a series of surveys and interviews conducted from steel traders and whole sellers, steel production companies, construction companies and engineers to determine the underlying reasons for the tendency towards building concrete structures rather than steel structures. First, whole sellers and steel traders were surveyed to find out the current market conditions, available products, prices and demand. It was found out that Afghanistan’s steel industry is not able to fulfill the market demand due to lack of sufficient productive investment in this sector. Afghan government needs to take serious steps to medicate risk to the investment. For example, illegal imports and illegal imports of steel goods are an alarming danger for domestic steel industry. An increase of tariff on imports of steel and controlling illegal imports can Support of domestic production. Moreover, it was determined that there are both domestic and imported products are present in the market but majority of buyers prefer imported products because either their needed products are not produced in the country and has to be imported or they are not aware of the domestic products at all. Steel production companies, therefore, must adopt new strategies to cover the market, increase awareness of domestic products and attract buyers. Additionally, expanding capacity and producing more varieties of products will undoubtedly make it easier for buyers and companies to access domestic products that they require and in turn support domestic companies back. Secondly, steel production companies were surveyed to discover the current production capacity, product types, investments, raw materials, technology, price affecting factors, market competition but faced challenges. After a thorough analysis of the data it was concluded that although a number of notable investments have been made since 2015, unfair market competition, electricity, and shortage of raw materials have created barriers for steel production companies in Afghanistan. These 17 Steel Structures Usage and Steel Production: A Case Study in Afghanistan factors affect the production capacity, product types and product cost substantially. To address these problems, mines should be explored to provide access to a huge source of raw materials. 7. Conclusion Electricity must be provided so that production companies could expand their capacity. Moreover, increasing tariffs on imports will make it easier for new companies to enter the market and it will lessen the unfair competition with regional countries. Additionally, 5 construction companies were surveyed and about 16 engineers were interviewed to determine the reasons why they prefer building concrete structures rather than steel structures. The main reasons provided were shortage of design professionals, skilled labor and specialized equipment. Moreover, 95% of the customers, demand concrete structures because they are not aware of the benefits of steel structures. To answer these concerns, investments should be done to import specialized steel erection equipment and technologies so that high quality materials could be produced inside the country. Opportunities should be provided for students to study abroad so that they may use their expertise in construction and steel industry to overcome the problem of shortage of professional’s. Additionally, people’s awareness should be increased about steel structures so that more demand is created in the market. In conclusion, the data obtained from the surveys and analyzed by SPSS indicates that Afghanistan has a huge potential and market for building steel structures. however, there are some problems that need to be addressed by the government, steel production companies, construction companies and engineers. [1] Fons, Bas. "Steel container, especially intended for the transport of bulk goods." U.S. Patent 6,783,032, issued August 31, 2004. [2] Al-Kodmany, Kheir. "The sustainability of tall building developments: A conceptual framework." Buildings 8, no. 1-7, 2018. [3] Nikiforova, Vira A. "World steel industry: current challenges and development trends (analytical overview)." Economy of Industry 1 (81) 86-114, 2018. [4] Taranath, Bungale S. Structural analysis and design of tall buildings: Steel and composite construction. CRC press, 2016. References [1] Fons, Bas. "Steel container, especially intended for the transport of bulk goods." U.S. Patent 6,783,032, issued August 31, 2004. 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https://openalex.org/W1922118979	https://seer.ufs.br/index.php/tomo/article/download/3191/2793	Portuguese	null	A Presença Negra nos Cursos de Graduação em Direito da Universidade Estadual de Mato Grosso do Sul: Análise do Percurso após Ingresso Pelas Cotas – Turma 2008	Tomo	2,014	cc-by	12,959	2 Doutora em Educação-Currículo pela Pontifícia Universidade Católica de São Paulo. Do- cente do Curso de Pedagogia e do Mestrado Profissional Ensino em Saúde na Unidade Universitária de Dourados e do Mestrado em Educação da Unidade de Paranaíba - Uni- versidade Estadual de Mato G. Mestre em Educação pela Universidade Estadual de Mato Grosso do Sul (UEMS). Docente e Coordenador do Núcleo de Prática Jurídica do Curso de Direito, Unidade Universitária de Paranaíba/UEMS. Membro do GEPPE – Grupo de Estu- dos e Pesquisas em Práxis Educacional, e-mail airesnpj@yahoo.com.br e endereço para correspondência na Rua Ana Luiza, n. 25, Centro, Prosso do Sul (UEMS). Líder do Grupo de Pesquisa em Educação, Gênero, Raça e Etnia – GEPEGRE/Cnpq, e-mail maju@uems. br e endereço para correspondência na Rua Oliveira Marques, n. 269, Jardim Tropical, Dourados-MS, CEP - 79820-040. 1 Mestre em Educação pela Universidade Estadual de Mato Grosso do Sul (UEMS). Docen- te e Coordenador do Núcleo de Prática Jurídica do Curso de Direito, Unidade Universitá- ria de Paranaíba/UEMS. Membro do GEPPE – Grupo de Estudos e Pesquisas em Práxis Educacional, e-mail airesnpj@yahoo.com.br A PRESENÇA NEGRA NOS CURSOS DE GRADUAÇÃO EM DIREITO DA UNIVERSIDADE ESTADUAL DE MATO GROSSO DO SUL Os resultados gerais apontam para um índice considerável de desistência ou evasão dos negros cotistas pelas razões mais di- versas e que serão analisadas neste trabalho. q Palavras-chave: Negros cotistas; UEMS; Curso de Direito; Con- quistas e dificuldades. A Presença Negra nos Cursos de Graduação em Direito da Universidade Estadual de Mato Grosso do Sul: Análise do Percurso após Ingresso Pelas Cotas – Turma 2008 Aires David de Lima1 Maria José de Jesus Alves Cordeiro2 Em 06/01/2003, foi publicada, em Mato Grosso do Sul, a Lei n. 2.605 estabelecendo as cotas para negros na Universidade Esta- dual de Mato Grosso do Sul – UEMS, sendo regulamentada para o vestibular do mesmo ano, com ingresso dos negros cotistas já no início de 2004. Passados nove anos de oferta, realizamos uma pesquisa no Mestrado em Educação/UEMS com o objeti- vo de avaliar os resultados gerais e específicos da presença dos negros cotistas nos quatro cursos de graduação em Direito, des- velando as conquistas e dificuldades enfrentadas no acesso e na permanência dos 47 (quarenta e sete) acadêmicos ingressantes em 2008. Para isso, utilizou-se a pesquisa qualitativa do tipo estudo de caso e fez-se uso de questionários semiestruturados. 238 239 Aires David de Lima; Maria José de Jesus Alves Cordeiro Abstract On January 6, 2003, Law n. 2605 was enacted. The law, which es- tablished quotas for blacks in the State University of South Mato Grosso – UEMS, came into effect in the same year, with the en- trance of Black students in early 2004. Nine years later, we con- ducted research on the Master’s Program in Education at UEMS in order to assess the general and specific results of the presen- ce of Black quota students in the four undergraduate degrees in law, revealing the achievements and difficulties in access and permanence of 47 freshmen students in 2008. We used the qua- litative research technique of case study and conducted semi- -structured questionnaires. The results reveal that Black quota students had significant attrition rates. This article analyzes the several reasons for that. Keywords: Black quota students; UEMS; Law School; achieve- ments and difficulties. Introdução Estudar o acesso e a permanência de negros cotistas no ensino superior, bem como, os percalços enfrentados e as conquistas re- alizadas por estes, constitui-se numa tarefa para a qual, apenas a revisão bibliográfica não é suficiente para responder a todas as indagações, pois ainda são poucas as produções sobre o negro e a maioria não traz, em suas palavras, os dramas particulares vividos por cada um, suas angústias, esperanças e perspectivas, principalmente educacionais. Para melhor compreendermos uma realidade muitas vezes apenas dedutível, realizamos uma pesquisa cujos resultados são aqui apresentados como uma pos- sibilidade de reflexão sobre o papel das cotas no ensino supe- rior, principalmente numa área de formação considerada de eli- te, algo que muito nos inquietava enquanto docente da mesma. Com estas inquietações, realizamos no Mestrado em Educação, uma pesquisa sobre a cota para negros na Universidade Esta- dual de Mato Grosso do Sul – UEMS que, conforme Cordeiro (2011), busca promover o princípio da igualdade em prol das mi- norias raciais e étnicas. Foram abordados os aspectos legais que proporcionam o ingresso na universidade, os que favorecem a permanência e a conclusão, especificamente nos quatro cursos de Direito ofertados pela UEMS, o que podemos chamar de “cota dos sonhos”, porque para muitos é considerado o passaporte para uma vida melhor. No entanto, cotas é um assunto interessante e instigador que, a cada discussão, novas polêmicas se levantam relacionadas aos critérios de oferta e seu aperfeiçoamento, mesmo depois da de- cisão judicial do Supremo Tribunal Federal – STF, em 26 de abril de 2012, que reconheceu sua constitucionalidade. A pesquisa realizada numa abordagem qualitativa, do tipo estu- 240 A PRESENÇA NEGRA NOS CURSOS DE GRADUAÇÃO EM DIREITO DA UNIVERSIDADE ESTADUAL DE MATO GROSSO DO SUL 240 A PRESENÇA NEGRA NOS CURSOS DE GRADUAÇÃO EM DIREITO DA UNIVERSIDADE ESTADUAL DE MATO GROSSO DO SUL A PRESENÇA NEGRA NOS CURSOS DE GRADUAÇÃO EM DIREITO DA UNIVERSIDADE ESTADUAL DE MATO GROSSO DO SUL do de caso, teve como objetivo avaliar os resultados da presença dos negros cotistas na graduação da UEMS, especialmente nos cursos de Direito ofertados nas Unidades Universitárias de Dou- rados (matutino), Naviraí (noturno) e Paranaíba (matutino e no- turno). Para isso, buscou-se, ainda, levantar as conquistas e as dificuldades para permanência, enfrentadas pelos acadêmicos que ingressaram em 2008 e que, supostamente, deveriam estar, em 2012, no quinto ano. Além disso, foram incluídos na pesqui- sa os coordenadores(as) de curso que atuaram nesse período. Para coleta dos dados aplicou-se um questionário semiestrutu- rado aos negros cotistas e outro aos coordenadores(as) de cur- so. A opção pela pesquisa qualitativa, na modalidade estudo de caso, deu-se por entendermos que esta busca dados nas experi- ências e vivências dos personagens pesquisados, através de uma interação mais direta, para se compreender essas vivências, sem deixar de lado os cuidados peculiares que uma pesquisa cientí- fica exige. Levando-se em conta o objetivo proposto, este tipo de meto- dologia é a que melhor pode oferecer subsídios para o desen- volvimento da pesquisa, pois para Ludke e André (1986, p. 11), [...] é cada vez mais evidente o interesse que os pesquisadores da área da educação vêm demonstrando pelo uso das metodologias qualitativas. Por isso, além dos dados dos questionários, foram analisadas as Atas de Resultados Finais de 2008 a 2012. Estas Atas têm como finalidade gerar as folhas do livro de resultado final dos cursos, registrando os resultados acadêmicos dos alunos. De acordo com Cordeiro (2012), as Atas são de responsabilidade da Direto- ria de Registro Acadêmico - DRA /UEMS disponibilizada, apenas, para pesquisadores com projeto sobre a temática registrado na UEMS. Por meio das análises dessas Atas detectou-se alto índice de evasão e de reprovações, bem como, a análise do PP em vigor das três Unidades onde o curso de Direito é oferecido, as norma- tizações que tratam das cotas, tanto internas da UEMS, quanto Aires David de Lima; Maria José de Jesus Alves Cordeiro 241 241 Aires David de Lima; Maria José de Jesus Alves Cordeiro leis, decretos e principais tratados internacionais que cobram uma política de inclusão. Todas estas informações serviram de parâmetro para algumas ponderações e sugestões relativas ao sistema de cotas na UEMS e ao curso de Direito. A PRESENÇA NEGRA NOS CURSOS DE GRADUAÇÃO EM DIREITO DA UNIVERSIDADE ESTADUAL DE MATO GROSSO DO SUL caso foi a transição do trabalho escravo para o trabalho livre, que ocorreu no final do século XIX, ganhando proporções maio- res de desigualdades educacionais e sociais no século XX, sendo que, no século XXI, o cenário pouco se modificou. Dessa forma, utilizando-se de um discurso pseudocientífico, dentre outros elementos de uma retórica racista, os intelectu- ais e gestores públicos influenciaram decisivamente nas polí- ticas educacionais, excluindo os negros do processo educati- vo já que eram tidos como doentes, indolentes e improdutivos (CARVALHO, 1989, apud PASSOS, 2012, n. p.). A educação tinha o papel de libertar a nação brasileira, com o fim de torná-la saudável, produtiva e civilizada, ou seja, uma justificativa para a exclusão do negro. Assim, a escravidão do negro no Brasil não pode ser vista como única explicação para as atuais desigual- dades entre brancos e negros, outros elementos colaboraram para a ocorrência desses fatos. A naturalização dessa exclusão dificulta o acesso ao ensino superior e a permanência, pois além das questões econômicas, as questões étnico-raciais têm peso significativo. 1 Cursos de Direito no Brasil e a presença do negro Os primeiros esboços de Universidades surgiram no final do sé- culo XI e início do século XII na Europa. A partir do século XII, começaram a se estruturar e se organizar sendo que, apenas na segunda metade do século XIX, foram esboçados os mode- los das atuais universidades nos padrões como as conhecemos atualmente. Com relação a esses períodos, não foi possível aferir dados sobre raça e cor dos primeiros estudantes, bem como da população em geral, tampouco se o negro esteve presente nos primeiros cursos superiores oferecidos. A ausência de um banco de dados sobre esse assunto deve-se à falta de interesse do Estado, bem como ao fato de as pesquisas sobre raça, naquela época, ainda se encontrarem em fase em- brionária, uma vez que estudos mais aprofundados sobre este tema remontam ao ano de 1775, elaborados pelo alemão Johann Friedrich Blumenbach (1752-1840). Ao analisar as primeiras ofertas de ensino superior no Brasil, ocorridas no começo do século XIX, por ocasião da transferência do trono português para o país, podemos dizer que a falta de in- teresse da Coroa retardou a educação superior no Brasil. Outros países da América do Sul anteciparam-se nesse aspecto, a exem- plo do Peru, que criou a Universidade de São Marcos em 1551. Com relação à instrução do negro, estudos demonstram que sua exclusão das instituições escolares desenvolveu-se no decorrer da história da educação brasileira. O marco inicial para esse des- 242 A PRESENÇA NEGRA NOS CURSOS DE GRADUAÇÃO EM DIREITO DA UNIVERSIDADE ESTADUAL DE MATO GROSSO DO SUL 242 Cota para negros nos cursos de Direito na UEMS Os primeiros cursos jurídicos no Brasil tiveram sua implantação por meio do Decreto Imperial de 11.08.1827, sendo criados em São Paulo e Olinda com a denominação original de Academias de Direito. O curso de São Paulo foi instalado no Largo de São Francisco, em março de 1828, e o de Olinda, em maio do mesmo ano, no Mosteiro de São Bento, ocorrendo, posteriormente, sua transferência para Recife. O novo país em formação precisava capacitar uma classe que iria dirigi-lo, pois esta era uma exigência premente da independên- cia, que necessitava substituir o aparelho administrativo metro- politano que partia (TREVISAN, 1987, p. 37). Assim, a formação Aires David de Lima; Maria José de Jesus Alves Cordeiro 243 243 Aires David de Lima; Maria José de Jesus Alves Cordeiro desse novo quadro que iria dirigir o país seria incumbência da educação, uma vez que a administração pública se encontrava desprovida de pessoal especializado para isso em fins do século XIX. Nessa conjuntura, entretanto, o negro novamente foi prete- rido, pois: O Estado brasileiro se preparava, então, para atender as transformações que a consolidação do capitalismo inter- nacional exigia dos Estados de economia dependente. A organização socioeconômica relativamente à realidade da existência de uma população negra que fatalmente iria se misturar nos espaços e no direito da sociedade branca - eli- te ou não - impõe aos poderes do Estado estabelecer, no seu braço regulador e ordenador social, qual seja, do direito, as diretrizes para a manutenção e desenvolvimento da nova nação que surgia a fim de adequá-la aos parâmetros já defi- nidos racialmente, qual seja, uma nação branca (BERTÚLIO, 2002 apud PASSOS, 2012, n.p.). Naquele momento histórico, uma grande preocupação em for- mar uma sociedade de “brancos”, propiciou o nascimento de um novo Estado, porém com as mesmas mazelas excludentes do an- terior. No entanto, relevantes transformações se aproximavam com a chegada do novo século, pois o Estado mudaria de domí- nio. Logo, as bases do ensino também precisariam ser revistas, para se adequarem a essas inovações, recebendo novas funções. As primeiras turmas dos cursos de Direito no Brasil formaram- -se em 1832. Em sua composição, havia jovens de diversas pro- víncias do Brasil e, também, de Portugal e Angola. Cota para negros nos cursos de Direito na UEMS Entretanto, essa proposta de Universidade foi elaborada, apenas para uma selecionada elite, pois à época, o ensino básico também era pri- vilégio de poucos, sendo esta, uma etapa de seleção daqueles que comporiam os poucos bancos universitários para, em mo- mento posterior, dirigir a nação brasileira nessa nova ordem im- plantada com a independência de Portugal. Desse modo, o negro, até mesmo o liberto, mais uma vez ficava 244 A PRESENÇA NEGRA NOS CURSOS DE GRADUAÇÃO EM DIREITO DA UNIVERSIDADE ESTADUAL DE MATO GROSSO DO SUL 244 A PRESENÇA NEGRA NOS CURSOS DE GRADUAÇÃO EM DIREITO DA UNIVERSIDADE ESTADUAL DE MATO GROSSO DO SUL de fora desse processo educacional, uma vez que outros fato- res, além da escravidão, o levavam a ocupar os piores postos e lugares, bem como auferir rendimentos inferiores aos brancos, herança que ainda carrega. Nos últimos 12 (doze) anos, são as ações afirmativas que vem oportunizando o acesso dos negros ao ensino superior e a UEMS vem contribuindo nesse sentido, tendo sido a terceira universidade no Brasil a implementar cotas para negros e a primeira a instituir cotas para indígenas. O sistema de cotas na universidade é uma Ação Afirmativa no sentido em que entende Munanga, (2001, p.31), pois [...] visam oferecer aos grupos discriminados e excluídos um tratamento di- ferenciado para compensar as desvantagens devidas à sua situa- ção de vítimas do racismo e de outras formas de discriminação. A UEMS iniciou suas atividades em 1994 com a oferta de vá- rios cursos, dentre estes o curso de Direito na cidade de Três Lagoas – MS, transferindo-o tempos depois para a cidade de Paranaíba – MS, onde vem sendo ofertado nos turnos matutino e noturno. Em seguida criou mais dois cursos de Direito, um na cidade de Dourados–MS, sede da UEMS, e outro na cidade de Naviraí-MS. Desde o ano de 2003, realiza processo seletivo com cotas de 20% para negros e 10% para indígenas, em todos os cursos de graduação oferecidos na sede e nas 14 (quatorze) Unidades Universitárias espalhadas em todo o estado de Mato Grosso do Sul. A primeira oferta de cotas na UEMS ocorreu em 2003, em cum- primento da Lei Estadual n. 2.605, de 06/01/2003, que dispõe sobre a reserva de, no mínimo, 20% das vagas para negros, mas até o presente momento ainda encontramos resistência em sua operacionalização, uma vez que, guiados pela ideolo- gia e por meio de discursos desinformados, docentes, gestores e a comunidade em geral continuam acreditando no mito da “democracia racial” e na hipótese utópica de que a melhoria da qualidade do ensino fundamental e médio possa resolver a 245 desigualdade no acesso ao ensino superior, desconsiderando questões sociais e raciais. A pouca representatividade negra nos bancos universitários no Brasil e, em especial, no curso de Direito, referendada por dados estatísticos, legitima a necessidade do sistema de cotas para o ensino superior. Com base na pesquisa elaborada em 2004, pelo Prof. A PRESENÇA NEGRA NOS CURSOS DE GRADUAÇÃO EM DIREITO DA UNIVERSIDADE ESTADUAL DE MATO GROSSO DO SUL um processo único no Brasil. [...] um processo de discussão e de- bate acadêmico e sociopolítico com todos os segmentos internos e externos à Universidade”. Atualmente, os quatro Cursos de Direito da UEMS oferecem 180 (cento e oitenta) vagas e deste quantitativo a UEMS disponibi- liza cota de 20% para negros que tenham estudado em escola pública ou como bolsista em escola particular. No vestibular de dezembro de 2007, para ingresso em 2008, foram oferecidas 10 (dez) vagas em Dourados, 10 (dez) em Naviraí e 16 (dezesseis) em Paranaíba, divididas em período matutino e noturno, ou seja, 36 (trinta e seis) vagas para os negros. Todas as vagas são pre- enchidas ano após ano, porém em Paranaíba, os negros ocupam 30% das vagas, pois os 10% destinados aos indígenas em ne- nhum ano foi preenchido, e, de acordo com as normas, são os negros que as ocupam. Hoje, as cotas na UEMS são uma realidade que não pode ser questionada quanto a sua legalidade e os estudos servem para subsidiar seu aprimoramento. Para isso, buscou-se informações junto daqueles que se encontram no fim desse trajeto, ou seja, cotistas negros que estavam concluindo o curso em 2012 e os coordenadores de curso que acompanharam de perto essa cami- nhada. São estes dados e informações que apresentamos aqui. A PRESENÇA NEGRA NOS CURSOS DE GRADUAÇÃO EM DIREITO DA UNIVERSIDADE ESTADUAL DE MATO GROSSO DO SUL Dilvo Ristoff, diretor do INEP, o curso de Direi- to é o sexto curso “mais branco” do Brasil, com 79,4% das vagas preenchidas por estes (SANTOS, 2006a). Diante desses dados, questionamos: E se nada fosse feito, como ficaria a questão do negro brasileiro? A resposta mais coerente sugere que o abis- mo entre negros e brancos permaneceria inalterado e o quadro de exclusão persistiria no futuro como historicamente vem sen- do comprovado. As cotas refletem a ideia de inclusão social, pois, levando em con- ta o respeito às peculiaridades de cada um, dão condições para o acesso, por meio de uma política diferenciada, de grupos de pes- soas que sofreram, e ainda sofrem, um histórico processo de dis- criminação e preconceito, a exemplo, os negros e indígenas. Nos estudos realizados por Cordeiro (2008), consta que em dezembro de 2003, o primeiro vestibular com cotas na UEMS, foram destinadas 328 (trezentos e vinte e oito) vagas para negros. Ao final das matrículas, em 2004, especificamente no curso de Direito, houve o ingresso de 40 (quarenta) negros, de- monstrando, já no primeiro ano de oferta, uma mudança no cenário desalentador anteriormente relatado. Todos os anos, desde então, todas as vagas são preenchidas e, as vagas da cota de indígenas não ocupadas por eles, são destinadas aos negros que as vem ocupando sistematicamente, com exceção do curso de Dourados-MS. De acordo com Cordeiro (2008, p. 17), a implementação das co- tas na UEMS, foi “uma oportunidade para construir e executar 246 248 A PRESENÇA NEGRA NOS CURSOS DE GRADUAÇÃO EM DIREITO DA UNIVERSIDADE ESTADUAL DE MATO GROSSO DO SUL comentados sobre os quais podemos ainda tecer comparações entre os cursos. 2. Dos resultados da pesquisa O sistema de cotas raciais envolve uma realidade que não pode passar despercebida pela própria Universidade nem por seus docentes e gestores. Dessa forma, faz-se necessário o compro- metimento de todos no aperfeiçoamento desta importante fer- ramenta de inclusão social. As cotas envolvem uma realidade que deve fazer parte das atividades de todos os docentes do cur- so, tendo em vista que o currículo e o fazer pedagógico devem se voltar para esta nova realidade multicultural, o que culminará 247 com uma melhor preparação do discente, pois estará antecipan- do o que o mesmo encontrará no mercado de trabalho. As análises que apresentamos é resultado dos dados obtidos com a aplicação do questionário aos negros cotistas, no qual abor- damos questões sobre dados pessoais, tais como sexo e idade, estado civil, se tem filhos, autodeclaração de raça e de cor, com quem reside, escolaridade do pai e da mãe, dados profissionais como profissão e salário, bem como renda individual e familiar, dados educacionais relacionados à trajetória acadêmica, ano em que está matriculado e se carrega dependência; e, caso isso ocorra, os motivos; onde cursou o ensino fundamental e médio; se é ou se foi bolsista; maiores dificuldades para permanência na Universidade; conhecimento e participação em projetos ou pro- gramas; motivo pelo qual optou pela cota, bem como sugestões para a UEMS relacionadas às cotas. Muitas foram as dificuldades nessa coleta de dados envolvendo três cidades equidistantes até 600 km uma da outra, além das cidades circunvizinhas a cada uma, local de residência de muitos dos alunos pesquisados que haviam desistido do curso, mas que suas respostas eram cruciais para o entendimentos dos fatores de permanência. De um universo de 47 negros cotistas que ingressaram em 2008, nos quatro cursos de Direito, foram coletados 40 questioná- rios, o que representa 85% (oitenta e cinco por cento), sendo 12 (doze) na sede em Dourados, 12 (doze) na Unidade de Pa- ranaíba matutino, 10 (dez) na Unidade de Paranaíba noturno e 6 (seis) na Unidade de Naviraí. Dos coordenadores de curso, foram coletados dados de 100% dos que exerceram essa função desde 2008, o que representa 2 (dois) questionários em cada unidade, totalizando 6 (seis) ao todo. A seguir, apresentamos alguns dados tabulados, analisados e 248 Dados pessoais, familiares e socioeconômicos Em relação ao item I, que trata dos dados pessoais, 23 (vinte e três) são mulheres e 17 (dezessete) homens, ou seja, 73.9% de mulheres, o que nos permite olhar com otimismo o cenário que se descortina à frente, em relação ao gênero, quando olhamos o histórico da mulher negra no Brasil a partir de sua entrada na condição de escrava. Dados recentes comprovam que o subem- prego afeta grande parte das mulheres negras, grupo étnico- -racial que se encontra em maior vulnerabilidade com relação ao turismo sexual e ao tráfico internacional de mulheres, no qual [...] meninas, jovens e mulheres não brancas, e especialmente da região Norte e Nordeste, são alvos fundamentais da indústria in- ternacional do sexo (BORGES, 2005, p. 64). Com relação à idade, podemos perceber que o maior número de negros cotistas se enquadra na faixa etária dos 21 (vinte e um) aos 25 (vinte e cinco) anos, com 16 (dezesseis) respostas a esta alternativa, seguida da faixa etária dos 26 (vinte e seis) aos 30 (trinta) anos, com 10 (dez) acadêmicos. No entanto, existe a pre- sença de acadêmicos das mais diversas faixas etárias, incluindo os de 46 (quarenta e seis) aos 50 (cinquenta) anos. Assim, essa composição variada demonstra que a possibilidade de concor- rer pelas cotas trouxe incentivos para aqueles que se encontra- vam há longos anos fora dos bancos escolares, ou concluíram o ensino médio na modalidade de Educação de Jovens e Adultos – EJA, justificando, também, algumas das dificuldades relaciona- das aos conteúdos do curso. 249 Com relação à autodeclaração racial, a maioria se autodeclarou pertencente à raça negra, com 39 respostas a este item e 1 (uma) como pertencente à raça branca. No entanto, nas declarações de cor, três negros cotistas se autodeclararam brancos. Muitas indagações podem surgir pelo fato de alguns questionados se considerarem de cor branca, mesmo que todos tenham sido considerados negros pela comissão que analisa as inscrições dos candidatos às cotas de negros. O fato de se considerarem brancos, bem como não se identificarem com a própria raça, muitas vezes, ainda está ligado à questão do status social ocupado pelo branco, no qual tudo isso faz com que a parcela da população negra se recu- se a se assumir como negra [...] (GUIMARÃES, 2006, p. 111). A PRESENÇA NEGRA NOS CURSOS DE GRADUAÇÃO EM DIREITO DA UNIVERSIDADE ESTADUAL DE MATO GROSSO DO SUL O item II, relacionado aos dados familiares, as informações sobre moradia e escolaridade dos pais são importantes para conhecer- mos o contexto sociocultural do qual os cotistas são originários. mos o contexto sociocultural do qual os cotistas são originários. Em relação à escolaridade do pai, a maioria está concentrada no “fundamental incompleto” com 9 (nove) respostas, seguido pela alternativa “sem instrução”, com 8 (oito). O que demonstra a pouca instrução e a importância da universidade na reversão deste quadro num futuro bem próximo. Encontramos dados se- melhantes quando tratamos sobre a escolaridade da mãe, uma vez que o maior número de respostas assinaladas foram os itens fundamental incompleto com 13 (treze) e sem instrução com 6 (seis). Porém, as genitoras apresentaram um número ligeira- mente maior de graduadas, ou seja, 6 (seis), contra 5 (cinco) dos pais. Estudos sobre esse ponto têm demonstrado que [...] a escolarização dos pais tem influência poderosa na de- terminação do sucesso acadêmico do estudante, apontando, deste modo, para o ensino superior como um espaço que concorre para a reprodução das desigualdades entre os se- guimentos sociais (PRIOR, 1984; BRITO E CARVALHO, 1978; BOURDIEU e PASSERON, 1973, apud QUEIROZ, 2002, p. 35). Henriques, quando tratou da desigualdade racial e da evolução das condições de vida na década de 90, concluiu que: Henriques, quando tratou da desigualdade racial e da evolução das condições de vida na década de 90, concluiu que: [...] a escolaridade média de um jovem negro com 25 anos de idade gira em torno de 6,1 anos de estudo; um jovem branco da mesma idade tem cerca de 8,4 anos de estudo. O diferencial de 2,3 anos de estudos entre jovens brancos e negros de 25 anos de idade é a mesma observada entre os pais desses jovens. E, de forma assustadoramente natural, 2,3 anos é a diferença entre os avós desses jovens. [...] esco- laridade entre brancos e negros, mantém-se perversamente estável entre as gerações (HENRIQUES, 2001, apud PASSOS, 2012, n.p.). No item III, que aborda os dados profissionais, as informações [...] a escolaridade média de um jovem negro com 25 anos de idade gira em torno de 6,1 anos de estudo; um jovem branco da mesma idade tem cerca de 8,4 anos de estudo. O diferencial de 2,3 anos de estudos entre jovens brancos e negros de 25 anos de idade é a mesma observada entre os pais desses jovens. Dados pessoais, familiares e socioeconômicos No que diz respeito à cor, as respostas foram mais diversificadas, sendo que a maioria se considera preta com 20 (vinte) respos- tas; seguida por pardo, com 16 (dezesseis), além dos 3 (três) brancos e um que não respondeu. Essa quantidade de pardos demonstra a alta miscigenação do povo brasileiro. Nesse item, acreditamos que algumas respostas foram tendenciosas pelo fato do ingresso pelas cotas, do que a simples e pura convicção de pertencimento a uma determinada cor, ocasionadas, em al- guns casos, pelo grau de dificuldade dos próprios sujeitos em se autodenominar. Os critérios de aferição da raça para ingresso pelas cotas foram construídos a partir das discussões durante a regulamentação da Lei Estadual n. 2.605, de 06/01/2003. Como eixo central das exigências para os negros. Foram estabelecidos os critérios de [...] escola pública, fenótipo e autodeclaração [...] (CORDEIRO, 2008, p. 61). Portanto, se declarar pertencente à raça negra e, posteriormente, se afirmar de cor branca, nos induz a pensar na falta de identidade étnico-racial desde a infância, pois é natural o orgulho da raça branca pela supremacia branca (GUIMARÃES, 2006, p. 105), afirmação que vem sendo desmistificada nos últi- mos tempos, com o reconhecimento da identidade da população negra. 250 A PRESENÇA NEGRA NOS CURSOS DE GRADUAÇÃO EM DIREITO DA UNIVERSIDADE ESTADUAL DE MATO GROSSO DO SUL E, de forma assustadoramente natural, 2,3 anos é a diferença entre os avós desses jovens. [...] esco- laridade entre brancos e negros, mantém-se perversamente estável entre as gerações (HENRIQUES, 2001, apud PASSOS, 2012, n.p.). No item III, que aborda os dados profissionais, as informações Aires David de Lima; Maria José de Jesus Alves Cordeiro 251 251 Aires David de Lima; Maria José de Jesus Alves Cordeiro obtidas sobre trabalho e renda dos negros cotistas nos possibili- taram justificar parte das dificuldades encontradas na trajetória acadêmica, bem como o alto número de desistências no curso. Perguntados sobre dados profissionais, 29 (vinte e nove) acadê- micos disseram trabalhar, enquanto 11 (onze) responderam ne- gativamente, informando manter-se na universidade com a aju- da dos pais. Nesse aspecto, alguns fatores podem interferir na seletividade dos acadêmicos, tais como o turno em que o curso é oferecido, como na sede em Dourados e em Paranaíba (turno matutino), há dificuldade para obtenção de emprego, somado ao fato de que, no quinto ano, cumprem a disciplina de Estágio Su- pervisionado junto ao Núcleo de Prática e Assistência Jurídica, o que torna qualquer carga horária de trabalho mais complicada. Entretanto, podemos afirmar que os estudantes do matutino, em sua maioria, vivem com os pais e apresentam a maior renda salarial. Dos que disseram trabalhar, a faixa salarial que mais concentra acadêmicos é até 1 (um) salário mínimo, com 12 (doze) respos- tas a esta alternativa, divididos igualmente entre os turnos ma- tutino e noturno. A faixa de 1 (um) a 3 (três) salários mínimos obteve 11 (onze) respostas, sendo a maioria no período matuti- no, o que nos faz acreditar que esses acadêmicos possuem bol- sas ou estágio extracurricular remunerado. Quando juntamos os dados sobre renda até 3 (três) salários mí- nimos, obtemos um total de 23 (vinte e três) alunos ou 57% de- les nessa condição, o mesmo acontecendo no que diz respeito à renda familiar com maior concentração nas faixas de até 1 (um) salário mínimo com 7 (sete) respostas e de 1 (um) a 3 (três) com 16 (dezesseis) respostas. Observamos que, na renda familiar de 4 (quatro) a 10 (dez) salários, dos 14 (quatorze) cotistas oriun- dos dessas famílias, 10 (dez) estudam no turno matutino. 252 252 A PRESENÇA NEGRA NOS CURSOS DE GRADUAÇÃO EM DIREITO DA UNIVERSIDADE ESTADUAL DE MATO GROSSO DO SUL Dados Educacionais: cotistas negros Com relação aos dados educacionais, 33 (trinta e três) acadê- micos declararam estar matriculados e frequentando o quinto ano, 6 (seis) responderam estar matriculados e frequentando o quarto ano, 1 (um) informou que havia desistido e 23 declara- ram carregar dependência contra 17 (dezessete) que disseram não possuir dependência. Questionados sobre os motivos que geraram a dependência, 11 (onze) assinalaram a alternativa “outros motivos”, justificando como sendo: - Na Unidade de Dourados: a) falta de mais esforço de minha parte; b) falta de compromisso com a disciplina; c) dificuldade com o estudo devido ao grande desgaste com o trabalho fora; - Unidade de Paranaíba Matutino: a) problemas pessoais; b) doença e perda de muitas aulas; c) falta de boa didática de al- guns professores; d) transferência interna; e) desgaste com o trabalho. - Unidade de Paranaíba Matutino: a) problemas pessoais; b) doença e perda de muitas aulas; c) falta de boa didática de al- guns professores; d) transferência interna; e) desgaste com o trabalho. - Unidade de Paranaíba Noturno: a) dificuldades econômicas, falta de tempo e estrutura para estudar; b) trabalho; - Unidade de Paranaíba Noturno: a) dificuldades econômicas, falta de tempo e estrutura para estudar; b) trabalho; - Unidade de Naviraí: a) perda de avaliação, não mais conseguin- do recuperar na optativa; b) envolvimento em movimentos so- ciais, carga extensa no trabalho; c) falta de tempo para estudo. - Unidade de Naviraí: a) perda de avaliação, não mais conseguin- do recuperar na optativa; b) envolvimento em movimentos so- ciais, carga extensa no trabalho; c) falta de tempo para estudo. As respostas foram bem diversificadas. Porém, em sua maioria, percebida pelo próprio cotista como falta de esforço pessoal e devido ao desgaste ocasionado pela jornada de trabalho. A gra- duação oferecida no período noturno favorece quem trabalha, Aires David de Lima; Maria José de Jesus Alves Cordeiro 253 253 Aires David de Lima; Maria José de Jesus Alves Cordeiro no entanto, o rendimento pode ser inferior àquele dos que não exercem qualquer atividade, pois o desgaste ocasionado é um desestímulo aos estudos. Nesse ponto, muitas vezes, o acadêmi- co se vê em uma encruzilhada: estudar ou sobreviver? Por isso, a necessidade da universidade oferecer junto com o acesso, po- líticas de permanência. Outro item que ganhou destaque na questão da dependência foi a alternativa “c” “relação professor(a)-aluno(a)”, com 7 (sete) respostas. Dados Educacionais: cotistas negros Para a compreensão desse item, oferecemos alguns exemplos genéricos (discriminação, perseguições, etc.) dentre os quais o(a) acadêmico(a) poderia enquadrar outras situa- ções semelhantes vividas durante o curso. Essa alternativa era objetiva e, por consequência, não cabia justificativa. O índice de respostas a esse quesito, embora pareça pequeno, na verdade é significativo e mostra uma situação que não deveria ocorrer na universidade, já que esta [...] necessita, pois, continuamente pen- sar o que ela quer ser e que tipo de contribuição deseja prestar à sociedade na qual se encontra inserida (BENICÁ, 2011, p. 36), ou seja, como espaço de diálogo e interações, a universidade deve minimizar a ocorrência dessa prática, com intervenção perma- nente, junto aos professores e aos acadêmicos, visando uma me- lhor conscientização e respeito ao outro. O item “dificuldade com os conteúdos básicos do curso” contou com 4 (quatro) repostas, embora saibamos que as dependências, geralmente, são frutos de avaliações nas quais o aluno não domi- nou os conteúdos e que pode ter suas raízes no processo de ensino- -aprendizagem na Educação Básica. As informações sobre onde cursou o ensino fundamental e médio não causaram surpresa, ten- do em vista ser este um critério para concorrer às cotas. Contudo, dos três que declararam ter estudado em escola particular, apenas dois informaram mediante bolsa e um deixou em branco a alterna- tiva, levando-nos a pensar que motivos este teria para ocultar esta informação. Será que apresentou falsa declaração de bolsa? É possí- vel, mesmo com todo o cuidado que a Comissão responsável adotou 254 [...] estima-se que a média de horas trabalhadas por sema- no deferimento das inscrições para a cota de negros. Nas informações sobre as bolsas oferecidas durante a gradua- ção, 33 (trinta e três) declararam que não recebem(eram) e 7 (sete) que recebe(eram). Dos que receberam, as respostas foram as mais diversificadas, sendo 3 (três) de permanência, 1 (uma) de extensão e 3 (três) de iniciação científica. Quem se benefi- ciou de bolsa o fez, em média, de 7 (sete) meses a 1 (um) ano. E apenas 1 (um) acadêmico disse ainda receber bolsa perma- nência. Essas bolsas têm prestado um importante auxílio para a permanência e formação acadêmica, pois possibilita ao estu- dante carente sua manutenção na graduação, além de iniciação à pesquisa e à extensão. Dessa forma, podemos avaliar que é amplo o sistema de bolsas oferecido pela UEMS e que muitas delas sequer são pleiteadas, sobrando bolsas, o que demonstra, muitas vezes, uma postura passiva de professores e coordenadores dos cursos de Direito, que não incentivam os alunos a concorrer. Por outro lado, os alu- nos só podem concorrer se os seus professores possuírem pro- jetos aprovados e em desenvolvimento, situação bastante rara entre os docentes da área do Direito. Como maiores dificuldades para manterem-se na universidade, no curso de Direito, a maioria aponta os fatores ligados ao tra- balho, bem como ao deslocamento até a Universidade como em Dourados, onde o curso está situado a mais de 15km do centro da cidade. Outro fator é a renda que, como vimos, é baixa, tanto a individual quanto a familiar. Sabemos, ainda, que as profissões que exigem maiores esforços físicos são as que geram menores rendimentos e maior cansaço, principalmente para os que es- tudam no período noturno, fatos já comprovados em pesquisas com dados nos quais, [...] estima-se que a média de horas trabalhadas por sema- Aires David de Lima; Maria José de Jesus Alves Cordeiro na seja de 43 horas e 24 minutos, todavia entre pretos a jornada de 44 horas e 49 minutos é superior à média e à jornada dos de cor branca, que trabalham 44 horas e 33 minutos. [...] embora a população negra trabalhe mais que a população branca, seu salário ainda é inferior, pois a par- tir da média total de R$ 573,23, constatou-se que os bran- cos recebem em média R$ 644,46 e os negros, R$ 506,48, uma diferença em favor dos brancos de 27% (MARTINI; SILVA, 2005, p. 97-98). Nesse ponto, a universidade tem que rever as bolsas oferecidas, bem como seus valores, e caso estas já estejam sendo suficientes para suprir as necessidades dos negros cotistas e demais alunos, exigir dos professores e coordenadores a divulgação e ofereci- mento, pois muitas não são utilizadas pelo desconhecimento. Entre os quatro cursos, foram assinalados ainda, como fatores dificultadores da permanência, a letra “a” que diz respeito a di- ficuldades de aprendizagem originadas no ensino fundamental e médio (leitura, interpretação etc.) com 8 (oito) e letra “d” - ou- tros motivos - com 9 (nove) respostas. Nesses dados, podemos ver a deficiência da escola pública e a importância das cotas para o ingresso na graduação, bem como a necessidade de cursos de nivelamento para suprir uma carência em decorrência da edu- cação básica. O item “d” foi elencado pelas mesmas razões que justificaram as dependências. Quando perguntados sobre os projetos e programas para negros cotistas que a universidade oferece, impressiona o número dos que não têm conhecimento uma vez que 36 (trinta e seis) deles responderam não conhecer, e apenas 3 (três) disseram ter ciên- cia, havendo uma resposta não preenchida, validando mais uma vez a afirmação de que os coordenadores de curso precisam fa- zer uma melhor divulgação, incentivar a participação, bem como os docentes se conscientizarem dessa necessidade premente, a fim de oferecerem projetos necessários ao atendimento dos edi- tais e programas disponíveis. 256 A PRESENÇA NEGRA NOS CURSOS DE GRADUAÇÃO EM DIREITO DA UNIVERSIDADE ESTADUAL DE MATO GROSSO DO SUL Os dados assustam, pois na Unidade de Paranaíba, turno matu- tino, doze questionados informaram não haver participado de projeto algum. Na Unidade de Naviraí, todos os questionados responderam não haver participado de projeto algum, situação que pode ser justificada pelo fato de muitos acadêmicos residi- rem em cidades vizinhas à universidade e até mesmo o desloca- mento até esta para poder estudar já é uma dificuldade, somado ao fato de todos os acadêmicos trabalharem, o que impossibilita o desenvolvimento dos projetos, que não é o caso de Paranaíba no turno matutino. No entanto, quando indagamos a todos sobre a necessidade de implantação de algum projeto específico para os acadêmicos ne- gros cotistas, que os ajudasse na permanência, 20 (vinte) pesso- as responderam ser esta providência desnecessária, enquanto que 19 (dezenove) entenderam que deveria ocorrer, sendo que um acadêmico não opinou. Aos que responderam afirmativamente sobre a necessidade de oferecimento de algum programa ou projeto, as respostas fo- ram bem diversificadas, dentre as quais sugeriram ações que: visassem a integração e a relação entre os cotistas; oferecessem reforço acadêmico; possibilitassem estágio dentro da universi- dade, de forma remunerada, focado no curso que o cotista faz; incentivassem a cultura, a inclusão do cotista na universidade, os Direitos Humanos; incentivo à leitura e à pesquisa; consciên- cia negra, histórico e política; incentivo ao desenvolvimento de pesquisas sobre sua cor e raça; orientação e/ou grupo de estu- do; incentivo financeiro pelo fato de a maioria dos negros cotis- tas vir de famílias humildes. Ainda em relação às sugestões, elencaram: melhor preparação com base no próprio curso; oferta e divulgação de bolsas; exten- são na comunidade; reforço didático dos conteúdos abordados nas disciplinas. Nota-se que as sugestões dos discentes estão re- lacionadas às dificuldades que podem ser reduzidas mediante a Aires David de Lima; Maria José de Jesus Alves Cordeiro 257 257 Aires David de Lima; Maria José de Jesus Alves Cordeiro Aires David de Lima; Maria José de Jesus Alves Cordeiro contemplação de bolsas. Entretanto, o que pudemos perceber é que estas não são distribuídas por ausência de projetos dos pro- fessores dos quatro cursos de Direito da UEMS e as razões dessa omissão, podemos atribuir ao fato de os advogados não possuí- rem formação e conhecimento didático-pedagógico necessários ao exercício da docência que contempla a pesquisa e a exten- são, se atendo apenas as atividades realizadas na sala de aula. A PRESENÇA NEGRA NOS CURSOS DE GRADUAÇÃO EM DIREITO DA UNIVERSIDADE ESTADUAL DE MATO GROSSO DO SUL A coordenação, que deveria ser o elo entre as determinações das pró-reitorias e demais encaminhamentos da universidade, divulgando as bolsas e incentivando os professores e alunos a realizarem projetos, também se mantém inerte nesse sentido. Os poucos projetos e bolsas que existem, talvez pela omissão dos gestores da universidade ou, ainda, pela falta de divulgação do oferecimento destes, demonstram que os alunos que realmen- te precisam não usufruem, fato que pode ter levado os negros cotistas a um grande número de dependências, evasões e re- provações. A falta de oferta de projetos, incentivo e auxilio na participação dos discentes faz com que a universidade continue sendo reprodutora das injustiças sociais, uma contradição aos objetivos do curso de Direito. Ao perguntarmos sobre os motivos que levaram os negros co- tistas a concorrerem pelo sistema de cotas, colocamos quatro alternativas: a) menor concorrência; b) exercício de um direi- to constitucional; c) sentimento de identidade e pertencimento racial; d) (genérica) outro fator, solicitando a citação destes. A alternativa que concentrou o maior número, com 20 (vinte) res- postas, foi a letra “b” - “exercício de um direito constitucional”; e as alternativas “a” - menor concorrência e “c” - sentimento de identidade e pertencimento racial ficaram empatadas, com 10 (dez) respostas cada uma. A razão de o maior número concentrar as respostas na alternati- va “b” talvez esteja ligada ao fato de que apenas os negros cotis- tas do curso de Direito foram questionados e por ser o Direito 258 A PRESENÇA NEGRA NOS CURSOS DE GRADUAÇÃO EM DIREITO DA UNIVERSIDADE ESTADUAL DE MATO GROSSO DO SUL 258 A PRESENÇA NEGRA NOS CURSOS DE GRADUAÇÃO EM DIREITO DA UNIVERSIDADE ESTADUAL DE MATO GROSSO DO SUL A PRESENÇA NEGRA NOS CURSOS DE GRADUAÇÃO EM DIREITO DA UNIVERSIDADE ESTADUAL DE MATO GROSSO DO SUL uma ciência quase toda positivada, os acadêmicos tem uma pre- dileção especial por argumentos legais. A menor concorrência (alternativa “a”) foi respondida por 10 (dez) acadêmicos, o que demonstra que as cotas proporcionaram incentivos para cursar a graduação, o que muitas vezes não ocorre na prática pois, em alguns anos, a concorrência entre os candidatos pelas cotas cos- tuma ser maior que entre as vagas gerais, dado o número pe- queno de vagas oferecidas. O sentimento de identidade e per- tencimento racial também contou com 10 (dez) respostas, o que demonstra progresso, uma vez que A universidade brasileira, ao se propor a adotar um plano de ações afirmativas, não se encontra tão somente a corri- gir os erros dos 500 anos de colonialismo e escravidão; está reconhecendo que a população negra precisa ser valorizada no plano social e individual. Existe a consciência de que a nossa identidade pode ser bem ou mal formada no curso de nossas relações com outros significantes. Mas é fundamen- tal que haja ações políticas ininterruptas de reconhecimen- to igualitário (SANTOS, 2006b, p. 20). As respostas aos questionários nos fazem olhar com otimismo o número de acadêmicos que marcaram pertencimento racial e a assunção como tal. Acreditamos que, também nesse ponto, as cotas são importantes, aumentam a autoestima e, por consequ- ência, o reconhecimento de uma identidade étnico-racial. Perguntados se o sistema de cotas havia facilitado a sua entrada na Universidade, 26 (vinte e seis) responderam afirmativamente e 14 (quatorze) negativamente. As explicações a esse questiona- mento foram as mais variadas. Os que responderam afirmativa- mente disseram, em sua maioria, que, por conta da concorrência ser menor, facilitou a entrada, uma vez que alguns acadêmicos ex- plicaram não possuir “bagagem” suficiente para concorrer com os alunos de escolas particulares. Outros afirmaram que o sistema de cotas possibilita a entrada em cursos com maior concorrên- Aires David de Lima; Maria José de Jesus Alves Cordeiro 259 259 Aires David de Lima; Maria José de Jesus Alves Cordeiro cia, aos quais, geralmente, a população negra tem menos acesso; a concorrência era menor do que para as vagas gerais, e outro fa- lou que a concorrência foi com outros alunos que se encontravam com o mesmo nível de conhecimento, ou seja, os oriundos de es- cola pública. A PRESENÇA NEGRA NOS CURSOS DE GRADUAÇÃO EM DIREITO DA UNIVERSIDADE ESTADUAL DE MATO GROSSO DO SUL Alguns afirmaram que se não fossem as cotas, jamais poderiam entrar em uma universidade, uma vez que sustentam a família e não teriam condições financeiras para isso. Quanto aos que responderam negativamente, as explicações foram que: havia disputado a vaga com outros cotistas; pre- pararam-se para concorrer de qualquer forma; após saber que poderia concorrer pela cota, foi que optaram pelo siste- ma; o vestibular é uma disputa, tendo se colocado em uma situação igualitária a todos ao disputar a vaga; as cotas não facilitam em nada, e o fato de ser pública gera muita discrimi- nação e, alguns disseram que poderiam ter passado mesmo que não tivessem concorrido pelas cotas, dada a pontuação obtida, mas que optaram por estas por se tratar de um direito constitucional. Indagados sobre a percepção que possuíam sobre se teriam ou não as mesmas condições e chances que os alunos não cotistas no mercado de trabalho, as respostas obtidas foram: 31 (trinta e um) sim e 09 (nove) não. Dos que responderam afirmativamente, justificaram que a par- tir do momento em que se consegue entrar na faculdade, todos devem buscar a melhor maneira de colher conhecimentos para concorrer no mercado de forma justa, apesar das diferentes di- ficuldades, confiando na dedicação e oportunidade, pois será o desempenho profissional do negro que vai dizer se está apto para realizar um bom trabalho; o mercado de trabalho tem es- paço para todos; a maioria dos negros cotistas já formados está inserida no mercado de trabalho; o fato de entrar pelo sistema de cotas não os torna menos competentes que os demais; todos têm chances iguais citando, inclusive, a presença de um negro 260 260 A PRESENÇA NEGRA NOS CURSOS DE GRADUAÇÃO EM DIREITO DA UNIVERSIDADE ESTADUAL DE MATO GROSSO DO SUL como Ministro do Supremo Tribunal Federal - STF. Acreditam nas mesmas condições e chances e entendem que o mercado de trabalho seleciona os melhores, independentemente de raça. como Ministro do Supremo Tribunal Federal - STF. Acreditam nas mesmas condições e chances e entendem que o mercado de trabalho seleciona os melhores, independentemente de raça. como Ministro do Supremo Tribunal Federal - STF. Acreditam nas mesmas condições e chances e entendem que o mercado de trabalho seleciona os melhores, independentemente de raça. A explicação dos que responderam negativamente foi de que nem sempre as condições serão as mesmas, tendo em vista que a situação socioeconômica traz uma maior dificuldade para ascensão profissional. Além disso, a maioria dos cotistas vem de família de baixa renda e, assim, possuem dificuldades enor- mes; além de concluir o curso na universidade, têm que pro- var à sociedade que são capazes; entendem que, infelizmente, ainda existe preconceito, e isto dificultará até na escolha do próprio cliente para a sua defesa. Acreditam que as cotas de- veriam se estender ao mercado de trabalho, pois este seleciona preconceituosamente os candidatos às vagas, já que no Brasil, este se encontra [...] estruturado com base na relação desigual entre capital e trabalho, também está apoiado na própria desigualdade racial. Nesse sentido, a construção da ponte entre as políti- cas de ações afirmativas e o mercado de trabalho pode criar patamares mais equânimes e condições leais de concorrên- cia e ascensão para a população negra brasileira (MARTINI; SILVA, 2005, p.96). Ademais, uma política de inclusão e promoção da igualdade ma- terial também vem prevista no Estatuto da Igualdade Racial, Lei n. 12.288, de 20 de julho de 2010, que prevê diversas medidas visando assegurar igualdade de oportunidade e tratamento para a população negra em matéria de trabalho e renda. A PRESENÇA NEGRA NOS CURSOS DE GRADUAÇÃO EM DIREITO DA UNIVERSIDADE ESTADUAL DE MATO GROSSO DO SUL Hoje, mostra-se mais importante o nível escolar e não mais a questão de raça; o fato de ser negro não está relacionado ao sucesso ou insucesso de uma pessoa, um dia já foi motivo, hoje não mais. A percepção dos acadêmicos quanto ao fato de ser negro não in- terferir no sucesso ou insucesso da pessoa, dizendo que esse es- tigma se encontra superado, demonstra um olhar otimista para o cenário que encontra à frente ou desconhecimento de dados que mostram que os cargos, empregos e renda dos negros ainda são inferiores aos do branco. O que pudemos observar é um grande otimismo e entusiasmo com relação às cotas, bem como a possibilidade de ascensão so- cial e melhores rendimentos com a graduação. Muito embora ainda reconheçam no racismo, ainda presente na nossa socieda- de, um entrave para o crescimento socioeconômico e cultural do negro, mas que está diminuindo ao longo dos anos com a pro- moção da cultura e políticas de respeito à diversidade, dentre elas as cotas. 260 A PRESENÇA NEGRA NOS CURSOS DE GRADUAÇÃO EM DIREITO DA UNIVERSIDADE ESTADUAL DE MATO GROSSO DO SUL Nesse item, concordamos com a afirmação de que o mercado de trabalho ainda é preconceituoso, pois, muito embora haja quem respondeu que o mercado de trabalho seleciona os me- lhores, independentemente da raça, não é o que presenciamos até os dias atuais, uma vez que a escravidão não é a única razão para explicar a situação de penúria que até hoje grande parte Aires David de Lima; Maria José de Jesus Alves Cordeiro 261 261 Aires David de Lima; Maria José de Jesus Alves Cordeiro Aires David de Lima; Maria José de Jesus Alves Cordeiro da população negra vive; outros fatores, tais como a discrimi- nação e o preconceito, interferiram e ainda interferem para essa ocorrência, uma vez que [...] o preconceito de cor [...] têm se perpetuado como componentes estruturantes da desigualdade social no Brasil, constituindo fortes entraves para a conquista da cidadania plena por parte do conjunto da população (VENTURI; BOKANY, 2005, p. 17). Algumas observações podem ser traçadas com relação às afir- mações anteriores, uma vez que o acadêmico que afirmou que há ministros no STF que são negros não atentou para o fato des- se órgão se encontrar instalado desde 9 de janeiro de 1829, sen- do o atual ministro Joaquim Barbosa o primeiro negro a ocupar a sua presidência. O acadêmico que entende que terá que estudar mais que o não cotista para se destacar além da cor, intrinsecamente está reco- nhecendo que o preconceito existe e que, se o desempenho após a universidade for igual ao do não negro, suas chances serão me- nores de se destacar, somando-se à estrutura e histórico fami- liar, que não auxiliam nessa ascensão, muitas vezes por ausência de referenciais. Em relação ao item 5, perguntamos se havia alguma observa- ção a fazer em relação ao sistema de cotas, projetos e outras iniciativas da UEMS com relação aos negros. As respostas, de modo geral, foram: o sistema de cotas implantado por nossa universidade, até agora, facilita a inserção do aluno no Ensino Superior, mas não há nenhum incentivo para que este per- maneça; há discriminação implícita de alguns profissionais da universidade (professores); o sistema de cotas serve para proporcionar a inserção e facilitar a entrada de pessoas, po- rém existe uma classificação muito ampla de cor e, com isso, existe a entrada de pessoas com “identidade duvidosa” que não parecem se encaixar nas características negras ou índias. 262 Das coordenações de cursos Passaremos agora à análise do questionário aplicado aos(às) coordenadores(as) dos quatro cursos, sendo que em cada Unidade Universitária foram respondidos 2 (dois) questionários, um pela coordenação atual e outro pela coordenação anterior, ou seja, 4 (quatro) professoras e 2 (dois) professores, totalizando 6 (seis) questionários. Destes, três eram coordenadores no ano de 2008, data colocada como início dessa pesquisa. Quatro acompanharam a implantação das cotas, sendo que um na condição de docente anteriormente. Entendemos, pelas respostas obtidas, que mesmo dizendo te- rem acompanhado a implantação das cotas, afirmam que elas Aires David de Lima; Maria José de Jesus Alves Cordeiro 263 263 Aires David de Lima; Maria José de Jesus Alves Cordeiro não foram discutidas e, sim, que vieram impostas e os coorde- nadores tiveram que aceitar já que é lei, numa demonstração de que não fizeram nada além de aceitar as determinações e encaminhamentos da reitoria e pró-reitorias, quando deveriam articular uma proposta mais eficiente e propor projetos e pro- gramas para atender às necessidades de aprendizagem e per- manência dos cotistas que iriam receber no curso, tanto como docente quanto como coordenadores de curso. Com relação às mudanças provocadas na UEMS pelas cotas, em especial no curso de Direito, apenas 2 (duas) entenderam que estas de fato proporcionaram um maior acesso a segmentos so- ciais como negros, indígenas e deficientes provenientes de esco- la pública. Dos que entendem negativamente, uma justificou que “este público foi sempre bem-vindo aos cursos de Direito onde trabalhei (Dourados, Paranaíba e Naviraí)”; outro coordenador afirmou como “... irrelevante. Discussões sobre o assunto não al- teraram as condições estruturais da Universidade”; uma ex-co- ordenadora disse “só consigo detectar o repúdio dos alunos, que alegam proteção desnecessária e opinando pelas cotas sociais”. Um coordenador atual disse que “pelo princípio da universalida- de houve posicionamentos favoráveis e contrários”. Pode-se perceber nos discursos, que pouco se inteiraram com relação à implantação do sistema de cotas, bem como não ti- veram qualquer preocupação em compreender as razões pelas quais foi criado e se limitaram a cumprir as determinações su- periores. Muitos disseram que acompanharam, estiveram pre- sentes nos fórum promovidos para discussão, no entanto, não trouxeram qualquer elemento concreto do que apreenderam com estes debates. A PRESENÇA NEGRA NOS CURSOS DE GRADUAÇÃO EM DIREITO DA UNIVERSIDADE ESTADUAL DE MATO GROSSO DO SUL car quais foram essas mudanças, uma vez que não apontou em que parte do Projeto Pedagógico ocorreu alteração. Das coordenações de cursos Perguntados se houve mudança no Projeto Pedagógico - PP do curso de Direito com a implantação das cotas, apenas uma co- ordenadora respondeu “sim, para adequação própria ao curso e não por conta das cotas”, resposta que não nos permite identifi- 264 car quais foram essas mudanças, uma vez que não apontou em que parte do Projeto Pedagógico ocorreu alteração. Foi feita uma pergunta pessoal acerca da posição dos coorde- nadores com relação ao sistema de cotas, obteve-se como uma das respostas que sempre “fui favorável a estas, por ser defen- sora da inclusão ao sistema de qualquer cidadão, por simples- mente ser pessoa humana, e, conforme pesquisas, esse público sempre fora excluído por razões econômicas e de cor”. Outro declara que: “sou contra, em princípio. Prefiro que as cotas fossem para crianças e jovens em escolas de bom nível. Mas como não foi escolhida esta alternativa, aceito como medida que resgata um pouco a situação desfavorável”. Entre as ex-co- ordenadoras foi dito: “já fui contra, por não me aprofundar no assunto e entendia que tudo que acontece de cima para baixo é um desrespeito; mas hoje sou uma fiel defensora das cotas, não existe como não nos posicionarmos nesse sentido frente à rea- lidade discriminatória de nosso país”. “É um mecanismo neces- sário, porém de duração temporária, para assegurar o acesso de negros em Universidades”. “Não se pode/deve desconside- rar a condição financeira e o mérito nas próximas décadas [...]”. Dentre os que ocupam a função foi dito: “considerando a igual- dade formal e material sou favorável ao sistema”, bem como, “considero importante, porém, apenas garantir o acesso não é suficiente, é preciso desenvolver métodos de acompanhamen- to p/ esses alunos”. Pelas respostas dos coordenadores percebemos que, à exceção de um coordenador, os demais são favoráveis às cotas. No en- tanto, nota-se que a implantação destas encontrou certa resis- tência, pois acreditavam em outras alternativas para a inclusão do negro na graduação, tais como a melhoria no ensino básico e fundamental público. É importante destacar as observações da coordenadora da uni- dade de Naviraí, que entende que as cotas devem ir além do Aires David de Lima; Maria José de Jesus Alves Cordeiro 265 265 Aires David de Lima; Maria José de Jesus Alves Cordeiro acesso, que a Universidade deve acompanhar e desenvolver mé- todos para a permanência desses cotistas. O que acreditamos que deveria ter acontecido em um segundo momento após a im- plantação das cotas, no entanto, até hoje, há pouca movimenta- ção institucional nesse sentido. car quais foram essas mudanças, uma vez que não apontou em que parte do Projeto Pedagógico ocorreu alteração. No assunto oferecimento de bolsas e outros programas de au- xílio na manutenção dos alunos cotistas, quando questionados se o que a UEMS oferece em matéria de bolsas e programas é suficiente para atender aos alunos cotistas que precisam, dos que responderam negativamente afirmam: “além do valor ver- gonhoso, deveria ser criada assessoria econômica para ajudar a organizar as despesas e gastos”; “o valor é ínfimo, bem como transforma o bolsista em trabalho barato. Precisa reavaliar os objetivos e valores das bolsas”; “o valor é insuficiente e as ações desenvolvidas precisam ser analisadas em função da necessida- de de se contribuir para a formação do acadêmico”; “adequação do valor à necessidade de cada aluno e acompanhamento para garantir o necessário à formação”. Todos os que responderam negativamente falaram do valor da bolsa, que é insuficiente e precisa ser revisto. No entanto, o ofe- recimento de bolsas e a quantidade, conforme visto, já represen- ta um grande avanço na formação acadêmica, pois proporciona ao aluno uma ajuda na sua manutenção, iniciação à pesquisa, mais tempo na universidade dedicado aos estudos. Dessa forma, é preciso conscientizar docentes, discentes e coordenadores de que as bolsas vão além da mera manutenção, auxiliando grande- mente na formação acadêmica do aluno, de modo a garantir que esta ocorra com qualidade. Com relação ao desempenho acadêmico, os dois coordenado- res informaram desconhecer as dificuldades enfrentadas pe- los negros cotistas durante o curso. Como sabemos, as cotas são de responsabilidade de toda a universidade, cabendo aos coordenadores e professores estarem mais atentos ao desen- 266 A PRESENÇA NEGRA NOS CURSOS DE GRADUAÇÃO EM DIREITO DA UNIVERSIDADE ESTADUAL DE MATO GROSSO DO SUL 266 A PRESENÇA NEGRA NOS CURSOS DE GRADUAÇÃO EM DIREITO DA UNIVERSIDADE ESTADUAL DE MATO GROSSO DO SUL volvimento dessa política, pois o desconhecimento informa- do demonstra total apatia e desinteresse com a inclusão que elas representam e, mesmo mantendo um discurso de con- cordância com elas, como já informado, são completamente indiferentes quanto à correção das injustiças históricas que elas proporcionam. Apontam as dificuldades com conteúdos básicos do curso como a maior dificuldade na vida acadêmi- ca do negro cotista ensejando, muitas vezes, sua evasão ou desistência do curso. Contudo, como docentes sabemos que esta é uma dificuldade geral dos alunos independente de co- tas, exigindo do professor estar atento às dificuldades iniciais dos negros cotistas e demais acadêmicos. Na resposta alusiva a “outros motivos”, uma coordenadora citou a “necessidade de o negro cotista trabalhar para custear suas despesas o que, às vezes, interfere na realização do curso”. Po- rém, acreditamos que essas dificuldades poderiam ser reduzi- das com um eficiente programa de acesso às bolsas, não apenas aos negros cotistas, mas a todos os alunos em condições socioe- conômicas desfavoráveis. O tópico relacionado à relação professor/aluno também obteve apenas uma resposta, o que nos leva a inferir que muitos casos não chegaram ao conhecimento da coordenação, uma vez que, se cruzarmos essa informação com a mesma pergunta elabora- da aos acadêmicos, 7 (sete) dos 23 (vinte e três) acadêmicos que carregam dependência assinalaram essa alternativa como um dos motivos para a sua reprovação. Quando instigados a citar as ações desenvolvidas para auxiliar no enfrentamento das dificuldades dos negros cotistas, assim como para os demais alunos no curso de Direito sob sua responsabi- lidade, responderam: “enquanto coordenadora sempre atuei de maneira a que todas as ações contemplassem todos os públicos, abrindo possibilidade de participação a viagens técnicas, con- gressos da UEMS ou externos, inclusive publicações científicas”. Aires David de Lima; Maria José de Jesus Alves Cordeiro 267 267 “Não faço diferença entre acadêmicos, em princípio, exceto os casos problemáticos (ameaça de morte, cantadas indevidas, comportamentos inadequados e outros) independentemente de raças, credo ou cultura”. “Não detectei nenhuma ação por parte da coordenação, dos professores do curso. Porque, para mim, criar projeto de ‘reforço’ são ações de auxílio específico aos co- tistas”. “Propostas de projetos de ensino (português) dependem de disponibilidade do corpo docente para, fora do horário, aten- der os discentes com dificuldades, parte dos professores o faz”. A PRESENÇA NEGRA NOS CURSOS DE GRADUAÇÃO EM DIREITO DA UNIVERSIDADE ESTADUAL DE MATO GROSSO DO SUL As respostas sugerem que alguns coordenadores não têm qual- quer conhecimento, interesse e preocupação com este siste- ma nem com seu aprimoramento, uma vez que as cotas foram, apenas, o primeiro passo para a graduação, sendo importante o empenho de todos os partícipes (professores, coordenadores, gestores) para que esses cotistas permaneçam na universidade até a regular formação. Um exemplo é quando mencionam a ne- cessidade de criar “reforço” aos negros cotistas como se, somen- te a estes, fosse necessária uma intervenção pedagógica, não considerando o fato de que os demais graduandos poderiam se encontrar nas mesmas situações. Solicitados a fazer algumas observações em relação ao sistema de cotas, projetos ou iniciativas para os alunos negros cotistas realizados na UEMS, responderam: “todas as ações que, de algu- ma forma, proporcionem a inclusão da pessoa devem ser reali- zadas”; “os cotistas têm obrigação institucional de auxiliar outros em semelhante situação, por exemplo, tutor de alunos de ensino médio”. A obrigação dos cotistas em auxiliar outros alunos não pode ser admitida, e ainda prova que alguns coordenadores não entenderam nada do que as cotas representam e seu papel de reparadoras de injustiças sociais, uma vez que, para esse coor- denador, aos demais acadêmicos não se exige semelhante en- cargo. Com os comentários citados, podemos perceber o total despreparo daqueles que ocupam a coordenação, uma vez que, devendo ser os articuladores das políticas emanadas dos órgãos 268 A PRESENÇA NEGRA NOS CURSOS DE GRADUAÇÃO EM DIREITO DA UNIVERSIDADE ESTADUAL DE MATO GROSSO DO SUL superiores e das pró-reitorias, denotam uma omissão na execu- ção desses encaminhamentos. abaixo com dados do quinto ano dos cursos. abaixo com dados do quinto ano dos cursos. Tabela 1 – Quinto ano - Curso de Direito - 2012 Tabela 1 – Quinto ano - Curso de Direito - 2012 Curso de Direito – UEMS – Quinto ano 2012 Unidades Matri- culados Aprovados Repro- vados Eva- didos Cursando séries anteriores s.d. c.d. 1º 2º 3º 4º 5º Dourados Mat. 11 8 0 3 0 0 0 0 1 0 Paranaíba Mat. 7 3 0 4* 4 0 0 0 1 0 Paranaíba Not. 9 7 0 2 3 0 0 0 0 0 Naviraí Not. 5 2** 0 4 3 0 0 0 2 0 Total 32 20 0 13 10 0 0 0 4 0 um acadêmico aproveitou estudos e em 2008 ingressou no segundo ano. Em 2011, estava no quinto ano, fazendo disciplina no quarto, reprovando em disciplina deste e do quinto. Na tabela acima está como reprovado. Este acadêmico foi considerado em todas as tabelas, tendo em vista o número de disciplinas que cursou na série regular. * O resultado final foi de 47 acadêmicos, visto que um acadêmico de Naviraí aproveitou estudos e se formou em 2011, sendo incluído na soma dos aprovados de 2012. um acadêmico aproveitou estudos e em 2008 ingressou no segundo ano. Em 2011, estava no quinto ano, fazendo disciplina no quarto, reprovando em disciplina deste e do quinto. Na tabela acima está como reprovado. Este acadêmico foi considerado em todas as tabelas, tendo em vista o número de disciplinas que cursou na série regular. * O resultado final foi de 47 acadêmicos, visto que um acadêmico de Naviraí aproveitou estudos e se formou em 2011, sendo incluído na soma dos aprovados de 2012. um acadêmico aproveitou estudos e em 2008 ingressou no segundo ano. Em 2011, estava no quinto ano, fazendo disciplina no quarto, reprovando em disciplina deste e do quinto. Na tabela acima está como reprovado. Este acadêmico foi considerado em todas as tabelas, tendo em vista o número de disciplinas que cursou na série regular. * O resultado final foi de 47 acadêmicos, visto que um acadêmico de Naviraí aproveitou estudos e se formou em 2011, sendo incluído na soma dos aprovados de 2012. Em 2012, no quinto ano, 32 (trinta e três) negros cotistas foram matriculados. superiores e das pró-reitorias, denotam uma omissão na execu- ção desses encaminhamentos. Muitas outras afirmações foram feitas. Alguns entendem que alguma coisa tem que ser feita com relação aos cotistas que já se encontram na graduação, em alguns momentos, falaram das bolsas, que são insuficientes e de reduzidos valores. No entanto, percebe-se que a maioria não as divulga aos acadêmicos e não incentiva o seu pleito, auxiliando na elaboração de projetos e oferecendo extensão que possam contemplar os negros cotistas, demonstrando, inclusive, desconhecimento do assunto. Essa falta de iniciativa dos gestores é geradora, em parte, dos percalços vividos pelos negros cotistas e demais alunos na mes- ma situação social, econômica e de aprendizagem, numa clara demonstração de que as oportunidades oferecidas pela univer- sidade são suficientes para os negros e pobres que se aventura- ram adentrar esse “nicho” de superioridade chamado Curso de Direito. Do desempenho acadêmico dos negros cotistas de 2008 a 2012: tecendo considerações finais Nesse tópico, apresentaremos o desempenho acadêmico dos ne- gros cotistas com os seguintes itens: matriculados; aprovados sem e com dependência; reprovados; evadidos, e aqueles que estão cursando séries anteriores. Como evadidos, considerare- mos aqueles que, muito embora tenham sido aprovados na sé- rie anterior, não renovaram a matrícula, bem como aqueles re- provados que também não se matricularam na mesma série. Os aprovados foram subdivididos em sem dependência (sd) e com dependência (cd). A análise do quinto ano pode dar um panora- ma geral do que sucedeu ao longo da jornada acadêmica. Para o propósito deste artigo, nos restringiremos, apenas, a tabela 269 270 A PRESENÇA NEGRA NOS CURSOS DE GRADUAÇÃO EM DIREITO DA UNIVERSIDADE ESTADUAL DE MATO GROSSO DO SUL 270 abaixo com dados do quinto ano dos cursos. Destes, apenas 20 foram aprovados sem depen- dências, somando-se um que aproveitou estudos, os quais cola- ram grau, ou seja, egressos da turma de 2008. Ao analisar toda a amostra (47 negros cotistas), verificamos que apenas 42,5% conquistaram a aprovação, concluindo seus estudos no tempo regular, ficando os demais retidos no quinto ano, ou seja, 13 (tre- ze) reprovados. Além disso, 4 (quatro) ficaram retidos em séries anteriores e, possivelmente, colarão grau em anos posteriores. Contudo, 10 (dez) foram considerados evadidos (desistentes) durante a trajetória, ou seja, 21,3%. Para estes, só resta a alter- nativa de um novo ingresso. A PRESENÇA NEGRA NOS CURSOS DE GRADUAÇÃO EM DIREITO DA UNIVERSIDADE ESTADUAL DE MATO GROSSO DO SUL Levando-se em conta as aprovações verificadas, notamos que, nessa trajetória, existem gargalos que a universidade precisa identificar e procurar solucionar para que haja maior perma- nência e conclusão de curso. Ao cotejar as tabelas, verificamos um número de dependências, reprovações e evasões que deve- ria ter sido motivo de preocupação da Universidade (Pró-Rei- toria de Ensino e Coordenação de Curso) a fim de se evitar esse alto índice de negros cotistas nessa situação. Uma vez que, como mencionado anteriormente, as cotas foram apenas o primeiro passo para o ingresso do negro na graduação, cabe zelar pela permanência e aprovação dos cotistas para que essa importante ação afirmativa possa cumprir seu papel. Neste trabalho, pudemos constatar que as condições socioeco- nômicas (necessidade de conciliar trabalho e estudo) interferem em muito no desempenho e permanência, sendo que as bolsas poderiam amenizar este problema. No entanto, os valores ofe- recidos por elas estão muito aquém do necessário para a manu- tenção desses alunos. Assim, cabe, muitas vezes, ao negro cotis- ta, optar entre sobreviver ou estudar. Dos 47 negros cotistas que ingressaram em 2008, os questio- nários foram aplicados a 40 deles e pela análise das respostas, não foi possível identificar um único fator como responsável pela reprovação e dependência, pois muitos fatores, em maior ou menor grau, interferem no desempenho e na autoestima do negro cotista, perpassando desde um referencial familiar até a falta de material didático para estudos. A universidade, muitas vezes, nem sequer tem conhecimento dessas carências, e to- mar ciência desses fatos é o primeiro passo para a intervenção. As bolsas podem servir de suporte para amenizar o problema mencionado. Para isso, os professores e coordenadores devem conhecer as necessidades dos acadêmicos, oferecer projetos e programas para a participação de quem realmente precisa. As di- ficuldades já verificadas nos anos iniciais devem ser sanadas, para Aires David de Lima; Maria José de Jesus Alves Cordeiro 271 271 Aires David de Lima; Maria José de Jesus Alves Cordeiro que as dependências não se acumulem e se tornem um desestí- mulo à permanência, não só dos negros cotistas, mas também dos demais acadêmicos que se encontram na mesma situação. que as dependências não se acumulem e se tornem um desestí- mulo à permanência, não só dos negros cotistas, mas também dos demais acadêmicos que se encontram na mesma situação. A PRESENÇA NEGRA NOS CURSOS DE GRADUAÇÃO EM DIREITO DA UNIVERSIDADE ESTADUAL DE MATO GROSSO DO SUL que entram as cotas nas universidades públicas, com o discurso acirrado de que estas eram inconstitucionais, o que perdurou até 26 de abril de 2012, oportunidade em que o STF reconheceu a constitucionalidade das cotas, o que vincula todos os outros tribu- nais do Brasil, nenhum deles podendo julgar de maneira diversa. A amostra selecionada na UEMS serviu para compreender o que significam as cotas e a trajetória vivenciada pelos negros cotis- tas na área do Direito. Além disso, as atas de resultados finais de 2008 a 2012 denunciam um índice de 21,2% de evasão dos ne- gros cotistas nos cursos pesquisados. Identificar os problemas e atuar para sua solução deveria ser um desdobramento natu- ral nas ações docentes e de gestores de uma universidade que oferta cotas étnico-raciais. Portanto, cabe à UEMS, assim como a outras instituições com cotas, buscar seu aperfeiçoamento pelo comprometimento de todos nesse processo de inclusão e demo- cratização do ensino superior, num claro movimento de respeito às diferenças e luta contra o racismo. Conhecer as dificuldades enfrentadas pelos negros cotistas e de- mais alunos e empenhar-se em solucioná-las devem ser motivos de preocupação dos coordenadores de curso, professores e ges- tores da universidade, como iniciativas que vão além das cotas, em um diálogo franco e direto com os sujeitos envolvidos. Só as- sim a universidade estará cumprindo seu papel de disseminado- ra de conhecimentos, respeitadora da diversidade e promotora da igualdade de direito e de fato. AIX Sistemas. Disponível em: <http.aix.com.br/wiki/index.php?titel=Ata_de_ Resultado>. Acesso em: 8 abr. 2012. A PRESENÇA NEGRA NOS CURSOS DE GRADUAÇÃO EM DIREITO DA UNIVERSIDADE ESTADUAL DE MATO GROSSO DO SUL Precisamos, no atual estágio de consolidação das cotas, pensar além do oferecimento destas (ingresso), como também na per- manência dos cotistas, para que haja formação (egresso regu- lar). Caso contrário, o trajeto universitário estará padecendo do mesmo problema que as cotas tentaram corrigir, ou seja, da ex- clusão da universidade para a exclusão na universidade. Não há como negar que, nessa trajetória, também houve con- quistas, uma vez que 20 (vinte) negros cotistas, que represen- tam 42,5%, colaram grau, o que proporciona grande alento a essa população, nos permitindo olhar com otimismo o cenário à frente, somado ao fato de que 4 (quatro) ainda se encontravam cursando o quarto ano, em 2012, quando do encerramento des- ta pesquisa. No Brasil, a escravidão sustentou a discriminação e a exclusão do negro, cujos frutos são perceptíveis até hoje. Para reverter essa situação uma das principais bandeiras de luta do Movimento Negro ainda é o acesso à educação, na crença de que o conheci- mento, fruto da formação superior, é o caminho para a ascensão social. O não reconhecimento do Brasil como país racista dificul- tou a tomada de medidas na tentativa de efetuar compensação pelos prejuízos sofridos pela raça negra desde que esta entrou no Brasil, na condição de escrava. A pressão internacional cobrou do Brasil uma postura mais ativa no enfrentamento dessas questões, uma vez que nenhuma justi- ficativa explicava os dados que mostram que, aos negros, foram reservados os piores trabalhos com os menores rendimentos, bem como o fato de a escolaridade destes, sua situação social e econômica serem inferior à do branco, muito embora a população negra seja numericamente superior à branca. É nessa conjuntura 272 A PRESENÇA NEGRA NOS CURSOS DE GRADUAÇÃO EM DIREITO DA UNIVERSIDADE ESTADUAL DE MATO GROSSO DO SUL 272 BENICÁ, Dirceu. Uma universidade em movimento. In: BENICÁ, Dirceu. (Org.) Uni- versidade e suas fronteiras. 1. ed. São Paulo: Outras Expressões, 2011. p. 31-63. Aires David de Lima; Maria José de Jesus Alves Cordeiro Aires David de Lima; Maria José de Jesus Alves Cordeiro Referências AIX Sistemas. Disponível em: <http.aix.com.br/wiki/index.php?titel=Ata_de_ Resultado>. Acesso em: 8 abr. 2012. AIX Sistemas. Disponível em: <http.aix.com.br/wiki/index.php?titel=Ata_de_ Resultado>. Acesso em: 8 abr. 2012. BENICÁ, Dirceu. Uma universidade em movimento. In: BENICÁ, Dirceu. (Org.) Uni- versidade e suas fronteiras. 1. ed. São Paulo: Outras Expressões, 2011. p. 31-63. 273 BORGES, Rosane da Silva. Pensando a transversalidade de gênero e raça. In: SANTOS, Gevanilda; SILVA, Maria Palmira da. Racismo no Brasil: percepções da discrimina- ção e do preconceito no século XXI.São Paulo: Fundação Perseu Abramo, 2005. BORGES, Rosane da Silva. Pensando a transversalidade de gênero e raça. In: SANTOS, Gevanilda; SILVA, Maria Palmira da. Racismo no Brasil: percepções da discrimina- ção e do preconceito no século XXI.São Paulo: Fundação Perseu Abramo, 2005. 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https://openalex.org/W2008349676	https://durham-repository.worktribe.com/preview/1393389/21063.pdf	English	null	The effects of screening length in the non-local screened-exchange functional	Journal of physics. Condensed matter	2,014	cc-by	9,073	PACS numbers: 71.15.Mb, 71.15.Dx, 71.20.Mq, 71.20.Nr The eﬀects of screening length in the non-local screened-exchange functional Yuzheng Guo1, John Robertson1 and Stewart J Clark2 1Engineering Department, Cambridge University, Cambridge CB2 1PZ, UK 2Physics Department, Durham University, Durham, DH1 3LE, UK 1. Introduction Density functional theory (DFT) has become the standard method to predict atomic and electronic properties in condensed matter physics and quantum chemistry[1, 2, 3]. The most widely used exchange-correlation functionals such as the local density approximation (LDA) or general gradient approximation (GGA) replace the exchange- correlation energy of the many-electron Schr¨odinger equation with a functional of the local electron density[4, 5]. DFT describes the ground state properties such as lattice constants and bulk modulus quite well at low computational cost. However, it is well known that both LDA and GGA underestimate the band gap in semiconductors and insulators as they do not give the energy discontinuity across the Fermi level as a function of electron occupancy[6, 7]. They also do not describe well localised states or strongly correlated systems such as Mott insulators[8] partly due to the absence of the self-interaction correction[9]. Various improvements have been suggested to overcome the drawbacks of LDA and GGA. One of the simplest is the DFT+U method[10, 11, 12]. This introduces a repulsive potential U on localised electrons as in the Hubbard model. This functional can be used for structural relaxation with low computational cost. However, this method strictly only applies to open shell systems such as the transition metal compounds, although it is now (often incorrectly) used as an empirical ﬁtting method well outside its true range of physical validity[12]. Most semiconductors are close shell systems, for which DFT+U gives only limited improvements. There are also more advanced methods such as the GW method based on the Greens function[13, 14, 15, 16, 17] and dynamical mean ﬁeld theory (DMFT)[18]. In the GW approximation, the quasi-particle energy is calculated by expanding the electron self- energy and dielectric function. GW can give accurate band structures. However, it is computationally expensive, so that often only the ﬁrst order perturbative version is used, referred to as G0W0. On the other hand, DMFT maps the many-body problem onto an impurity model without the approximation of independent electrons. It can cure some of the problems of DFT such as band gap under estimation and electron localisation, and has been widely used for actinides and Mott insulators. However, it is more costly than GW. This makes it computationally expensive to apply these methods to complex systems such as surfaces, interfaces, or defect supercells with hundreds of atoms. E-mail: s.j.clark@durham.ac.uk E-mail: s.j.clark@durham.ac.uk Abstract. The screened exchange (sX) hybrid functional can give good band structures for simple sp bonded semiconductors and insulators, charge transfer insulators, Mott-Hubbard insulators, two dimensional systems and defect systems. This is particularly attributed to the sX hybrid scheme ﬁxing the self-interaction problem associated with local functionals. We investigate the eﬀect of varying the screening parameter of the exchange potential on various material properties such as the band gap. The Thomas Fermi screening scheme in which the screening parameter varies with an average valence electron density leads to a weak dependence of the band gap on valence electron density, so that a ﬁxed screening parameter could be applied to heterogeneous systems like surfaces, interfaces and defects. PACS numbers: 71.15.Mb, 71.15.Dx, 71.20.Mq, 71.20.Nr The eﬀects of screening length in the sX functional 2 2 1. Introduction Thus, it is still desirable to ﬁnd an accurate yet reasonably low cost DFT-style functional. It is well known that the non-local Hartree-Fock (HF) potential tends to over- estimate band gaps while LDA tends to under-estimate band gaps[5, 6, 9]. Also, HF over- estimates localisation while LDA under-estimates localisation[9]. Hybrid functionals are a type of functional which empirically mixes a fraction of the HF potential with a local exchange-correlation functional and they can give reasonably correct band gaps and electron/hole localisations[19, 20]. Various hybrid functionals have been proposed such as B3LYP[20, 21], PBE0[22], and the Heyd-Scuseria-Ernzerhof (HSE) The eﬀects of screening length in the sX functional 3 functional[23, 24, 25]. These hybrid functionals are generalised Kohn-Sham functionals that can be used variationally for geometry optimisation just like LDA[20, 26]. These functionals are found to give good descriptions of the band gap in a variety of systems[28, 29, 30, 31]. Here, we discuss the screened exchange (sX) hybrid functional of Bylander and Kleinman[32]. This replaces the short range part of the LDA by a short-range screened Hartree-Fock exchange energy[26, 32, 33, 34, 35, 36, 37]. sX has been implemented for a plane wave basis within the Castep code[34]. Geometry optimisation was helped by a fast algorithm to calculate the Hellman-Feynman stress[38]. sX has been widely used in many band structure and defect calculations[33, 34, 35, 36, 37, 39, 40, 41, 42, 43]. The band gaps from sX of semiconductors are improved with a mean relative error of 7.4%[34]. The early papers on hybrid functionals used a ﬁxed mixing parameter of α=0.25 (meaning 25% HF exchange) based on Becke’s rationalisation[20]. HSE kept this value of α and varied the screening parameter between the HSE03 and HSE06 versions[24, 25]. On the other hand, some recent papers have varied the mixing fraction, noting that this parameter might vary with the band gap or the dielectric constant[44, 45] and that HSE under-estimates the band gap of very wide gap insulators[30]. On the other hand, Moussa et al[46] considered the optimisation of both parameters for some solids. The hybrid functionals have been analysed[47, 48, 49] and criticised[50] with respect to GW. In the case of sX, the mixing fraction is kept at 1, and the screening parameter is chosen in terms of the screening arising from the valence electron density[34]. 1. Introduction Nevertheless, it is interesting to see how the structural properties and band gap would vary if the screening parameter is allowed to vary. At one level, we ﬁnd that the calculated band gap is rather insensitive to the valence electron density in this approximation. At another level, HSE chose a ﬁxed screening parameter independent of system[24], a useful simpliﬁcation when treating heterogeneous systems such as surfaces or interfaces between systems of electron density. Diﬀerent approximations have been proposed to calculate the screening eﬀects[50] but we ﬁnd that the band gap is rather insensitive to screening parameter. This paper is organised as follows. In the next section, we present the methods used in this work with special attention paid to the screening parameter. In section 3, we give detailed description of the eﬀect of screening parameter on total energy, lattice constant, band structure, and defect calculation. In section 4, we discuss the results and ﬁnally draw conclusions from the results obtained in this work. 2. Methods The sX functional is a non-local Schr¨odinger equation in which the exchange-correlation energy depends on both the electron density and electron orbitals, −1 2∇2 + VH[n(r)] + Eext[n(r)] + V XC loc [n(r)] ψi(r) + The eﬀects of screening length in the sX functional The eﬀects of screening length in the sX functional 4 Z V XC nl (r, r′)ψi(r′)dr′ = ϵiψi(r) Z V XC nl (r, r′)ψi(r′)dr′ = ϵiψi(r) (1) (1) Z V XC nl (r, r′)ψi(r′)dr′ = ϵiψi(r) (1 where the term in brackets is the kinetic energy, the Hartree energy, the external potential, and the local XC energy such as the LDA or GGA part. The second part is the non-local part of XC energy which depends explicitly on electron orbitals. sX assumes that the HF exchange is screened by a Thomas-Fermi (TF) screening parameter, so the potential decays exponentially with the distance, EXC nl = −1 2 X ij,kq Z Z drdr′φ∗ ik(r)φik(r′)φ∗ jq(r′)φjq(r) \|r −r′\| exp (−kTF \|r −r′\|) ( (2) where i and j label the electron bands, k and q for k-points, and kTF is the TF screening parameter. The local part is modiﬁed to avoid the double counting of the local part of the screening potential, EXC loc = EXC HEG [ρ(r)] −EXC NL−HEG [ρ(r)] (3) (3) where the ﬁrst term on the right is just the LDA functional. The second term is the non- local exchange-correlation energy of a homogeneous electron gas (HEG) with a density ρ(r). Combining the local and non-local part gives the full exchange-correlation energy where the ﬁrst term on the right is just the LDA functional. The second term is the non- local exchange-correlation energy of a homogeneous electron gas (HEG) with a density ρ(r). Combining the local and non-local part gives the full exchange-correlation energy EXC = EXC nl + EXC loc . (4) EXC = EXC nl + EXC loc . (4) The TF screening constant represents the screening range of the electron interaction. If kTF = 0, the non-local XC becomes the full HF potential and the local part becomes the LDA correlation energy. Thus the result from HF limit should not be interpreted as comparable to the pure HF method or advanced methods such as exact exchange method (EXX). If kTF is inﬁnite, there is full screening. The HF part is screened away and the local part returns to LDA. 2. Methods Therefore sX gives the correct asymptotic limit of the free electron gas. Figure 1 compares the screening in the diﬀerent hybrid functionals. Figure 1(a) shows the screened fraction of the HF potential as a function of inter-electron distance. The HF fraction is a constant for PBE0. For the sX, the screening parameter is varied to show how this aﬀects the HF fraction. As the increases the fraction of HF decreases and vice versa. The medium range, from 2 to 4˚A, is an important range for this parameter because most inter-atomic distances fall into this range. Figure 1(b) compares the screening in reciprocal space. The HSE screening and sX screening with various screening parameters are compared with the dielectric function of GaAs[51]. We see that a TF screening parameter of 1-2˚A−1 is close to that of the dielectric function. In principle, we can achieve a good description of the electronic structure if the screening parameter is allowed to vary. Ideally, the screening parameter should be ﬁxed by some procedure. If the TF screening model[52, 53] is used, the screening parameter is given by kTF = 2 s kF π (5) The eﬀects of screening length in the sX functional 5 Figure 1. (a) Comparison of HF potential fraction in diﬀerent hybrid schemes. Diﬀerent TF screening parameters are used in sX (green lines). For PBE0 (blue line) α = 0.25. For HSE (red line), α = 0.25 and µ = 0.2. (b) Comparison of the inverse dielectric constant for diﬀerent functionals. The same parameters as (a) have been used. The inverse dielectric function of GaAs has been plotted (black line) for comparison[51]. Note the similarity of sX screening to that of the experimental inverse dielectric function. (a) 0 2 4 6 8 10 Distance (Å) 0.0 0.2 0.4 0.6 0.8 1.0 Fraction of Hartree-Fock sX (kTF=0.1Å -1) sX (kTF=2.5Å -1) sX (kTF=10.0Å -1) HSE PBE0 sX (kTF=1.0Å -1) (b) 0 2 4 6 8 10 Wavenumber, Q (Å -1) 0.0 0.2 0.4 0.6 0.8 1.0 Inverse dielectric, 1/ε sX (kTF=10.0Å -1) sX (kTF=1.0Å -1) sX (kTF=0.1Å -1) sX (kTF=2.5Å -1) GaAs HSE where kF is the electron Fermi wavevector We can also write kTF in terms of electron Figure 1. (a) Comparison of HF potential fraction in diﬀerent hybrid schemes. Diﬀerent TF screening parameters are used in sX (green lines). For PBE0 (blue line) α = 0.25. 2. Methods The screening parameter used for diﬀerent materials in units of ˚A−1. The plane wave basis pseudopotential package Castep is used to carry out all the calculations in this work[54]. The norm-conserving pseudo potentials are deﬁned in Ref. 21, where most of them are the default pseudopotentials while some more transferable potentials are generated using the Opium code. A Monkhorst-Pack (MP) k-point mesh is used for the integration over the Brillouin zone[55]. The convergence of the total energy diﬀerences with respect to the mesh size and cutoﬀis better than 0.01 eV/atom. The density mixing scheme is used for electronic energy minimisation for most sp semiconductors, while a preconditioned conjugate-gradient scheme is used for most transition metals because the density mixing method can be unstable due to the orbital dependence of the functional[26]. 2. Methods For HSE (red line), α = 0.25 and µ = 0.2. (b) Comparison of the inverse dielectric constant for diﬀerent functionals. The same parameters as (a) have been used. The inverse dielectric function of GaAs has been plotted (black line) for comparison[51]. Note the similarity of sX screening to that of the experimental inverse dielectric function. ( ) (a) 0 2 4 6 8 10 Distance (Å) 0.0 0.2 0.4 0.6 0.8 1.0 Fraction of Hartree-Fock sX (kTF=0.1Å -1) sX (kTF=2.5Å -1) sX (kTF=10.0Å -1) HSE PBE0 sX (kTF=1.0Å -1) (b) 0 2 4 6 8 10 Wavenumber, Q (Å -1) 0.0 0.2 0.4 0.6 0.8 1.0 Inverse dielectric, 1/ε sX (kTF=10.0Å -1) sX (kTF=1.0Å -1) sX (kTF=0.1Å -1) sX (kTF=2.5Å -1) GaAs HSE F is the electron Fermi wavevector. We can also write kTF in terms of electron ρ(r), ( ) 0 2 4 6 8 10 Distance (Å) 0.0 0.2 0.4 0.6 0.8 1.0 Fraction of Hartree-Fock sX (kTF=0.1Å -1) sX (kTF=2.5Å -1) sX (kTF=10.0Å -1) HSE PBE0 sX (kTF=1.0Å -1) (b) (b) 0 2 4 6 8 10 Wavenumber, Q (Å -1) 0.0 0.2 0.4 0.6 0.8 1.0 Inverse dielectric, 1/ε sX (kTF=10.0Å -1) sX (kTF=1.0Å -1) sX (kTF=0.1Å -1) sX (kTF=2.5Å -1) GaAs HSE where kF is the electron Fermi wavevector. We can also write kTF in terms of electron density ρ(r), where kF is the electron Fermi wavevector. We can also write kTF in terms of electron density ρ(r), kTF = 2 3ρ π 1 6 (6 kTF = 2 3ρ π 1 6 (6) so the screening parameter is proportional to the one sixth order of valence electron density, which explains why the kTF is a slowly varying function of ρ(r). Unless stated otherwise, all the values used in this work is summarised in Table 1. so the screening parameter is proportional to the one sixth order of valence electron density, which explains why the kTF is a slowly varying function of ρ(r). Unless stated otherwise, all the values used in this work is summarised in Table 1. The eﬀects of screening length in the sX functional 6 Table 1. The screening parameter used for diﬀerent materials in units of ˚A−1. Material kT F C 2.50 Si 2.09 Ge 2.06 GaAs 2.04 Al2O3 2.48 HfO2 2.54 Ti2O3 2.43 Cr2O3 2.52 Fe2O3 2.56 BN 2.56 Table 1. 3.1. Wave function It has been pointed out by several groups that the sX wave function can be almost identical to that from LDA/GGA in many cases[26]. We have conﬁrmed this similarity. First of all the LDA/PBE wave function can be a good starting point for sX calculation. This greatly reduces the computational time. Furthermore the LDA/GGA wave function can be used for a sX band structure calculation: ϵsX n,k = D ψsX n,k V sX hn ψLDA n′,k′ oi ψsX n,k E . (7) (7) The band structure from an LDA/PBE basis with an sX potential shares the similar curve as the self-consistent sX band structure. But the band gap is lower compared to a self-consistent sX calculation. Figure 2 compares the Si band gap as a function of the screening parameter from LDA/GGA with the self-consistent sX band gaps. The same 6 × 6 × 6 MP grid has been used for reciprocal space integrations. The experimental crystal structure has been used for all cases. The diﬀerence between the sX and the LDA/GGA bands increases as the screening parameter decreases. At the HF limit, the error could be as be large as 25% of the band gap. However, the error is about 2%-3% The band structure from an LDA/PBE basis with an sX potential shares the similar curve as the self-consistent sX band structure. But the band gap is lower compared to a self-consistent sX calculation. Figure 2 compares the Si band gap as a function of the screening parameter from LDA/GGA with the self-consistent sX band gaps. The same 6 × 6 × 6 MP grid has been used for reciprocal space integrations. The experimental crystal structure has been used for all cases. The diﬀerence between the sX and the LDA/GGA bands increases as the screening parameter decreases. At the HF limit, the error could be as be large as 25% of the band gap. However, the error is about 2%-3% The eﬀects of screening length in the sX functional 7 7 Figure 2. The calculated band gap of Si as function of screening paramete diﬀerence wave functions from LDA (orange line), PBE (black line), and sX line). The ﬁgures shows that LDA and PBE wave function gives similar when screening parameter is larger than 1. The diﬀerence becomes signiﬁcant approaching HF limit. The dashed line shows the experimental band gap. 3.1. Wave function 10 -2 10 -1 10 0 Screening length, kTF (Å -1) 0 1 2 3 4 5 6 Band gap (eV) sX PBE LDA Table 2. sX band gaps (in eV) based on diﬀerent wave functions. Material sX wavefunction LDA wavefunction Diﬀerence C 5.32 5.04 0.28 Si 1.09 1.07 0.02 Ge 0.69 0.60 0.09 GaAs 1.52 1.47 0.05 Ti2O3 0.22 0.00 0.22 Cr2O3 3.56 3.48 0.08 f bl i t f 1 8 2 5˚A−1 hi h t th t th LDA/ Figure 2. The calculated band gap of Si as function of screening parameter with diﬀerence wave functions from LDA (orange line), PBE (black line), and sX (green line). The ﬁgures shows that LDA and PBE wave function gives similar results when screening parameter is larger than 1. The diﬀerence becomes signiﬁcant when approaching HF limit. The dashed line shows the experimental band gap. 6 10 -2 10 -1 10 0 Screening length, kTF (Å -1) 0 1 2 3 4 5 6 Band gap (eV) sX PBE LDA Table 2. sX band gaps (in eV) based on diﬀerent wave functions. Table 2. sX band gaps (in eV) based on diﬀerent wave functions. Table 2. sX band gaps (in eV) based on diﬀerent wave functions. Material sX wavefunction LDA wavefunction Diﬀerence C 5.32 5.04 0.28 Si 1.09 1.07 0.02 Ge 0.69 0.60 0.09 GaAs 1.52 1.47 0.05 Ti2O3 0.22 0.00 0.22 Cr2O3 3.56 3.48 0.08 for reasonable screening parameters of 1.8-2.5˚A−1, which suggests that the LDA/GGA wave function could be almost identical to the sX wave function, in agreement with previous results. for reasonable screening parameters of 1.8-2.5˚A−1, which suggests that the LDA/GGA wave function could be almost identical to the sX wave function, in agreement with previous results. Table 2 shows the band gap of several solids with their respect the TF screening parameter using the LDA wave function or the sX wave function. The error induced by the LDA wave function is usually less than 0.2eV. Therefore, it is acceptable for most semiconductors and insulators. However, the LDA wave function should not be used for small band gap or correlated semiconductors. Ti2O3, a paramagnetic semiconductor, whose band gap is only 0.1-0.2eV, can be well described by the self-consistent sX with a band gap 0.22eV, but a LDA/GGA wave function will give a metallic band structure. The eﬀects of screening length in the sX functional 8 Figure 3. 3.2. Lattice constant We used sX to relax the lattice constant of Si to see the inﬂuence of various screening lengths, kTF. The LDA is known to under-estimate lattice constants while GGA tends to over-estimate it (5.40˚A and 5.46˚A respectively). The sX hybrid functional mixes the screened HF potential and LDA local potential. The results are shown in Figure 3. The LDA gives 5.40˚A which is below the experimental value, while the HF limit gives 5.355˚A. This underestimate of lattice parameter using HF is expected given standard molecular results[27]. We used sX to relax the lattice constant of Si to see the inﬂuence of various screening lengths, kTF. The LDA is known to under-estimate lattice constants while GGA tends to over-estimate it (5.40˚A and 5.46˚A respectively). The sX hybrid functional mixes the screened HF potential and LDA local potential. The results are shown in Figure 3. The LDA gives 5.40˚A which is below the experimental value, while the HF limit gives 5.355˚A. This underestimate of lattice parameter using HF is expected given standard molecular results[27]. 3.1. Wave function Variation of lattice parameter of Si with screening length (black line). The dashed line marks the experimental lattice parameter of 5.41˚A. We see the usual LDA (large kT F ) overbinding and HF (small kT F ) overbinds further. 0.001 0.01 0.1 1 10 100 Screening length, kTF (Å -1) 5.36 5.37 5.38 5.39 5.40 5.41 Si lattice parameter (Å) 3.2. Lattice constant We used sX to relax the lattice constant of Si to see the inﬂuence of various screening lengths, kTF. The LDA is known to under-estimate lattice constants while GGA tends to over-estimate it (5.40˚A and 5.46˚A respectively). The sX hybrid functional mixes the screened HF potential and LDA local potential. The results are shown in Figure 3. The LDA gives 5.40˚A which is below the experimental value, while the HF limit gives 5.355˚A. This underestimate of lattice parameter using HF is expected given standard molecular results[27]. Figure 3. Variation of lattice parameter of Si with screening length (black line). The dashed line marks the experimental lattice parameter of 5.41˚A. We see the usual LDA (large kT F ) overbinding and HF (small kT F ) overbinds further. 0.001 0.01 0.1 1 10 100 Screening length, kTF (Å -1) 5.36 5.37 5.38 5.39 5.40 5.41 Si lattice parameter (Å) Figure 3. Variation of lattice parameter of Si with screening length (black line). The dashed line marks the experimental lattice parameter of 5.41˚A. We see the usual LDA (large kT F ) overbinding and HF (small kT F ) overbinds further. 0.001 0.01 0.1 1 10 100 Screening length, kTF (Å -1) 5.36 5.37 5.38 5.39 5.40 5.41 Si lattice parameter (Å) Figure 3. Variation of lattice parameter of Si with screening length (black line). The dashed line marks the experimental lattice parameter of 5.41˚A. We see the usual LDA 3.3. Band structure The most important application of the sX hybrid functional is to correct the band gap problem in DFT. Here, the band structures are calculated with various screening parameters for various materials ranging from the simple sp semiconductors, to transition metal oxides with d electrons, Mott-Hubbard systems and 2-dimensional systems. The data is summarised in Figure 4. The gaps are normalised to the experimental band gap as 1 and to the band gap in the HF limit as 2, for each material. The experimental value of band gap gathers round a screening length of 1.8-2.5˚A−1 which conﬁrms that TF screening scheme gives a correct band gap. It has been shown previously that the sX functional can give much better band gaps in many sp semiconductors[32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43]. Here we show the band gap variance as a function of the screening parameter. In order to eliminate the eﬀect of lattice constants, we use the experimental lattice constants. Figure 4 shows the results from Si, C (diamond), Ge, and GaAs. The electron conﬁguration and crystal structure are the same for these semiconductors, thus their TF screening parameters are similar. We see that the slope of the variation of band gap with screening parameter is smaller The eﬀects of screening length in the sX functional 9 Figure 4. The band gaps versus screening parameter for Si, C (diamond), Ge, GaAs, Al2O3, HfO2, BN, Ti2O3, Cr2O3, Fe2O3, and Si(111)-(2 × 1) surface. The data is normalised by two limits, the experimental band gap and HF limit band gap (respectively, 1.0 and 2.0 on the vertical axis). Figure 4. The band gaps versus screening parameter for Si, C (diamond), Ge, GaAs, Al2O3, HfO2, BN, Ti2O3, Cr2O3, Fe2O3, and Si(111)-(2 × 1) surface. The data is normalised by two limits, the experimental band gap and HF limit band gap (respectively, 1.0 and 2.0 on the vertical axis). Figure 4. The band gaps versus screening parameter for Si, C (diamond), Ge, GaAs, Al2O3, HfO2, BN, Ti2O3, Cr2O3, Fe2O3, and Si(111)-(2 × 1) surface. The data is normalised by two limits, the experimental band gap and HF limit band gap (respectively, 1.0 and 2.0 on the vertical axis). 3.3. Band structure 0.01 0.1 1 Screening length, kTF (Å -1) 1.0 1.5 2.0 Normalised band gap Si C Ge GaAs HfO2 Al2O3 Fe2O3 Cr2O3 Ti2O3 BN Si(111) than the experimental band gap, so that the band gap is not so sensitive to the screening parameter. The error induced by a small deviation from the optimum TF value is quite small. GaAs, Al2O3, HfO2, BN, Ti2O3, Cr2O3, Fe2O3, and Si(111) (2 1) surface. Th data is normalised by two limits, the experimental band gap and HF limit band ga (respectively, 1.0 and 2.0 on the vertical axis). 0.01 0.1 1 Screening length, kTF (Å -1) 1.0 1.5 2.0 Normalised band gap Si C Ge GaAs HfO2 Al2O3 Fe2O3 Cr2O3 Ti2O3 BN Si(111) 0.01 0.1 1 Screening length, kTF (Å -1) 1.0 1.5 2.0 Normalised band gap Si C Ge GaAs HfO2 Al2O3 Fe2O3 Cr2O3 Ti2O3 BN Si(111) than the experimental band gap, so that the band gap is not so sensitive to the screening parameter. The error induced by a small deviation from the optimum TF value is quite small. Figure 4 also shows the band gaps for the wide gap, closed-shell, insulators BN, HfO2 and Al2O3. These insulators are important from both the scientiﬁc and technological points of view. HfO2 and Al2O3 are the widely used as gate insulators in ﬁeld eﬀect transistors. Hexagonal BN is now used as a substrate for 2D systems such as graphene and MoS2. We see that the normalised trend of band gap versus screening parameter is similar in all these systems, and that the optimum value is close to the TF value. Another problem for the LDA/GGA functional is over delocalisation of semi-core states such as the d and f electrons. For the sp semiconductors only the band gap is under-estimated but the band structure is qualitatively correct. However LDA/GGA fails qualitatively for localised, open-shell, d and f electron systems. The LDA band gap disappears in systems such as the Mott-Hubbard insulators Ti2O3. We previously found that sX gives good band gaps for several transition metal oxides[41, 42] and that hybrid functionals in general can treat the transition metal and lanthanides well[31, 43]. Here, we have calculated the band gap of Ti2O3, Cr2O3, and Fe2O3 as a function of screening parameter as also shown in Figure 4. 3.3. Band structure These transition metal oxides range from small band gap semiconductor to wide band gap semiconductor with both paramagnetic and anti-ferromagnetic ordering. sX was found to successfully describe all these systems. The experimental lattice constants are used for the primitive cells. Ti2O3 is a paramagnetic semiconductor with band gap of about 0.1eV. We noted above that sX with an LDA wavefunction will give a metallic band structure. The band gap does not Another problem for the LDA/GGA functional is over delocalisation of semi-core states such as the d and f electrons. For the sp semiconductors only the band gap is under-estimated but the band structure is qualitatively correct. However LDA/GGA fails qualitatively for localised, open-shell, d and f electron systems. The LDA band gap disappears in systems such as the Mott-Hubbard insulators Ti2O3. We previously found that sX gives good band gaps for several transition metal oxides[41, 42] and that hybrid functionals in general can treat the transition metal and lanthanides well[31, 43]. Here, we have calculated the band gap of Ti2O3, Cr2O3, and Fe2O3 as a function of screening parameter as also shown in Figure 4. These transition metal oxides range from small band gap semiconductor to wide band gap semiconductor with both paramagnetic and anti-ferromagnetic ordering. sX was found to successfully describe all these systems. The experimental lattice constants are used for the primitive cells. Ti2O3 is a paramagnetic semiconductor with band gap of about 0.1eV. We noted above that sX with an LDA wavefunction will give a metallic band structure. The band gap does not The eﬀects of screening length in the sX functional 10 open up until the screening parameter is less than 5˚A−1. Cr2O3 is an anti-ferromagnetic insulator, intermediate between a charge transfer and Mott-Hubbard insulator. Fe2O3 is an anti-ferromagnetic Mott-Hubbard insulator. We see here that TF screening can also give a correct band gap for these correlated d electron systems. Surface band structures might be quite diﬀerent from the bulk band structures. Quantum conﬁnement eﬀects are quite important in quasi-2D surface band structures. It has been claimed that the hybrid functionals could not give the correct prediction of 2D and 1D systems[50]. However we ﬁnd here that the sX functional with a similar TF screening can give a reasonable band gap of low-dimension systems. We previously found that sX will reproduce the Fermi velocity of graphene[40], which is under-estimated by LDA. 3.3. Band structure Here we calculate the Si(111)-(2 × 1) surface as an example of a 2D system. The Si(111)-(2 × 1) surface is a good test of a 2D system because of its well known atomic and electronic structure[56, 57, 58, 59, 60, 61]. Figure 5(a) shows that the (2×1) reconstruction causes the surface dangling bonds to form a π-bonded chain along the [011] direction. In our calculation, the same pseudopotential and screening parameter from the bulk Si is used for the surface calculation and the bulk band structures. The surface is constructed from a 16-layer Si slab with 20˚A of vacuum. Hydrogen is used to passivate the opposite side of the slab. The Brillouin zone is sampled with a 2×4×1 k-point mesh. This supercell is found to converge the energy to within 0.01 eV with respect to the slab thickness. In order to eliminate the eﬀects of geometry relaxation, the sX-relaxed structure with the bulk TF screening parameter is used during all calculations. The force is relaxed to less than 0.02eV/˚A. The surface atomic structure from sX is almost the same as the PBE-relaxed structure as long as the same bulk lattice constant is used. Figure 5(b) shows the surface band structure calculated in sX, compared to the surface states found by photoemission and inverse-photoemission[57], and also to the GW calculations of Northrup et al[61]. The band gap of the surface π-bonded chain is direct and at J. Most bulk semiconductors and insulators that we have discussed so far have small exciton binding energies, so that it is not necessary to distinguish between the optical gap and quasi-particle gaps. However, the exciton binding energy of the Si(111) surface is 0.28eV, much larger than the bulk binding energy of 0.015 eV[58]. Thus, the quasi-particle gap is signiﬁcantly diﬀerent from the optical gap. The calculated sX surface band gap is 0.45 eV. Interestingly, this is closer to the experimental optical band gap of 0.47 eV, but is 0.2 eV less than the experimental photoemission gap of 0.65 eV. This behaviour is unusual, but it is consistent with the observation of Scuseria et al[62] that the HSE band gap might be identiﬁed more with the optical gap. Figure 6 shows the band gaps of Si(111)-(2 × 1) as a function of sX screening parameter. The ﬁgure shows similar trends as other systems. 3.3. Band structure The error by TF screening with average electron density is less than 0.05eV. The slope of band gap versus screening parameter is relatively small around the experimental gap. In order to compare the screening eﬀects in 2D and 3D systems, we also consider the electronic structure and screening in bulk (3D) and monolayer (2D) MoS2[63, 64, 65, 66]. 11 The eﬀects of screening length in the sX functional Figure 5. (a) Geometric structure of Si(111)-(2 × 1) surface. (b) Band structure of the Si(111)-(2 × 1) surface from sX (black lines), comparing to band energies from photoemission (PE) and inverse photoemission (IPE) (triangle)[57], and GW quasi- particle energies (cross) from Northrup et al[61]. ( ) Figure 5. (a) Geometric structure of Si(111)-(2 × 1) surface. (b) Band structure of the Si(111)-(2 × 1) surface from sX (black lines), comparing to band energies from photoemission (PE) and inverse photoemission (IPE) (triangle)[57], and GW quasi- particle energies (cross) from Northrup et al[61]. ( ) Figure 5. (a) Geometric structure of Si(111)-(2 × 1) surface. (b) Band structure of the Si(111)-(2 × 1) surface from sX (black lines), comparing to band energies from photoemission (PE) and inverse photoemission (IPE) (triangle)[57], and GW quasi- particle energies (cross) from Northrup et al[61]. ( ) Figure 5. (a) Geometric structure of Si(111)-(2 × 1) surface. (b) Band structure of the Si(111)-(2 × 1) surface from sX (black lines), comparing to band energies from photoemission (PE) and inverse photoemission (IPE) (triangle)[57], and GW quasi- particle energies (cross) from Northrup et al[61]. ( ) particle energies (cross) from Northrup et al[61]. (a) (b) -2.0 -1.6 -1.2 -0.8 -0.4 0.0 0.4 0.8 1.2 1.6 2.0 Energy ,eV J J' PE/IPE GW ( ) (b) -2.0 -1.6 -1.2 -0.8 -0.4 0.0 0.4 0.8 1.2 1.6 2.0 Energy ,eV J J' PE/IPE GW J' The eﬀects of screening length in the sX functional 12 Figure 6. Variation of the calculated surface band gap of Si(111)-(2 × 1) with sX l h Figure 6. Variation of the calculated surface band gap of Si(111)-(2 × 1) with sX screening length. 0.01 0.1 1 10 Screening length, kTF (Å -1) 1.0 1.5 2.0 2.5 3.0 Band gap (eV) Figure 6. Variation of the calculated surface band gap of Si(111)-(2 × 1) with sX screening length. 3.3. Band structure 0.01 0.1 1 10 Screening length, kTF (Å -1) 1.0 1.5 2.0 2.5 3.0 Band gap (eV) Here, the exciton binding energy is small in bulk MoS2, whose band gap is 1.27 eV and indirect. In contrast, the exciton energy is large, of order 0.9 eV, in monolayer MoS2[63, 64]. The minimum optical gap of monolayer MoS2 is 1.88 eV and direct. The Figure 6. Variation of the calculated surface band gap of Si(111)-(2 × 1) with sX screening length Here, the exciton binding energy is small in bulk MoS2, whose band gap is 1.27 eV and indirect. In contrast, the exciton energy is large, of order 0.9 eV, in monolayer MoS2[63, 64]. The minimum optical gap of monolayer MoS2 is 1.88 eV and direct. The quasi-particle gap of monolayer and bulk MoS2 is taken to be 2.7 eV from refs [63, 64]. This is consistent again with comment of Scuseria et al [62] that the HSE band gap might be identiﬁed more with the optical gap, not the quasi particle gap. Our sX calculations of MoS2 use the same pseudopotential and calculation parameters as in our previous work[66]. Figure 7(a) shows that the variation of band gap with screening parameter for 3D and 2D MoS2. We see that the variation is the same when normalised to the optical gap (Figure7(b)). However, it is diﬀerent if it is normalised to the quasi-particle gap, where the eﬀect of low screening particularly aﬀects the 2D system. These results illustrate that low dimensional systems are quite useful to study the eﬀects of low screening, without going to wide gap systems. To summarise, the TF average density method gives quite good band structures for all the systems studied so far. The band gap varies slowly for screening parameters around the TF value, 1.8-2.5˚A−1. This should allow us to use an average screening parameter for mixed systems such as interfaces or surfaces. 3.4. Charge transition level The defect levels of semiconductors are an important application of hybrid functionals. The charge transition level is deﬁned as the energy where the two charge states of the defect have the same formation energy. First-principles calculations are a useful tool to determine defect properties. However, semi-local XC functional are unlikely to get transition levels correct, due to the band gap problem. Moreover, the electron delocalisation problem of semi-local DFT can lead to the incorrect ground state for some The eﬀects of screening length in the sX functional 13 Figure 7. Dependence with respect to screening length of (a) minimum band gaps of 2D (curved dashed line) and 3D (curved solid line) MoS2, and (b) normalised to the experimental optical gap. Experimental gaps are shown in (a) by the horizontal lines for 2D (dashed line, 1.88 eV) and 3D (solid line, 1.27 eV) materials and the quasiparticle gap also indicated (dot-dash line, 2.7 eV). Note in (b) the similar dependence of 2D and 3D when normalised to the optical gap (1.0 on the vertical axis). Both diagrams indicate that the same screening is suitable for both 2D and 3D materials. ( ) ( ) 0.01 0.1 1 10 Screening length, kTF (Å -1) 0 1 2 3 4 5 6 Band gap (eV) Monolayer sX MoS2 Bulk sX MoS2 Monolayer Expt Bulk Expt Quasiparticle Gap (b) 0.01 0.1 1 10 Screening length, kTF (Å -1) 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 Normalised band gap Bulk MoS2 Monolayer MoS2 ( ) 0.01 0.1 1 10 Screening length, kTF (Å -1) 0 1 2 3 4 5 6 Band gap (eV) Monolayer sX MoS2 Bulk sX MoS2 Monolayer Expt Bulk Expt Quasiparticle Gap Screening length, kTF (Å -1) TF (b) 0.01 0.1 1 10 Screening length, kTF (Å -1) 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 Normalised band gap Bulk MoS2 Monolayer MoS2 (b) 2 0.1 1 Screening length, kTF (Å -1) 0.1 1 Screening length, kTF (Å -1 The eﬀects of screening length in the sX functional 14 defects[67, 68, 69, 70]. The sX hybrid functional is known to correct these problems in several semiconductors and insulators[35, 39, 66, 71, 72]. HSE has been widely used in defect calculations. Here we investigate the eﬀects of the sX screening parameter on the charge transition levels for the As anti-site centre in GaAs. 3.4. Charge transition level There are not so many cases where the defect is fully identiﬁed experimentally and the transition level is known. The As anti-site or EL2 centre in GaAs has been selected for this study. It is responsible for the Fermi level pinning in the mid band gap. It has been conﬁrmed to give two transition states at 0.5eV (+ + /+) and 0.7eV (+/0) by experiments such as scanning tunneling spectroscopy, and electron paramagnetic resonance[72, 73]. Its atomic structure is also known from both experiments and DFT calculations[72, 73, 74, 75]. It is a rare case where both the atomic and electronic structure of the defect are known from experiments. We used a 64-atom cubic super cell with one As anti-site. A 2 × 2 × 2 MP grid is used for Brillouin zone integrations. The geometry is relaxed with the sX hybrid functional for each diﬀerent screening parameter. The formation enthalpy is calculated for the 0, +, and ++ states. The total energy of the defect cell and the perfect cell are calculated for each charge state. The defect formation energy is then calculated as, Hq (EF, µ) = [Eq −EH] + q (EV + ∆EF) + X α nα µ0 α + ∆µα (8) (8) where Eq is the defect cell energy, EH is the perfect cell energy, qEV is the change in energy of the Fermi level when charge q is added and nα is the number of atoms of species α. The charge correction process follows the procedure of our previous calculations[39]. Figure 8(a) plots the two defect transition levels as a function of the screening parameter. The diﬀerence between these two states remains constant at 0.1eV. As the HF part becomes larger, the transition level rises, just like the band gap. The sX hybrid functional gives a slightly higher transition level compared to experiment. However other hybrid functionals, such as HSE, also give higher transition levels for EL2[74, 75]. The error could be due to the small size of supercell and pseudopotentials without d electrons. However we note that the charge transition level is not shifted signiﬁcantly for screening parameters of 1.8-2.3˚A−1. Figure 8(b) plots the transition energies as a fraction of the calculated band gap. 4. Discussion We ﬁnd that the sX hybrid functional works well for such a wide range of materials. Its success is based on the following two facts. First the screened exchange term can give the correct band structure for these materials due to the introduction of the long-range screened HF terms. Compared to LDA+U or LDA+DMFT where the weak correlation is still based on LDA, the sX hybrid functional could give a better description for the simple sp semiconductor and the strong correlated systems such as transition metal oxides at the same time. Secondly, the TF screening parameter which determines the fraction of HF exchange has only a weak eﬀect on the band gap, around its optimum 15 The eﬀects of screening length in the sX functional 15 Figure 8. (a) Calculated sX band gap (solid line) and the 0/+ (dashed line) and +/ + + (dotted line) transition levels of the As anti-site in GaAs as a function of the sX screening parameter, in eV. (b) Transition levels (labels as in (a)) normalised to the GaAs band gap, as a function of screening length, kT F . Note the levels fall as a fraction of the total band gap towards to HF limit on the left. (a) 0.1 1 10 Screening length, kTF (Å -1) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 Band gap/Transition level (eV) sX band gap 0/+ transition +/++ transition (b) 0.1 1 10 Screening length, kTF (Å -1) 0.55 0.60 0.65 0.70 0.75 Normalised transition level 0/+ transition +/++ transition Figure 8. (a) Calculated sX band gap (solid line) and the 0/+ (dashed line) and +/ + + (dotted line) transition levels of the As anti-site in GaAs as a function of the sX screening parameter, in eV. (b) Transition levels (labels as in (a)) normalised to the GaAs band gap, as a function of screening length, kT F . Note the levels fall as a fraction of the total band gap towards to HF limit on the left. ( ) ( ) 0.1 1 10 Screening length, kTF (Å -1) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 Band gap/Transition level (eV) sX band gap 0/+ transition +/++ transition (b) 0.1 1 10 Screening length, kTF (Å -1) 0.55 0.60 0.65 0.70 0.75 Normalised transition level 0/+ transition +/++ transition The band gap changes slow near the correct value. 4. Discussion kTF is proportional to the one ower of the valence electron density, so the screening parameter is always in the f 1.8-2.5˚A−1. This slow dependence allows a ﬁxed value of screening parameter to d if desired, for example in heterogeneous situations such as surfaces or interfaces. ( ) 0.1 1 10 Screening length, kTF (Å -1) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 Band gap/Transition level (eV) sX band gap 0/+ transition +/++ transition sX band gap 0/+ transition +/++ transition Screening length, kTF (Å -1) Screening length, kTF (Å -1) (b) 0.1 1 10 Screening length, kTF (Å -1) 0.55 0.60 0.65 0.70 0.75 Normalised transition level 0/+ transition +/++ transition Screening length, kTF (Å -1) value. The band gap changes slow near the correct value. kTF is proportional to the one sixth power of the valence electron density, so the screening parameter is always in the range of 1.8-2.5˚A−1. This slow dependence allows a ﬁxed value of screening parameter to be used if desired, for example in heterogeneous situations such as surfaces or interfaces. value. The band gap changes slow near the correct value. kTF is proportional to the one sixth power of the valence electron density, so the screening parameter is always in the range of 1.8-2.5˚A−1. This slow dependence allows a ﬁxed value of screening parameter to be used if desired, for example in heterogeneous situations such as surfaces or interfaces. The eﬀects of screening length in the sX functional 16 5. Conclusions To summarise, we analysed the performance of the sX hybrid functional calculation of band structures in this work. The TF screening with average valence electron density is found to give good agreement with experiment. Due to the nature of screening and similar valence density in materials, the screening parameter always falls into a similar range of 1.8-2.5˚A−1. This TF screening parameter guarantees that the sX functional are reliable as in the simple systems. 6. Acknowledgements The authors would like the thank EPSRC for partial funding of this work via the HPC facility Hector under grant EP/K013718/1. This work also made use of the facilities of N8 HPC provided and funded by the N8 consortium and EPSRC grant EP/K000225/1. 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https://openalex.org/W2578122321	https://journals.openedition.org/brussels/pdf/1253	Dutch	null	Regionalisering van de woonbonus: naar een beleid op maat van het Brussels Gewest?	Brussels studies	2,015	cc-by	11,066	Brussels Studies La revue scientifique pour les recherches sur Bruxelles / Het wetenschappelijk tijdschrift voor onderzoek over Brussel / The Journal of Research on Brussels Collection générale \| 2015 Éditeur Université Saint-Louis Bruxelles Regionalisering van de woonbonus: naar een beleid op maat van het Brussels Gewest? La régionalisation du « bonus logement » : vers une politique adaptée au contexte bruxellois ? The regionalisation of the “housing bonus”: towards a policy adapted to the Brussels context? Nicolas Bernard et Valérie Lemaire Traducteur : Ben Winant Traducteur : Ben Winant Cet article est une traduction de : La régionalisation du « bonus logement » : vers une politique adaptée au contexte bruxellois ? - URL : https://journals.openedition.org/brussels/1251 [fr] Référence électronique q Nicolas Bernard en Valérie Lemaire, «Regionalisering van de woonbonus: naar een beleid op maat van het Brussels Gewest?», Brussels Studies [Online], Algemene collectie, nr 83, Online op 26 janvier 2015, geraadpleegd op 10 octobre 2024. URL: http://journals.openedition.org/brussels/1253 ; DOI: https:// doi.org/10.4000/brussels.1253 The text only may be used under licence CC BY 4.0. All other elements (illustrations, imported files) are “All rights reserved”, unless otherwise stated. Brussels Studies is gepubliceerd met steun van Innoviris (Brussels Instituut voor Onderzoek en Ontwikkeling) Brussels Studies is gepubliceerd met steun van Innoviris (Brussels Instituut voor Onderzoek en Ontwikkeling) Nicolas Bernard, +32(0)2 211 78 44, nicolas.bernard@usaintlouis.be Benjamin Wayens (Secrétaire de rédaction), +32(0)2 211 78 22, bwayens@brusselsstudies.be Regionalisering van de woonbonus: naar een beleid op maat van het Brussels Gewest? Nicolas Bernard en Valérie Lemaire Vertaling: Ben Winant Regionalisering van de woonbonus: naar een beleid op maat van het Brussels Gewest? Nicolas Bernard en Valérie Lemaire Vertaling: Ben Winant Vertaling: Ben Winant In het kader van de zesde staatshervorming is de federale bevoegdheid voor de ﬁscaliteit van de hypothecaire kredieten voor de « enige eigen woning” onlangs overgedragen aan de Gewesten. Het systeem werd doorgaans “woonbonus” genoemd en toegekend aan een zeer groot aan- tal belastingplichtigen. Het is evenwel vatbaar voor diverse kritische opmerkingen. Het belastingvoordeel, dat in totaal verschillende tienduizen- den euro’s per persoon bedraagt, wordt toegekend zonder enige inkomensvoorwaarde. Ook de meest welgestelden krijgen het voordeel, terwijl lf d d d j i d i In het kader van de zesde staatshervorming is de federale bevoegdheid voor de ﬁscaliteit van de hypothecaire kredieten voor de « enige eigen woning” onlangs overgedragen aan de Gewesten. Het systeem werd doorgaans “woonbonus” genoemd en toegekend aan een zeer groot aan- tal belastingplichtigen. Het is evenwel vatbaar voor diverse kritische opmerkingen. Het belastingvoordeel, dat in totaal verschillende tienduizen- den euro’s per persoon bedraagt, wordt toegekend zonder enige inkomensvoorwaarde. Ook de meest welgestelden krijgen het voordeel, terwijl ze zelfs zonder dat duwtje in de rug eigenaar zou- den zijn geworden. Tegelijkertijd maken de achter- gestelde gezinnen er amper gebruik van om de eenvoudige reden dat de prijzen op de vastgoed- markt voor hen onbetaalbaar zijn. De ﬁscale steun stimuleert de vraag en drijft de verkoopprijzen op, zodat uiteindelijk de verkoper en niet de koper het voordeel op zak steekt Tot slot discrimineert de Nicolas Bernard is hoogleraar in de rechten aan de Université Saint-Louis — Bruxelles. H lid van verschillende representatieve instanties (zoals de Adviesraad voor Huisvesting van he Hoofdstedelijk Gewest en de Conseil supérieur du logement de Wallonie). Hij werkte als d voor ministers van verschillende politieke partijen. Thans staat hij mede aan het hoofd van de recherches interdisciplinaires sur Bruxelles (IRIB). Hij is auteur van een groot aantal werk kels over huisvesting en publiceerde onlangs een boek getiteld La dé-fédéralisation du ba tion : quel(s) levier(s) pour les Régions ? (Larcier 2014) In het kader van de zesde staatshervorming is de fe woning” onlangs overgedragen aan de Gewesten. He tal belastingplichtigen. Het is evenwel vatbaar voor d den euro’s per persoon bedraagt, wordt toegekend z ze zelfs zonder dat duwtje in de rug eigenaar zou- den zijn geworden. www.brusselsstudies.be het elektronisch wetenschappelijk tijdschrift voor onderzoek over Brussel Regionalisering van de woonbonus: naar een beleid op maat van het Brussels Gewest? Nicolas Bernard en Valérie Lemaire Vertaling: Ben Winant Tegelijkertijd maken de achter- gestelde gezinnen er amper gebruik van om de eenvoudige reden dat de prijzen op de vastgoed- markt voor hen onbetaalbaar zijn. De ﬁscale steun stimuleert de vraag en drijft de verkoopprijzen op, zodat uiteindelijk de verkoper en niet de koper het voordeel op zak steekt. Tot slot discrimineert de woonbonus alleenstaanden tegenover koppels, want ze krijgen maar de helft van het voordeel. Voor de Gewesten is het belangrijk, vooral in een periode van budgettaire besparingen, om de woonbonus te hervormen en onder meer om te vormen tot een socialer instrument. In de toekomst zou de woonbonus ook kunnen worden gekoppeld aan eisen op het vlak van gezondheid of energie- prestaties van de woning of worden gereserveerd voor degenen die voor de eerste keer een woning kopen... Nicolas Bernard is hoogleraar in de rechten aan de Université Saint-Louis — Bruxelles. Hij is of was lid van verschillende representatieve instanties (zoals de Adviesraad voor Huisvesting van het Brussels Hoofdstedelijk Gewest en de Conseil supérieur du logement de Wallonie). Hij werkte als deskundige voor ministers van verschillende politieke partijen. Thans staat hij mede aan het hoofd van het Institut de recherches interdisciplinaires sur Bruxelles (IRIB). Hij is auteur van een groot aantal werken en arti- kels over huisvesting en publiceerde onlangs een boek getiteld La dé-fédéralisation du bail d'habita- tion : quel(s) levier(s) pour les Régions ? (Larcier, 2014). Als juriste en voormalig onderzoeker aan de Université Saint-Louis — Bruxelles heeft Valérie Lemai- re verschillende publicaties over huisvesting op haar naam, onder meer over gemeenschappelijk wo- nen (solidair wonen) (Jurimpratique 2013/3). Thans is ze directeur van het sociaal verhuurkantoor van Schaarbeek. Nicolas Bernard, +32(0)2 211 78 44, nicolas.bernard@usaintlouis.be Benjamin Wayens (Secrétaire de rédaction), +32(0)2 211 78 22, bwayens@brusselsstudies.be Nicolas BERNARD, Valérie LEMAIRE, Regionalisering van de woonbonus: naar een beleid op maat van het Brussels Gewest?, Brussels Studies, Nummer 83, 26 januari 2015, www.brusselsstudies.be 1. Kritiek 1.1. de steun komt voornamelijk ten goede aan degenen die er geen behoefte aan hebben… 1. Sinds 1 januari 20051 mag degene die een “enige eigen” woning koopt (in die zin dat de koper de woning zelf moet betrekken en geen andere woning – in eigendom – mag hebben) en de aankoop ﬁnanciert met een hypothecair krediet dat een looptijd van minstens 10 jaar heeft, in zijn belastingaangifte de interesten, kapitaalaﬂossingen en andere verzekeringspremies die hij voor zijn lening betaalt2, aftrekken van al zijn inkomsten tijdens de hele looptijd van het krediet. Deze regeling wordt doorgaans “woonbonus” genoemd en is in de plaats gekomen van een ander belastingaftreksysteem – het “woonsparen” – dat er in grote lij- nen op lijkt (maar iets minder voordelig is voor de belastingplichtige). 1 Programmawet van 27 december 2004, B.S., 31 december 2004, err. 18 januari 2005. 2 Voor het aanslagjaar 2014 bedraagt de aftrek maximum 2.260 euro, verhoogd met 750 euro tijdens de eerste tien belastbare perioden en met 80 euro wanneer de belastingplichtige drie kinderen ten laste heeft op 1 januari van het jaar dat volgt op het jaar waarin de hypothecaire lening werd gesloten. 3 Art. 5/5, § 4 en 81quater van de bijzondere wet van 16 januari 1989 met betrekking tot de ﬁnanciering van de Gemeenschappen en Gewesten, B.S., 17 januari 1989, ingevoegd bij de artikelen 11 en 75 van de bijzondere wet van 6 januari 2014 houdende hervorming van de ﬁnanciering van de Gemeenschappen en Gewesten, uitbreiding van de ﬁscale autonomie van de Gewesten en ﬁnanciering van de nieuwe bevoegdheden, B.S., 31 januari 2014. 1.2. … maar blijft ontoegankelijk voor degenen die er wel nood aan hebben restvoeten, worden gewezen. In ieder geval is volgens de recentste statistieken het percentage eigenaars in het Brussels Gewest – dat is een primeur – lichtjes gedaald (39 % in 2011 tegenover 43 % tien jaar geleden)5. Dat is een extra bewijs dat de woonbonus niet efﬁciënt ge- noeg is, ondanks de hoge kosten voor de overheid. 5. Het mattheuseffect (het overheidsgeld gaat naar degenen die so- wieso de stap zouden hebben gezet, ongeacht eventuele overheids- steun) zou geen al te groot probleem zijn als het voordeel evenwichtig verdeeld zou zijn over de verschillende sociale klassen. Dat is helemaal niet het geval: hoe hoger het inkomen, hoe intensiever het instrument wordt gebruikt. Niet minder dan 50 % van de beschikbare middelen wordt aldus ingepalmd door de twee hoogste inkomensdecielen4, ter- wijl de eerste vijf decielen er slechts 15 % van gebruiken. De achterge- stelde gezinnen maken dus in zeer geringe mate gebruik van het sys- teem (amper 1 % voor het eerste inkomensdeciel tegenover 58 % voor het hoogste inkomensdeciel) [Valenduc, 2008]… Daar is een reden voor: de inﬂatie van de vastgoedprijzen, vooral in Brussel! Aangezien de wetgever (met de goedkeuring van de woonbonus) de bedoeling had ervoor te zorgen dat iedereen een eigendom kan verwerven, kan de efﬁciëntie van het systeem in twijfel worden getrokken, aangezien hele bevolkingsgroepen er helemaal niet in slagen een eigendom te verwer- ven. Als we er niet meer dan een lichte karikatuur van maken, zouden we kunnen stellen dat de maatregel veeleer dient om het onroerend goed te onderhouden dan om het aan te kopen. Degene die niet de middelen heeft om het belastingvoordeel (dat pas twee jaar later wordt uitgekeerd) te “preﬁnancieren”, heeft het zeer moeilijk om eigenaar te worden. 4 Het laatste deciel (van de tien) omvat de 10 % hoogste belastingaangiften en het eerste deciel de 10 % laagste belastingaangiften. 5 Vergelijking van de telling 2001 met de Census 2011, uitgevoerd door de FOD Economie. 1.1. de steun komt voornamelijk ten goede aan degenen die er geen behoefte aan hebben… 4. Ten eerste wordt het belastingvoordeel toegekend zonder inko- mensvoorwaarde; de rijksten hebben er recht op, net als de armsten. Deze universaliserende benadering is ongetwijfeld vrijgevig, maar brengt het traditionele mattheuseffect teweeg: de maatregel komt vooral ten goede aan gezinnen die er niet echt behoefte aan hebben en die hun onroerend goed zeer waarschijnlijk ook zouden hebben ge- kocht – en eigenaar zouden zijn geworden – zonder dat duwtje in de rug door de overheid. Voor mensen met een middelgroot of groot in- komen is de overheidssteun helemaal niet of nauwelijks doorslagge- vend bij de aankoop. Een bewijs daarvan is trouwens het feit dat de betrokkene het geldelijk voordeel pas ongeveer twee jaar na de trans- actie krijgt, aangezien de belastingen met zo'n tijdverschil worden te- rugbetaald. Een bijkomend bewijs (van het feit dat het belastingvoor- deel niet meespeelt wanneer een eventuele aankoop wordt besproken) is het feit dat de banken zelf geen rekening houden met de woonbonus om de terugbetalingscapaciteit - en de solvabiliteit – van een kandi- daat-lener te beoordelen. 2. In de praktijk is het ﬁnanciële voordeel van de woonbonus vrij aan- zienlijk voor de belastingplichtige. De steun bedraagt gemiddeld 1.348 euro per jaar in Brussel (tegenover 1.395 euro in Vlaanderen en 1.310 euro in Wallonië) en wordt elk aanslagjaar toegekend zolang de lening wordt terugbetaald. Aangezien een hypothecair krediet gemid- deld een looptijd van 20 jaar heeft, moet het belastingvoordeel worden vermenigvuldigd met 20 (waardoor het in totaal groter is dan 25.000 euro of 50.000 euro voor een koppel). 3. Er zij onmiddellijk op gewezen dat de woonbonus ontegenzegge- lijk tot discussies leidt om een groot aantal redenen die hieronder wor- den toegelicht. Op 1 juli 2014 zijn de Gewesten in het kader van de zesde staatshervorming3 bevoegd geworden voor die aangelegenheid, die tot dan onder de federale overheid ressorteerde. Dat is een regel- rechte revolutie. Dit artikel strekt er bijgevolg toe enkele voorstellen te formuleren (die onder meer uit onze kritische opmerkingen doorsche- meren) voor de heroriëntering van een ﬁscaal instrument dat erg in trek is bij de bevolking, maar tegelijk zeer omstreden is. 2 Nicolas BERNARD, Valérie LEMAIRE, Regionalisering van de woonbonus: naar een beleid op maat van het Brussels Gewest?, Brussels Studies, Nummer 83, 26 januari 2015, www.brusselsstudies.be Nicolas BERNARD, Valérie LEMAIRE, Regionalisering van de woonbonus: naar een beleid op maat van het Brussels Gewest?, Brussels Studies, Nummer 83, 26 januari 2015, www.brusselsstudies.be 1.4. de steun benadeelt ongehuwden 11. Qua sociale rechtvaardigheid is dat kenmerk van de woonbonus op zijn minst omstreden. De woonbonus is dus niet erg efﬁciënt, maar bovendien niet erg billijk. Kortom, het feit dat het systeem de facto be- stemd is voor de meest welgestelde gezinnen moet de overheden tot nadenken stemmen: is dat een optimaal gebruik van het geld van de gemeenschap? 9. Een ander kenmerk van de woonbonus is dat het belastingvoor- deel groter wordt als het ten goede komt aan elk van de twee leden van het gezin. Dan rijst een (terechte) vraag over de gelijkheid van be- handeling in zoverre de federale ﬁscale steun groter blijkt te zijn wan- neer het gezin dat de lening heeft gesloten, uit twee in plaats van één persoon bestaat, ook al zouden het gekochte pand en de gesloten le- ning volledig dezelfde zijn. Heeft een alleenstaande meestal niet minder middelen dan een koppel? Is de ﬁnanciële inspanning die hij(zij) moet leveren om een woning te verwerven dan verhoudingsgewijs niet gro- ter6? Zou het bijgevolg niet opportuner zijn om de steun voor alleen- staanden te verhogen in plaats van het omgekeerde te doen? Er zij in elk geval op gewezen dat, in tegenstelling tot wat de meeste mensen denken, vrij veel alleenstaanden een hypothecair krediet sluiten (bijna een derde van de leningen7), wat een juist beeld van de kwestie geeft. 1.3. de steun is groter voor de hoge inkomens (wat duidelijk in strijd is met het principe van de progressiviteit van de belasting) 3 Nicolas BERNARD, Valérie LEMAIRE, Regionalisering van de woonbonus: naar een beleid op maat van het Brussels Gewest?, Brussels Studies, Nummer 83, 26 januari 2015, www.brusselsstudies.be Nicolas BERNARD, Valérie LEMAIRE, Regionalisering van de woonbonus: naar een beleid op maat van het Brussels Gewest?, Brussels Studies, Nummer 83, 26 januari 2015, www.brusselsstudies.be 11. Qua sociale rechtvaardigheid is dat kenmerk van de woonbonus op zijn minst omstreden. De woonbonus is dus niet erg efﬁciënt, maar bovendien niet erg billijk. Kortom, het feit dat het systeem de facto be- stemd is voor de meest welgestelde gezinnen moet de overheden tot nadenken stemmen: is dat een optimaal gebruik van het geld van de gemeenschap? 1.6. de steun heeft als averechts (en zeer contraproductief) ge- volg dat de vastgoedprijzen worden opgedreven, wat vooral in het voordeel van de verkopers speelt 12. 9. Het is een ijzeren wet van de markteconomie dat de prijs van het aanbod wordt bepaald door de vraag. Degene die een woning te koop stelt, is meestal op de hoogte van het belastingvoordeel dat de kandidaat-koper zal krijgen. De verkoper zou bijgevolg geneigd kunnen zijn om dat voordeel op voorhand te verrekenen in de vraagprijs; de koper (die het belastingvoordeel pas twee jaar later krijgt) is zich daar- van niet altijd bewust, maar de tegenpartij lijkt daarvan beter op de hoogte te zijn. Men kan zich bijgevolg terecht de vraag stellen of de woonbonus als (nieuw) (averechts) gevolg heeft dat de vastgoedprijzen worden opgedreven. Die vraag is helemaal niet controversieel en kwam aan bod in een wetenschappelijke studie [Huyghebaert, 2012], die erin geslaagd is de invloed van de woonbonus onder de traditionele inﬂatoi- re factoren (zoals de demograﬁsche explosie) te isoleren. De studie be- sluit dat de extra koopkracht inderdaad verrekend is in de prijzen [zie in dezelfde zin, Albrecht en Van Hoofstat, 2012]. Dat gebeurde misschien niet in de jaren onmiddellijk na de invoering van de woonbonus, maar de vastgoedverkopers hebben zich snel aangepast en rekening gehou- den met dat gegeven. Die vaststelling wordt ofﬁcieel bevestigd door de Vlaamse Woonraad, die van oordeel is dat de woonbonus, een “extra inkomen”, geleid heeft tot een stijging van de prijzen “in zoverre de vraag toeneemt, terwijl het woningaanbod niet in evenredige mate stijgt” (en de woningmarkt in België in het algemeen “weinig ﬂexibel” 6 De maximale aftrek voor een koppel met drie kinderen ten laste bedraagt 6.180 euro (aanslagjaar 2014). Dat bedrag wordt gehalveerd (3.090 euro) voor een eenoudergezin dat eveneens drie kinderen ten laste heeft. 7 Precies 29 % [Huyghebaert, 2012]. 8 Zie supra voetnoot nr. 2. 6 De maximale aftrek voor een koppel met drie kinderen ten laste bedraagt 6.180 euro (aanslagjaar 2014). Dat bedrag wordt gehalveerd (3.090 euro) voor een eenoudergezin dat eveneens aar 2014). Dat bedrag wordt gehalveerd (3.090 euro) voor een eenoudergezin dat eveneens 1.3. de steun is groter voor de hoge inkomens (wat duidelijk in strijd is met het principe van de progressiviteit van de belasting) 7. Het is niet alleen zo dat de meest welgestelden – veruit – het meest een beroep doen op de woonbonus, maar op de koop toe moet de overheid voor hen meer uitgeven. Waarom? Gewoon omdat de be- lastingaftrek gebeurt tegen de marginale aanslagvoet. Enige uitleg is hier op zijn plaats. De aftrek gebeurt vóór de berekening van de belas- ting en bestaat erin dat de belastingplichtige bepaalde ﬁscaal aftrekbare uitgaven van zijn belastbaar bedrag aftrekt. De aftrek gebeurt op de hoogste inkomensschijf van de belastingplichtige, dus op de schijf die in een progressief belastingsysteem zoals het onze onderworpen is aan de hoogste aanslagvoet (met een maximum van 50 % – aangezien de lagere inkomensschijven worden belast tegen 45 %, 40 %, 30 % en 25 %). Bijgevolg is die werkwijze voordelig voor belastingplichtigen met hoge inkomens, aangezien hun belastingbesparing groter is dan die van belastingplichtigen met een bescheiden inkomen, ook al zou het- zelfde bedrag worden afgetrokken. Vanuit het standpunt van de Staat zou de belasting op het bedrag dat aldus van de inkomens wordt afge- trokken, proportioneel gezien veel hebben opgebracht (aangezien de PB-voet stijgt in verhouding tot de ﬁnanciële middelen) en in ieder geval meer dan als de belastingplichtige een lager inkomen had gehad. 6. Er zij tot slot op gewezen dat het percentage eigenaars in België in enkele decennia toch gestegen is. De Vlaamse Woonraad zelf verklaart evenwel in zijn ofﬁcieel advies over de regionalisering van de woonbo- nus dat hij “geen enkele zekerheid” heeft over een correlatie tussen die evolutie en de invoering van ﬁscale stimuli ten gunste van de eigenaars. De Woonraad baseert zich daarvoor zelfs op internationale studies die bevestigen dat er “geen enkel verband” kan worden aangetoond [Vlaamse Woonraad, 2012]. Er moet veeleer op andere factoren, zoals de verbetering van de levensstandaard en de forse daling van de inte- 8. Vanuit dat oogpunt kan de aftrek onbetwistbaar als “regressief” worden beschouwd, waarbij het belastingvoordeel toeneemt in verhou- ding tot de rijkdom van de belastingplichtige. Ons belastingsysteem wordt geacht progressief te zijn en verbiedt in theorie dat een belas- tingplichtige met een hoog inkomen een ﬁscale stimulans krijgt die pro- portioneel interessanter is dan de ﬁscale stimulans die een minder wel- gestelde burger kan krijgen. 1.7. de steun weegt zeer zwaar op de overheidsbegroting 15. De woonbonus wordt toegekend zonder inkomensvoorwaarde (maxima bijvoorbeeld). Het ontbreken van sociale selectiviteit – en varia- tie van het maximum van de aftrekbare uitgaven – heeft natuurlijk een prijskaartje, dat hoog oploopt. In 2011 was de woonbonus 2005 op zich goed voor een inkomstenverlies voor de ﬁscus van 1,227 miljard euro voor het hele land, verdeeld als volgt: 828 miljoen euro voor Vlaanderen, 330 miljoen euro voor Wallonië en 69 miljoen euro voor Brussel9. Als de (talrijke) uitgaven die nog heel wat gezinnen krachtens de ﬁscale regeling van vóór 2005 kunnen aftrekken, daaraan worden toegevoegd, bedragen de totale kosten bijna 2,2 miljard … elk jaar! Dat is drie keer meer dan de begrotingen van alle Gewesten die speciﬁek bestemd zijn voor huisvesting [Monnier en Zimmer, 2008]! 13. Met andere woorden, de woonbonus ondersteunt de vraag. Ho- gerop in dit artikel werd gesteld dat de woonbonus niet voldoende sti- mulerend – en bekend was – om achtergestelde gezinnen ervan te overtuigen “de sprong te wagen” (en een woning te kopen), al was het maar omdat ze de steun moeten preﬁnancieren, wat ze ﬁnancieel niet kunnen. Voor de zogenaamde middenklasse, die niet ongevoelig is voor dat duwtje in de rug door de Staat (en misschien beter geïnfor- meerd is), kan er in zekere mate een aanstekelijkheidseffect worden vastgesteld. De onrust die zich heeft meester gemaakt van een deel van de burgers naar aanleiding van de plannen om de woonbonus (in minder gunstige zin) te hervormen, komt tot uiting in de forse toename van het aantal aanvragen voor een hypothecair krediet in september 2014 (+ 85 % ten opzichte van september 2013, aldus Febelﬁn). In de praktijk haasten de mensen zich om te kopen zolang er enigszins nog tijd is, wat natuurlijk de prijzen de hoogte injaagt (+ 13 % in dezelfde periode). 16. Laten we dieper ingaan op het veelbetekenende budgettaire as- pect. In feite is de federale woonbonus helemaal niet de enige steun die aan de eigenaars wordt toegekend. 1.5. de steun is niet erg billijk 10. Om de aftrek tegen de marginale aanslagvoet enigszins te com- penseren (waarbij de rijkeren worden bevoordeeld, zoals hierboven werd vermeld), had men ervoor kunnen zorgen dat het maximumbe- drag van de uitgaven die van de inkomsten kunnen worden afgetrok- ken, er omgekeerd evenredig mee daalt; met andere woorden, een welgesteld gezin zou niet hetzelfde bedrag mogen aftrekken als een gezin met een kleiner inkomen. Dat is echter helemaal niet het geval, aangezien het maximumbedrag gelijk is voor iedereen, ongeacht de middelen van de betrokkenen8. Ook vanuit dat standpunt krijgt de be- lastingplichtige die er minder rooskleurig voorstaat, ﬁscaal gezien niet meer dan de anderen. Dan hebben we het nog niet over degenen die te arm zijn en geen belasting betalen en daardoor de facto het ﬁnan- cieel voordeel niet kunnen krijgen (… gesteld natuurlijk dat zij erin ge- slaagd zouden zijn een bank ervan te overtuigen hun geld voor te schieten om een woning te kopen). Nicolas BERNARD, Valérie LEMAIRE, Regionalisering van de woonbonus: naar een beleid op maat van het Brussels Gewest?, Brussels Studies, Nummer 83, 26 januari 2015, www.brusselsstudies.be Nicolas BERNARD, Valérie LEMAIRE, blijkt te zijn) [Vlaamse Woonraad, 2012]. In vergelijking met de andere Europese landen is het tempo waarin het woningenbestand in België toeneemt, “ongelooﬂijk laag sinds 1980” [OESO, 2013]. Het onthutsen- de gevolg daarvan is dat in feite vooral de verkopers de ﬁscale steun op zak steken. de woonbonus uiteindelijk op zak steekt. Ook vanuit dat standpunt is de woonbonus inefﬁciënt en zelfs contraproductief. 9 Eind 2013 bedroeg het bedrag voor Brussel al 101 miljoen. 10 Art. 46bis van het Wetboek van registratie-, hypotheek- en grifﬁerechten, ingevoegd bij artikel 2 van de ordonnantie van de Brusselse Hoofdstedelijke Raad van 20 december 2002, B.S., 31 december 2002. 11 Art. 2, 1°, van de ordonnantie van het Brussels Hoofdstedelijk Parlement van 10 februari 2006, B.S., 15 februari 2006. 11 Art. 2, 1°, van de ordonnantie van het Brussels Hoofdstedelijk Parlement van 10 februari 2006, B.S., 15 februari 2006. 9 Eind 2013 bedroeg het bedrag voor Brussel al 101 miljoen. 1.7. de steun weegt zeer zwaar op de overheidsbegroting Het Brussels Gewest bijvoorbeeld heeft een reeks steunmaatregelen ingevoerd om de verwerving van eigendom te bevorderen: leningen bij het Woningfonds tegen een voor- keurtarief (gemiddeld 2 % lager dan het tarief van de klassieke banken), productie van woningen die door Citydev (de voormalige GOMB) ge- subsidieerd worden en bestemd zijn voor gezinnen met een middenin- komen (die dan nog maar ongeveer twee derde van de verkoopwaarde van het pand moeten betalen), gedeeltelijke vrijstelling van registratie- rechten (abattement)10 (dit voordeel werd achteraf nog uitgebreid11), 14. Kortom, het ﬁnanciële voordeel dat verondersteld wordt ten goede te komen aan de koper, blijkt onbestaand, aangezien het (volledig) “op- geslokt” wordt door de meerprijs. Het belastingvoordeel bestaat dus niet of komt, anders gezegd, niet ten goede aan degene voor wie het oorspronkelijk is bestemd, aangezien niet de koper, maar de verkoper 5 Nicolas BERNARD, Valérie LEMAIRE, Regionalisering van de woonbonus: naar een beleid op maat van het Brussels Gewest?, Brussels Studies, Nummer 83, 26 januari 2015, www.brusselsstudies.be Nicolas BERNARD, Valérie LEMAIRE, Regionalisering van de woonbonus: naar een beleid op maat van het Brussels Gewest?, Brussels Studies, Nummer 83, 26 januari 2015, www.brusselsstudies.be vrijstelling van successierechten op de gezinswoning voor de langstle- vende echtgenoot (daardoor hoeft het pand niet te worden verkocht om de successierechten te kunnen betalen)12, ﬁnanciering van “com- munity land trusts”, enz. Dan wordt nog geen rekening gehouden met het feit dat de koper van een door Citydev gesubsidieerde woning sinds kort een verlaagd btwtarief geniet (6 %)13. 18. Bovendien kunnen die steunmaatregelen voor de eigenaars meestal gecumuleerd worden. Het volgende voorbeeld spreekt in dat verband boekdelen [Mathieu, 2014]. Een gezin (met bijvoorbeeld een jaarlijks inkomen van 42.000 euro) dat een door Citydev gesubsidieerd appartement met 3 slaapkamers voor 260.000 euro (met verlaagde btw) koopt, krijgt een lening op 30 jaar tegen een voorkeurtarief van het Woningfonds en ﬁscale steun van de federale overheid, waarbij het to- taalbedrag van de overheidssteun oploopt tot 240.240 euro15! Dat is bijna evenveel als het startbedrag. 17. In totaal (federale ﬁscale aftrek en gewestelijke steunmaatregelen samen) wordt bijna 219 miljoen euro bestemd voor de Brusselse eige- naars. In vergelijking met de – lage – bedragen voor de steun aan de huurders (op de Brusselse privémarkt) stemt dat bedrag tot nadenken. 12 Ordonnantie van 30 januari 2014 tot wijziging van het Wetboek van successierechten, B.S., 6 maart 2014. 13 Art. XXXVI van bijlage A van het koninklijk besluit van 20 juli 1970 tot vaststelling van de tarieven van de belasting over de toegevoegde waarde en tot indeling van de goederen en dien- sten bij die tarieven, B.S., 31 juli 1970 zoals gewijzigd bij artikel 3 van het koninklijk besluit van 21 december 2013, B.S., 31 december 2013. 14 Deze sector geeft toch onderdak aan 38.000 gezinnen, maar is slechts goed voor 8 % van de Brusselse woningen. Als daar de overige woningen onder openbaar beheer of voor sociale doeleinden (zoals de sociale verhuurkantoren) worden bijgeteld, palmt die sector 78,2 % van de Brusselse begroting 2014 voor huisvesting in, zijnde 202 miljoen euro. 15 Namelijk 163.000 euro voor de Citydev-subsidie (de woning heeft de overheid in werkelijkheid 423.000 euro gekost, maar, zoals reeds uitgelegd, betaalt de koper er slecht twee derde van), 34.800 euro voor de btw-verlaging, 77.230 euro aan besparingen op de interesten dankzij de lening van het Woningfonds en 49.480 euro aan federale ﬁscale aftrek. 1.7. de steun weegt zeer zwaar op de overheidsbegroting Afgezien van de sociale huisvesting14, trekt het Brussels Gewest aldus minder dan 12 miljoen euro uit voor de begunstigden (voornamelijk via de “verhuis-, installatie- en huurtoelagen” die bestemd zijn om de extra kosten ten gevolge van de verhuizing van een ongezonde naar een ge- zonde woning te compenseren en de huurtoelagen die dienen om het aandeel van de huurprijs in het gezinsbudget, los van verhuizingen, te doen dalen). Dat bedrag is goed voor amper 5 % van de uitgaven die in totaal worden gedaan voor de eigenaars. De lage ﬁnanciële steun van overheidswege is al opmerkelijk op zich en lijkt niet afgestemd te zijn op de realiteit, aangezien – laten we dat niet vergeten – de huurders de meerderheid in het Brussels Hoofdstedelijk Gewest vormen (44,75 % tegenover 41,45 % eigenaars en 13,8 % huurders van een woning on- der openbaar beheer of voor sociale doeleinden). 19. Er zij nog gewezen op een laatste paradox. Ondanks het zeer ho- ge prijskaartje, werd de woonbonus tot nu toe weinig besproken en nog minder ter discussie gesteld. Waarom? Precies omdat het gaat om een belastingaftrek die als dusdanig niet erg duidelijk of ﬁnancieel ob- jectiveerbaar is voor de burger. Het is inderdaad zo dat het inkomsten- verlies voor de Staat in het algemeen moeilijker te bevatten is dan een bruto-uitgave (een premie bijvoorbeeld). Voorts hoeft de reden waarom het belastingvoordeel zo goed ligt bij de politieke klasse niet verder ge- zocht te worden; het procedé is inderdaad gemakkelijk (maar niet pijn- loos voor de overheidsﬁnanciën …). 0 tot vaststelling van de tarieven van de belasting over de toegevoegde waarde en tot indeling van de goederen en dien- an het koninklijk besluit van 21 december 2013, B.S., 31 december 2013. 00 euro voor de Citydev-subsidie (de woning heeft de overheid in werkelijkheid 423.000 euro gekost, maar, zoals reeds uitgelegd, betaalt de koper er slecht twee derde voor de btw-verlaging, 77.230 euro aan besparingen op de interesten dankzij de lening van het Woningfonds en 49.480 euro aan federale ﬁscale aftrek. r is slechts goed voor 8 % van de Brusselse woningen. Als daar de overige woningen onder openbaar beheer of voor socia , palmt die sector 78,2 % van de Brusselse begroting 2014 voor huisvesting in, zijnde 202 miljoen euro. 1.9. men kan zoveel als men wil proﬁteren van de steun (die bij- gevolg niet voorbehouden is voor de “eerstekeerkopers”) dan niet toe aangemoedigd schulden te maken? Is het systeem, op de keper beschouwd, vooral niet in het voordeel van de banken en andere kredietinstellingen? Het uitstaand bedrag aan hypothecaire leningen is zeer groot in België, aangezien het eind 2013 tot 188 miljard euro op- liep (en het is trouwens verdrievoudigd in de laatste veertien jaar). Van de 28 lidstaten van de Europese Unie hebben trouwens slechts vijf lan- den een hogere hypothecaire schuld per inwoner dan België. Daarbij komt nog dat de huidige regeling van de ﬁscale steunmaatregelen niet beperkt is in de tijd (een hypothecaire lening kan een looptijd van 20 jaar, 30 jaar en zelfs meer hebben en de overheidssteun zal heel die periode lang uitgekeerd blijven worden) en de kopers aanspoort om schulden op lange termijn aan te gaan, wat een bijkomend nadelig ge- volg heeft: de prijzen worden nog meer opgedreven [Bigot en Hoibian, 2009]. 22. Indien de woonbonus behouden zou blijven, zou het opportuun kunnen zijn om hem te reserveren voor de “eerstekeerkopers” om on- der meer de kritiek te pareren dat de steun ten goede komt aan dege- nen die er geen behoefte aan hebben. Het is immers van cruciaal be- lang om degenen die een pand kopen, een duwtje in de rug te geven, vooral wanneer ze jong zijn. Een gevolg daarvan zou zijn dat het belas- tingvoordeel slechts één keer zou worden toegekend. Dat is thans niet het geval: op voorwaarde dat het eerste pand vooraf werd doorver- kocht, kan een individu bij zijn volgende aankoop opnieuw het belas- tingvoordeel krijgen enzovoort. De mogelijkheid om meerdere keren overheidssteun te krijgen bij de verwerving van eigendom bestaat eveneens op het vlak van de registratierechten: om een abattement te krijgen, mag de koper op de datum van de verkoopovereenkomst geen ander onroerend goed dat geheel of gedeeltelijk voor bewoning is be- stemd, volledig in volle eigendom hebben. Niets belet echter dat hij overgaat tot opeenvolgende verkopen en aankopen18. 21. De Europese Commissie heeft zich al kritisch uitgelaten over het Belgische beleid ter zake, aangezien ze van oordeel is dat de vrij gun- stige ﬁscale behandeling van de hypothecaire leningen een van de fac- toren is die bijdragen tot zowel de schuldgraad van de gezinnen en de overinvestering in vastgoed (wat tot een vastgoedzeepbel kan leiden) [European Commission, 2012]. 1.8. zet de steun aan om schulden te maken? 20. Er moet nog een andere vraag gesteld worden: als het belasting- voordeel wordt voorbehouden voor degenen die een hypothecaire le- ning sluiten om hun woning te kopen, wordt de belastingplichtige er 6 Nicolas BERNARD, Valérie LEMAIRE, naar een beleid op maat van het Brussels Gewest?, Brussels Studies, Nummer 83, 26 januari 2015, www.brusselsstudies.be 16 In Frankrijk bijvoorbeeld zijn de interesten van hypothecaire leningen nog enkel aftrekbaar indien de lening is gesloten vóór 1 januari 2011. Ierland werkt aan een geleidelijke afschafﬁng van de aftrekbaarheid van de interesten, die niet meer zal bestaan in 2017; ook Portugal heeft een geleidelijk uitdovingsscenario. Finland heeft beslist om het aandeel van de aftrekbare hypothecaire interesten te verminderen van 85 % in 2012 tot 80 % in 2013 en tot 75 % in 2014. Spanje, tot slot, heeft (na verschillende opeenvolgende hervormingen) de aftrekbaarheid van de leningen die vanaf 1 januari 2013 zijn gesloten, volledig afgeschaft. 16 In Frankrijk bijvoorbeeld zijn de interesten van hypothecaire leningen nog enkel aftrekbaar indien de lening is gesloten vóór 1 januari 2011. Ierland werkt aan een geleidelijke afschafﬁng van de aftrekbaarheid van de interesten, die niet meer zal bestaan in 2017; ook Portugal heeft een geleidelijk uitdovingsscenario. Finland heeft beslist om het aandeel van de aftrekbare hypothecaire interesten te verminderen van 85 % in 2012 tot 80 % in 2013 en tot 75 % in 2014. Spanje, tot slot, heeft (na verschillende opeenvolgende hervormingen) de aftrekbaarheid van de leningen die vanaf 1 januari 2013 zijn gesloten, volledig afgeschaft. 17Duitsland, Cyprus, Malta, Oostenrijk, Slowakije, Slovenië, Litouwen, Letland, Hongarije, Polen, Roemenië en het Verenigd Koninkrijk. 18 Artikel 46bis van het Wetboek van registratie hypotheek en grifﬁerechten 17Duitsland, Cyprus, Malta, Oostenrijk, Slowakije, Slovenië, Litouwen, Letland, Hongarije, Polen, Roemenië en het Verenigd Koninkrijk. 18 Artikel 46bis van het Wetboek van registratie-, hypotheek- en grifﬁerechten. 1.9. men kan zoveel als men wil proﬁteren van de steun (die bij- gevolg niet voorbehouden is voor de “eerstekeerkopers”) Sommige van onze buurlanden hebben precies hun wetgeving onlangs gewijzigd om de stimulans (soms in aanzienlijke mate) terug te schroeven en die soms gewoonweg afge- schaft16. Dan spreken we niet van de (vele) landen die geen ﬁscale voordelen bieden om de verwerving van de eigendom te bevorderen17. 23. België heeft, in tegenstelling tot de meerderheid van zijn buurlan- den, beslist om geen belasting te heffen op de meerwaarden uit de verkoop van een bebouwd onroerend goed, indien het om de hoofd- verblijfplaats van de verkoper gaat of het pand bij wege van erfenis werd verworven (en, zelfs buiten die gevallen, als de herverkoop meer dan vijf jaar na de initiële aankoop plaatsheeft). Voorts wordt de even- tuele belasting (tegen 16,5 %) enkel geheven op de meerwaarde en niet op het totaalbedrag van de transactie. Aangezien de vastgoedprij- zen in Brussel bijvoorbeeld bijna verviervoudigd zijn in amper 2 decen- nia tijd [Kahane, Staelens en Franck, 2012], krijgt men een idee van de Nicolas BERNARD, Valérie LEMAIRE, Regionalisering van de woonbonus: naar een beleid op maat van het Brussels Gewest?, Brussels Studies, Nummer 83, 26 januari 2015, www.brusselsstudies.be Nicolas BERNARD, Valérie LEMAIRE, Regionalisering van de woonbonus: naar een beleid op maat van het Brussels Gewest?, Brussels Studies, Nummer 83, 26 januari 2015, www.brusselsstudies.be belastingvrije winst19 die men al die jaren heeft binnengehaald met dat soort verrichtingen. gekozen hebben zich in de stadscentra te vestigen, moeten verhogen. Ze hebben dat niet gedaan. 24. In ieder geval rijst de vraag of ons huidig belastingstelsel – dat gebaseerd is op de woonbonus – speculatie aanmoedigt, aangezien de eigenaar op geen enkele manier “ontmoedigd” wordt om een pand te kopen en het daarna, bijvoorbeeld in een periode van prijshausse, door te verkopen. Dat fenomeen zou zelf bijdragen tot de stijging van de prijzen en aldus de voornoemde hypothese bevestigen. 29. Een ander aspect doet vragen rijzen. Zelfs als de aftrek geldt voor de “enige eigen” woning, staat niets een eigenaar in de weg om zijn woning vervolgens te verhuren en het belastingvoordeel te blijven ge- nieten, op voorwaarde dat hij de woning heeft bewoond op 31 decem- ber van het jaar waarin hij de lening heeft gesloten (en geen ander pand heeft gekocht). 1.10. diverse kritische opmerkingen 25. In verband met de woonbonus kunnen nog verschillende kritieken en voorstellen geformuleerd worden, die misschien minder met de es- sentie te maken hebben. 26. Eerst had men het belastingvoordeel meer kunnen toespitsen op de primaire markt (nieuwbouw), met als uitdrukkelijk doel het wo- ningaanbod en de bouw te stimuleren. Onze bestuurders hebben dat idee echter nooit opgepikt. 30. Nu men energiebesparingen wenst te bevorderen, kan het, tot slot, verrassend overkomen dat het belastingvoordeel (dat in de over- grote meerderheid van de gevallen wordt gebruikt op de secundaire markt, namelijk de bestaande woningen in tegenstelling tot de primaire markt, de nieuwbouw) kan worden verkregen zonder enige verbintenis inzake energierenovatie. Men zou op zijn minst moeten overwegen om de omvang van de steun te koppelen aan de inspanningen die op dat vlak worden geleverd. 27. Het Franse model voor ﬁscale steun aan de huursector werd evenmin gevolgd. In Frankrijk bestaat er een breed spectrum maatrege- len om degene die een pand koopt en het vervolgens te huur stelt, vrij te stellen van belasting (wetten van Robien, Borloo, Scellier, Duﬂot, …). Soms zijn er averechtse effecten, aangezien de (in een uitsluitend ﬁnan- ciële optiek) gebouwde woningen niet altijd voldoen aan de bestaande behoeften. In België is de situatie helemaal omgekeerd: ingeval een pand te huur wordt gesteld, wordt het kadastraal inkomen verhoogd met 40 %. 1.9. men kan zoveel als men wil proﬁteren van de steun (die bij- gevolg niet voorbehouden is voor de “eerstekeerkopers”) Als hij zou beslissen om de woning te verhuren, is de- gene die de ﬁscale steun van de overheid krijgt, er geenszins toe ver- plicht om achteraf, als tegenprestatie, enige gematigdheid aan de dag te leggen bij de vaststelling van de huurprijs. De ontvangen steun kan in zekere zin worden omgezet in zuivere privéwinst; de eigenaar mag zo- veel mogelijk proﬁjt halen uit de woning die hij (ten dele) met geld van de gemeenschap heeft gekocht. 19 Tenzij het gaat om een activiteit van speculatieve aard (in tegenstelling tot “normaal beheer van een privépatrimonium dat uit onroerende goederen bestaat […]”, volgens art. 90, 1°, van het Wetboek van Inkomstenbelastingen). Voor een recent voorbeeld, zie Cass., 21 november 2013, Fiscoloog, 2013, nr. 1365, p. 11. 2.2. Brussel in nauwe budgettaire schoentjes? oplossing zal echter enkel (by default) worden toegepast als de Gewesten geen eigen regeling hebben uitgewerkt. oplossing zal echter enkel (by default) worden toegepast als de Gewesten geen eigen regeling hebben uitgewerkt. 34. De kosten van de woonbonus zijn zeer zwaar. Dat is niet alles: al die reeds grote bedragen dreigen, ingeval het Brussels Gewest het be- leid van de federale Staat ongewijzigd voortzet, te worden vermenig- vuldigd met 2,5 op 15 jaar tijd! Alleen al voor de regeling 2005 gaat men immers uit van een factuur van 185 miljoen euro tegen 2027 en zelfs van 274 miljoen euro [Brusselse Bond voor het Recht op Wonen, 2014]. De bijgewerkte laatste prognoses (mei 2014) van de uitgaven voor belastingaftrek met betrekking tot vastgoed in Brussel bevestigen die vrees, aangezien er tussen de belastingjaren 2008 en 2019 een verdubbeling van de kosten wordt verwacht (van 79,63 naar 154,75 miljoen euro, huisvesting en gevolgen van het vorige systeem samen) [FOD Financiën, 2014]. Op nationaal niveau zouden die uitgaven 4,9 miljard euro bereiken (en 2,7 miljard euro alleen al voor de regeling 2005)20. Aangezien de middelen die de federale Staat toekent om de nieuwe bevoegdheid te ﬁnancieren, gebaseerd zijn op de huidige kos- ten en niet op de toekomstige kosten, zijn ze verre van voldoende. 32. Wat is het verschil tussen belastingvermindering en belastingaf- trek? Belastingvermindering wordt pas toegepast zodra het bedrag van de belasting is vastgesteld, dus na de berekening ervan. De verminde- ring wordt afgetrokken van de verschuldigde belasting en neemt in concreto de vorm van een breuk aan – dezelfde voor alle belasting- plichtigen – die wordt toegepast op de totale aanslag (45 % in Brussel 32. Wat is het verschil tussen belastingvermindering en belastingaf- trek? Belastingvermindering wordt pas toegepast zodra het bedrag van de belasting is vastgesteld, dus na de berekening ervan. De verminde- ring wordt afgetrokken van de verschuldigde belasting en neemt in concreto de vorm van een breuk aan – dezelfde voor alle belasting- plichtigen – die wordt toegepast op de totale aanslag (45 % in Brussel – Wallonië en Vlaanderen hebben onlangs beslist om die drempel te verlagen tot 40 %, voor de eerste keer vanaf 1 januari 2015), ongeacht de ﬁnanciële middelen waarover de betrokkene beschikt. Om die reden kan de belastingvermindering als meer egalitair worden beschouwd dan de belastingaftrek (tegen de marginale aanslagvoet), aangezien het belastingvoordeel voor de rijkeren relatief gezien niet groter is. 2.1. van aftrek naar vermindering 28. Voorts bevestigen de overheden dat ze de stadsuitbreiding onder controle willen houden en de verdichting van de bestaande kernen be- vorderen om redenen die zowel te maken hebben met energie, leefmi- lieu, mobiliteit en instandhouding van het landschap. Als ze consequent zouden zijn, dan zouden ze de ﬁscale steun voor gezinnen die ervoor 31. In feite bestaat de woonbonus niet meer sinds 1 januari 2015. Om technische redenen die het bestek van deze tekst te buiten gaan, kun- nen de deelgebieden juridisch gezien geen belastingaftrek toestaan. Daarom hebben de federale overheden beslist om, alvorens de be- voegdheid aan de Gewesten over te dragen, de belastingaftrek om te vormen tot een belastingvermindering (van 45 %) vanaf die datum. Die 8 Nicolas BERNARD, Valérie LEMAIRE, Nicolas BERNARD, Valérie LEMAIRE, Regionalisering van de woonbonus: naar een beleid op maat van het Brussels Gewest?, Brussels Studies, Nummer 83, 26 januari 2015, www.brusselsstudies.be naar een beleid op maat van het Brussels Gewest?, Brussels Studies, Nummer 83, 26 januari 2015, www.brusselsstudies.be 20 Cf. Vlaams Parlement, Commissie voor Algemeen Beleid, Financiën en Begroting, vergadering van 24 januari 2012, Doc. 61 (2011-2012). 21 Art. 35decies van de bijzondere wet van 16 januari 1989 met betrekking tot de ﬁnanciering van de Gemeenschappen en de Gewesten, B.S., 17 januari 1989, ingevoegd bij artikel 32 van de bijzondere wet van 6 januari 2014 tot hervorming van de ﬁnanciering van de Gemeenschappen en de Gewesten, tot uitbreiding van de ﬁscale autonomie van de Gewesten en tot ﬁnan- ciering van de nieuwe bevoegdheden, B.S., 31 januari 2014. 2.2. Brussel in nauwe budgettaire schoentjes? Zodoen- de komt er iets meer ﬁscale rechtvaardigheid. Blijft nog dat deze oplos- sing (nog) niet volledig strookt met het principe van de progressiviteit van de belasting. 35. Hoe kan die stijgende tendens worden verklaard? De bij de bij- zondere ﬁnancieringswet bepaalde berekeningswijze zou weliswaar voordelig uitdraaien voor Brussel, in zoverre ze gebaseerd is op de PB- bijdrage en niet op de mate waarin men gebruikmaakt van de woonbo- nus. Die begrotingsmiddelen worden immers verdeeld tussen de Gewesten volgens de verdeelsleutel van de personenbelasting21; aan- gezien het Brussels aandeel van die belasting bijna 8,4 % (voor 2015) bedraagt voor een gebruikspercentage van de belastingaftrek dat rond 6,7 % blijft hangen, is de regeling van de ﬁnancieringswet onbetwist- baar gunstig voor het Brussels Hoofdstedelijk Gewest. 33. Het is natuurlijk veel te vroeg om de verminderingsregeling, die pas van kracht is geworden, al wetenschappelijk te beoordelen. Onze heroriënteringsvoorstellen, die gebaseerd zijn op kritische opmerkingen over de belastingaftrek, zijn volkomen haalbaar in de nieuwe institutionele context. Het is echter nu al verbazend dat het Brussels Gewest niet van de gelegenheid heeft gebruikgemaakt om al was het maar het federale percentage van de belastingvermindering te verlagen (zoals het noorden en het zuiden van het land hebben gedaan). Dat zou het mattheuseffect al hebben afgezwakt. Als er al een wijziging wordt aangebracht (er is een evaluatie aan de gang), zal dat niet gebeuren vóór het aanslagjaar 2017. Dat lijkt ons laat. 36. In dat verband moeten er evenwel twee nuances worden aange- bracht. In de eerste plaats zijn de uitgetrokken begrotingsmiddelen vastgesteld volgens berekeningsregels (aftrek tegen marginale aanslag- voet) die achterhaald zijn. Ze blijken voor het Brussels Gewest minder 9 Nicolas BERNARD, Valérie LEMAIRE, Regionalisering van de woonbonus: naar een beleid op maat van het Brussels Gewest?, Brussels Studies, Nummer 83, 26 januari 2015, www.brusselsstudies.be 9 Nicolas BERNARD, Valérie LEMAIRE, Regionalisering van de woonbonus: naar een beleid op maat van het Brussels Gewest?, Brussels Studies, Nummer 83, 26 januari 2015, www.brusselsstudies.be Nicolas BERNARD, Valérie LEMAIRE, Regionalisering van de woonbonus: naar een beleid op maat van het Brussels Gewest?, Brussels Studies, Nummer 83, 26 januari 2015, www.brusselsstudies.be duur uit te vallen dan de nieuwe regels (aangezien de marginale aan- slagvoet in het Brussels Gewest ver onder 45 % blijft – het percentage van de belastingverminderingen). 2.2. Brussel in nauwe budgettaire schoentjes? Met andere woorden, degenen die thans worden belast tegen een aanslagvoet van minder dan 45 % – de overgrote meerderheid in Brussel – erbij winnen … ten koste van de overheidsﬁnanciën. Vervolgens – wat nog fundamenteler is – zullen de ﬁscale uitgaven elk jaar exponentieel blijven stijgen tot de woonbonus op kruissnelheid is gekomen (pas in 2024), aangezien de uitgaven cu- mulatief zijn (zowel voor de nieuwe leners als voor de bestaande leners wier belastingvoordeel verder uitbetaald wordt). Dat is niet meer dan logisch: elke begunstigde krijgt het belastingvoordeel tijdens de hele looptijd van de lening en elk jaar sluiten nieuwe gezinnen een lening. In 2005 waren de ﬁscale uitgaven beperkt tot de voordelen die waren toegekend aan de eerste leners, maar vanaf 2006 moesten de overhe- den niet alleen de woonbonus betalen van de leners die een lening had gesloten tijdens het lopende jaar, maar ook het belastingvoordeel van degenen die hun lening het jaar daarvoor hadden gesloten enzovoort. Pas in 2024 zal er een fase komen waarin er een evenwicht zal worden bereikt tussen degenen die in het systeem stappen en degenen die eruit stappen (die hun lening zullen hebben terugbetaald en bijgevolg het belastingvoordeel niet meer zullen krijgen). Dat zou nog steeds in het voordeel van de gemiddelde Brusselaar zijn (in vergelijking met de vorige regeling), aangezien de gemiddelde mar- ginale aanslagvoet in de loop van de jaren in ieder geval onder 40 % zal blijven. De overheden hebben echter beslist om geen enkele wijziging aan te brengen in het systeem dat ze van de federale overheid hebben overgeërfd (in elk geval niet vóór 2017). Dat zou nog steeds in het voordeel van de gemiddelde Brusselaar zijn (in vergelijking met de vorige regeling), aangezien de gemiddelde mar- ginale aanslagvoet in de loop van de jaren in ieder geval onder 40 % zal blijven. De overheden hebben echter beslist om geen enkele wijziging aan te brengen in het systeem dat ze van de federale overheid hebben overgeërfd (in elk geval niet vóór 2017). 22 Met ongeveer 25 % volgens economist Julien Manceaux bij de ING-bank (L'Echo, 25 juni 2014, p. 26). Nicolas BERNARD, Valérie LEMAIRE, Regionalisering van de woonbonus: naar een beleid op maat van het Brussels Gewest?, Brussels Studies, Nummer 83, 26 januari 2015, www.brusselsstudies.be 3.1. afschafﬁng, aanpassing of verlenging? 10 Nicolas BERNARD, Valérie LEMAIRE, Regionalisering van de woonbonus: naar een beleid op maat van het Brussels Gewest?, Brussels Studies, Nummer 83, 26 januari 2015, www.brusselsstudies.be Nicolas BERNARD, Valérie LEMAIRE, Regionalisering van de woonbonus: naar een beleid op maat van het Brussels Gewest?, Brussels Studies, Nummer 83, 26 januari 2015, www.brusselsstudies.be mentaire regels inzake rechtszekerheid verbieden dat de spelregels achteraf gewijzigd worden voor degenen die een pand hebben gekocht en daarbij de huidige woonbonus in hun ﬁnanciële berekening hebben geïntegreerd. vorm van dat belastingvoordeel, het bedrag of nog de toekenningsre- gels zouden bijgevolg op zijn minst moeten worden herzien om de maatschappelijke evoluties te volgen die werden vastgesteld sinds de invoering van dat ruggensteuntje voor de mensen met de spreekwoor- delijke “baksteen in de maag”. 45. Er zij in dat verband op gewezen dat veel Belgen (een derde) op dit ogenblik al minstens één hypothecair krediet hebben lopen23. In Vlaanderen waren er in 2011 594.400 belastingaftrekken met betrek- king tot vastgoed (15,4 % van het totale aantal aangiften24) en in Wallo- nië 250.200 (11,5 %), maar dat cijfer daalt tot 51.200 in Brussel (7,2 %). Dat verschil moet gerelateerd worden aan het veel kleinere aandeel eigenaars in het Brussels Gewest (39 %) dan in de overige twee Gewesten (71 % in Vlaanderen en 66 % in Wallonië). Het aandeel eige- naars is traditioneel kleiner in een stedelijke omgeving dan in rurale ge- bieden, maar het verschil in het aantal aftrekken wordt ook verklaard door het peil (dat bovendien snel stijgt, zoals reeds vermeld) van de aankoopprijzen van woningen in Brussel25 in combinatie met de lage inkomens van een groot deel van de Brusselse bevolking26. 42. In dat opzicht kan men tussen de regels van de voornoemde kriti- sche opmerkingen een aantal voorstellen voor de heroriëntering van de woonbonus lezen. Men zou de steun voor de lage inkomens kunnen verhogen (en er zelfs de welgestelden van uitsluiten), het belastingvoor- deel voor alleenstaanden verhogen, het belastingvoordeel enkel toe- kennen aan de “eerstekeerkopers”, het belastingvoordeel koppelen aan eisen op het vlak van leefmilieu, energie of huurprijs enz. Volgens ons zou een koppeling van de steun aan een aantal redelijke voorwaarden de werking van dat uitstekend instrument voor sociale promotie (op papier) geenszins in het gedrang brengen. Het moet gewoon de be- leidsprioriteiten van het moment beter weerspiegelen. 43. 23 31,9 % eind 2012. Ze genieten echter niet allemaal de woonbonus, aangezien hun woning misschien niet de “enige eigen” woning is. 24 Dit – vrij – lage percentage hoeft niet te verbazen: zeer veel gezinnen hebben in feite hun hypothecaire lening al terugbetaald of hebben vroeger hun woning geërfd (als ze het al niet volledig met eigen middelen hebben gekocht, maar dat gebeurt niet zo vaak). 25 In 2012 bedroeg bijvoorbeeld de gemiddelde prijs van een appartement 218.000 euro in Brussel tegenover 207.000 euro in Vlaanderen en 162.000 euro in Wallonië. Het verschil wordt groter als het gaat om huizen in Brussel, die gemiddeld 354.000 euro kosten; de prijs stagneert daarentegen in Vlaanderen (207.000 euro) en is zelfs lager in Wallonië (147.000 euro) om de eenvoudige reden dat dergelijke panden veeleer gelegen zijn in een ruraal gebied, waar de prijzen lager liggen (terwijl de appartementen meestal gelegen zijn in stedelijke gebieden, waar de prijzen hoger zijn). 26 Het gemiddelde inkomen per inwoner bedraagt 17.146 euro in Vlaanderen, 15.277 euro in Wallonië en slechts 12.885 euro in Brussel. Bron: FOD Economie, Fiscale statistiek van de inkomens. 3.1. afschafﬁng, aanpassing of verlenging? Een andere oplossing bestaat erin de door de federale Staat aan- bevolen regeling “by default” (net vóór de regionalisering), namelijk een eenvormige belastingvermindering met 45 %, gewoon te verlengen. Die oplossing zou echter nog meer kosten (dan de vorige aftrekregeling) voor het Brussels Gewest, aangezien de gemiddelde marginale aan- slagvoet in Brussel ver onder die drempel van 45 % blijft. Het is even- wel toegestaan om het verminderingspercentage te variëren volgens de inkomens, wat de regeling tegelijk “progressiever” zou maken. 3.1. afschafﬁng, aanpassing of verlenging? 39. De toekomstige gewestelijke wetgever heeft duidelijk geen gebrek aan denksporen. Bij de integratie van de nieuwe bevoegdheid hebben de overheden op papier een zeer grote speelruimte. Welke bakens zouden ze bijgevolg kunnen uitzetten? 40. In het algemeen is gematigdheid aangewezen. In de eerste plaats zou een afschafﬁng (of een te forse vermindering) van de woonbonus de vastgoedmarkt in zijn geheel in min of meer vrije val storten22. De kwestie wordt echter besproken. In dat opzicht komt het Nederlandse tegenvoorbeeld steeds in beeld, maar in het licht van wat er in andere Europese landen wordt vastgesteld, zijn sommigen veeleer van oordeel dat die vrees “niet mag worden overdreven” [De Decker, 2014]. Indien een dergelijke keuze zou worden gemaakt, dan moeten de overheden er in ieder geval op zijn minst op toezien dat het effect van de maatre- gel in de tijd wordt gespreid en geleidelijk uitdooft (“phasing out”). Tege- lijk moeten er dan misschien meer doelgerichte alternatieven worden ingevoerd om de verwerving van eigendom te blijven steunen, zoals de toekenning van een lager btw-tarief, maar dan niet volgens het statuut van de operator, maar volgens de hoogte van de prijs van de woning (bij verkoop of verhuur), de verlaging van de registratierechten (of het heroriënteren van het abattement naar de gezinnen met weinig midde- len), … 37. Duidelijker gesteld, de aan de deelgebieden toegekende bedragen zullen in de komende jaren niet overeenkomen met de reële kosten die ze zullen moeten dragen, in de eerste plaats voor het Brussels Gewest, dat voor 2015 evenwel een voorlopig bedrag van ongeveer 120 miljoen euro zal krijgen. Dat lijkt weinig. Achteraf zal er een deﬁnitief referentie- bedrag worden berekend in het licht van een rapport van het Rekenhof (dat rekening zal houden met de daadwerkelijke ﬁscale uitgaven – bij ongewijzigd beleid – voor dat aanslagjaar). Het rapport zal uiterlijk tegen 31 december 2016 klaar zijn op basis van de gegevens die uiterlijk op 31 oktober 2016 zullen worden overgezonden. 38. Het Brussels Gewest had de budgettaire “ontsporing” enigszins in bedwang kunnen houden met de beslissing om de belastingverminde- ring te verlagen tot bijvoorbeeld 40 % (zoals in Vlaanderen en Wallonië). 41. Waarschijnlijk zal de woonbonus evenwel behouden blijven (en niet afgeschaft worden) om zowel politieke als symbolische redenen. De 22 Met ongeveer 25 % volgens economist Julien Manceaux bij de ING-bank (L'Echo, 25 juni 2014, p. 26). 3.3. de belastingaftrek omzetten in een premie? looptijd van de lening het gewicht van de maandelijkse terugbetaling – die constant is – in ieder geval ten opzichte van het inkomen, aange- zien de inkomens onvermijdelijk stijgen onder invloed van de gezond- heidsindex, zelfs als de betrokkene geen loonopslag krijgt. De tweede reden is dat de beroepssituatie van particulieren volgens de statistieken meestal mettertijd verbetert (opslag, promotie, verandering van werk, …), zodat de gezinnen in het midden van de loopbaan meer ﬁnanciële middelen hebben dan bij het begin. 49. Een laatste voorstel luidt als volgt: om bij voorkeur jonge gezinnen te steunen, zou het interessant zijn om de belastingaftrek (waarvan de positieve effecten zich, zoals reeds vermeld, pas later laten gevoelen) om te vormen tot een soort van investeringspremie die onmiddellijk ter beschikking wordt gesteld. Dat zou voordelig zijn voor degenen die wegens hun bescheiden middelen geen twee jaar kunnen wachten “om steun te krijgen” en die zonder de steun niet de middelen hebben om hic et nunc eigenaar te worden. Die oplossing werd niet gekozen. Men mag niet vergeten dat de armsten sowieso geen belasting betalen, waardoor de aftrek van de interesten voor hen niet interessant is. 47. Om die redenen zou het bedrag van de toekomstige woonbonus volgens ons veel hoger moeten zijn bij het begin van de terugbetaling van de lening dan aan het einde en bijvoorbeeld jaarlijks afnemen. Van- daag bestaat er weliswaar een vergelijkbare regeling (stopzetting van de verhoging met 750 euro na de tiende belastbare periode), maar een meer lineaire en geleidelijke aanpak lijkt ons beter. Een meer ingrijpende oplossing zou erin bestaan de woonbonus te beperken in de tijd (bij- voorbeeld de eerste tien jaar van de lening en daarna niet meer). 50. Men mag echter niet uit het oog verliezen dat die oplossing, die erop neerkomt een belastingvoordeel dat over verschillende jaren is gespreid, om te zetten in rechtstreekse en eenmalige steun (one shot), impliceert dat de overheden in één keer een aanzienlijk bedrag zouden moeten uitkeren. 48. Eigenlijk zou in de eerste plaats de verwerving van eigendom en misschien minder het onderhoud van de woning moeten worden on- dersteund. Voor het overige blijft het aantal in gebreke blijvende eige- naars (die hun maandelijks bedrag niet kunnen terugbetalen en daar- door het risico op vastgoedbeslag lopen) in België vrij laag27 ten op- zichte van het totale aantal leners. 3.2. meer degressiviteit inbouwen 46. Om de analyse in een ruimer kader te plaatsen is er nood aan een ander soort voorstellen. Wat als de gewestelijke overheden in de eerste plaats de ﬁscale steun degressiever (in de tijd) zouden maken? Het is immers in het begin van de terugbetaling van de hypothecaire lening dat de grootste ﬁnanciële inspanning moet worden geleverd en wel om minstens twee redenen. Gesteld dat de betrokkene tegen een vaste interestvoet heeft geleend (wat het geval is voor de overgrote meerder- heid van de leningen) en zijn werk niet heeft verloren, daalt tijdens de 44. Het lijkt in ieder geval belangrijk om de eventueel aangepaste re- geling enkel toe te passen op de nieuwe vastgoedtransacties. De ele- 11 Nicolas BERNARD, Valérie LEMAIRE, Nicolas BERNARD, Valérie LEMAIRE, Regionalisering van de woonbonus: naar een beleid op maat van het Brussels Gewest?, Brussels Studies, Nummer 83, 26 januari 2015, www.brusselsstudies.be Regionalisering van de woonbonus: naar een beleid op maat van het Brussels Gewest?, naar een beleid op maat van het Brussels Gewest?, Brussels Studies, Nummer 83, 26 januari 2015, www.brusselsstudies.be 3.3. de belastingaftrek omzetten in een premie? Voorts zorgen de deelgebieden al voor een bescherming tegen wanbetaling door de leners een gratis verzekering tegen inkomensverlies aan te bieden28. Het is in ieder geval onder meer op die voorwaarde dat men erin zal slagen de woonbonus enigszins socialer te maken. 28 Zie onder meer art. 112, § 2, van de Brusselse Huisvestingscode (het uitvoeringsbesluit laat echter op zich wachten). In Wallonië geldt de gratis verzekering tegen inkomensverlies gedu- rende drie jaar ten belope van 6.200 euro per jaar, indien de niet-terugbetaling van de hypothecaire lening rechtstreeks kan worden toegeschreven aan het verlies van de betrekking of arbeidsongeschiktheid (besluit van de Waalse regering van 21 januari 1999 tot invoering van een verzekering tegen het risico van inkomensverlies wegens verlies van betrekking of arbeids- ongeschiktheid, B.S., 25 februari 1999). 27 8.000 [P. De Decker en V. Geurts, 2004]. 3.4. een principe van budgettaire realiteit 51. Hoewel die voorstellen als adequaat kunnen worden beschouwd, dient er rekening te worden gehouden met de budgettaire realiteit. Zo- als reeds vermeld werd, zouden de middelen die samen met de be- voegdheden worden overgedragen, wel eens ontoereikend kunnen zijn, in zoverre die middelen niet enkel dienen om nieuwe kopers te steunen, maar ook om de belastingaftrek voor de bestaande leningen te blijven ﬁnancieren. De omvang van het belastingvoordeel zal misschien moe- ten worden beperkt, ook voor degenen die al een woning hebben ge- kocht (… ongeacht wat de politici beweren). 52. Nochtans “zal de regering daarom sterkere ondersteuning bieden om de toegang tot eigendom te bevorderen”, aldus een stoutmoedige 12 Nicolas BERNARD, Valérie LEMAIRE, Regionalisering van de woonbonus: naar een beleid op maat van het Brussels Gewest?, Brussels Studies, Nummer 83, 26 januari 2015, www.brusselsstudies.be 12 Nicolas BERNARD, Valérie LEMAIRE, Regionalisering van de woonbonus: naar een beleid op maat van het Brussels Gewest?, Brussels Studies, Nummer 83, 26 januari 2015, www.brusselsstudies.be uitspraak van de nieuwe Brusselse regering, die klare wijn schenkt in verband met de toekomstige woonbonus: “De bestaande ﬁscale stimuli zullen op zijn minst behouden blijven, en in de mate van het mogelijke versoepeld en zelfs uitgebreid worden […] Verder zal de ﬁscale aftrek- baarheid van de « woonlening » behouden blijven en geëvalueerd wor- den in het kader van de (globale) ﬁscale hervorming die in 2017 inge- voerd zal worden”29. Er zij terloops op gewezen dat men niet kan weten of de uitbreiding waarvan sprake is, betrekking heeft op de doelgroep dan wel op het bedrag van de ﬁnanciële steun zelf. 55. Uiteindelijk is men er zich van bewust dat vastgoedﬁscaliteit een geheel vormt met een complexe architectuur, waarvan de bestanddelen in grote mate onderling afhankelijk zijn. Als één bestanddeel wordt ge- wijzigd zonder compensaties in de andere bestanddelen, bestaat het gevaar dat het algemene evenwicht wordt verstoord. In deze aangele- genheid zijn eenzijdige aanpassingen dus uit den boze. 56. Dat neemt niet weg dat deze argumenten volgens ons een serene en objectieve discussie over de vastgoedﬁscaliteit zonder vooroordelen of doctrinaire taboes niet in de weg zouden mogen staan. Die discussie uit principe uit de weg gaan zou kunnen worden beschouwd als een corporatistische reﬂex. 53. Zonder extra begrotingsmiddelen is het al een illusie te geloven dat men de vorige regeling ongewijzigd zal kunnen verlengen. 3.4. een principe van budgettaire realiteit Wat valt er dan te zeggen over de aangekondigde uitbreidingen van het systeem? Uiteindelijk draait het in de politiek altijd om prioriteiten. Voor Brussel zal het op zijn minst niet evident zijn om zo'n algemene ambitie te verzoe- nen met een andere, socialere doelstelling die openlijk werd aangekon- digd: “Voor gezinnen met een bescheiden of middelgroot inkomen zal de eerste aankoop van een eigen woning ﬁscaal aantrekkelijker worden gemaakt”30. De kwadratuur van de cirkel is niet veraf.... 30 Deel 2 van de Bijlage bij de Algemene Toelichting van de middelen- en uitgavenbegroting van het Brussels Hoofdstedelijk Gewest voor het begrotingsjaar 2015, Doc. Parl. Brussels Hoofdstedelijk Gewest, gewone zitting 2014-2015, nr. A-51/2, p. 114. 29 Meerderheidsakkoord 2014/2019, p. 95 30 Deel 2 van de Bijlage bij de Algemene Toelichting van de middelen- en uitgavenbegroting van het Brussels Hoofdstedelijk Gewest voor het begrotingsjaar 2015, Doc. Parl. Brussels Hoofdstedelijk Gewest, gewone zitting 2014-2015, nr. A-51/2, p. 114. 29 Meerderheidsakkoord 2014/2019, p. 95 Nicolas BERNARD, Valérie LEMAIRE, Regionalisering van de woonbonus: naar een beleid op maat van het Brussels Gewest?, Brussels Studies, Nummer 83, 26 januari 2015, www.brusselsstudies.be n de Bijlage bij de Algemene Toelichting van de middelen- en uitgavenbegroting van het Brussels Hoofdstedelijk Gewest voor het begrotingsjaar 2015, Doc. Parl. Brussels k Gewest gewone zitting 2014 2015 nr A 51/2 p 114 30 Deel 2 van de Bijlage bij de Algemene Toelichting van de middelen- en uitgavenbegroting van het Brussels Hoofds Hoofdstedelijk Gewest, gewone zitting 2014-2015, nr. A-51/2, p. 114. Besluit 57. 29. De woonbonus is onder meer niet billijk en efﬁciënt genoeg. De zesde staatshervorming is voor de Gewesten een unieke kans om de woonbonus te hervormen ten dienste van de algemene doelstellin- gen van hun huisvestingsbeleid. Door de (ﬁscale en nog andere) voor- delen voor de eigenaars te verbeteren, is men misschien het uiteindelij- ke doel van de belasting (en van het aftrek/verminderingssysteem) uit het oog verloren: de rijkdommen zo rechtvaardig mogelijk te herverde- len en niet om een bepaalde levenswijze fervent (en enigszins blind) aan te moedigen en een andere levenswijze niet. In dat opzicht is het mis- schien raadzaam erop te wijzen dat de woonbonus niet bedoeld is als een rechtstreekse steun bij eigendomsverwerving noch als een regeling op zich. De woonbonus is niet meer dan een bijsturing van de perso- nenbelasting via aftrek/vermindering. 54. In hun reﬂectie mogen de overheden evenmin uit het oog verliezen dat buitensporige bevordering van de verwerving van eigendom (hier langs ﬁscale weg) altijd risico's inhoudt, namelijk minstens drie: toena- me van de exodus van de middenklasse uit het Brussels Gewest (zoals werd vastgesteld in de laatste decennia), stijging van de prijzen op de koopmarkt (veroorzaakt door een verhoging/solvabilisering van de vraag, zoals men enkele jaren geleden in Vlaanderen heeft vastgesteld toen de registratierechten werden verlaagd van 12,5 % naar 10 %) en, tot slot, de toename van de schuldenlast van de gezinnen. Bijgevolg is ook hier voorzichtigheid geboden... 13 Nicolas BERNARD, Valérie LEMAIRE, Regionalisering van de woonbonus: naar een beleid op maat van het Brussels Gewest?, Brussels Studies, Nummer 83, 26 januari 2015, www.brusselsstudies.be 58. Tegelijkertijd moet een Gewest zoals Brussel ervoor zorgen dat zijn vastgoedmarkt vitaal blijft, want het haalt meer dan 40 % 31 van zijn ei- gen ﬁscale ontvangsten uit de registratierechten (de ﬁscale ontvangsten ﬁnancieren op hun beurt het Gewest voor 39 % 32). Een forse daling van het aantal transacties, die misschien zou worden veroorzaakt door al te radicale aankondigingen, dreigt de budgettaire basis van het Gewest ernstig te ondermijnen en bijgevolg ook zijn vermogen om de heden- daagse uitdagingen, in de eerste plaats de demograﬁsche explosie, aan te nemen. 31 503 miljoen euro op een belastingtotaal van 1,245 miljard (begroting van ontvangsten en uitgaven van het Brussels Hoofdstedelijk Gewest voor het begrotingsjaar 2014, Parl. Doc., Brussels Hoofdstedelijk Gewest, gewone zitting 2013-2014, nr. A-463/1, p. 142). 32 Ibid 142 32 Ibidem, p. 142. Bibliograﬁe ALBRECHT, Johan en VAN HOOFSTAT, Rob, 2012. De woonbonus mildert de ﬁscale druk op nieuwbouw van 106% tot 60%, maar slaagt er niet in de nieuwbouwproductie op een voldoende hoog peil te houden en zou moeten worden vervangen door een btw-ver- laging. In: Itinera institute Analyse, 2012, nr. 3, pp. 1-23. BIGOT, Régis en HOIBIAN, Sandra, 2009. Les difﬁcultés des Français face au logement. In: Cahiers de recherche du CREDOC, 2009, n° 265, pp. 1-170. 59. De gulden middenweg is aangewezen. Is het dan niet zo dat in de politiek, zoals op andere gebieden, beperking tot meer creativiteit leidt? BRUSSELSE BOND VOOR HET RECHT OP WONEN, 2014. « Hoeveel kost de woonbonus aan de overheid? » In: Art. 23, 2014, nr. 56, p. 6. DE DECKER, Pascal en GEURTS, Veerle, 2004. Home ownership at risk in Belgium, Antwerpen, Universiteit Antwerpen. DE DECKER, Pascal, 2014. Afbouw woonbonus In : Sampol, nr. 1, ja- nuari 2014, pp. 64-75. EUROPEAN COMMISSION, 2012. Tax Reform in EU Member States – Report 2012, In : Working Paper, n°34. FOD FINANCIEN, 2014. Bijgewerkte prognoses van de ﬁscale uitgaven na integratie van de belastingjaren 2012 en 2013. Meerjarenraming van de overdracht van ﬁscale middelen voor de periode 2015-2019. HUYGHEBAERT, Nancy, 2012. Percentage hypothecaire kredieten met één kredietnemer. Studie naar het belang van het consumentenkre- diet en het hypothecair krediet voor de Belgische economie. Leu- ven, Katholieke Universiteit Leuven. KAHANE, Lucien, STAELENS, Pieter en FRANCK, Pierre-Alain, 2012. Observatiecentrum van de verkoop van residentieel vastgoed in het 03 miljoen euro op een belastingtotaal van 1,245 miljard (begroting van ontvangsten en uitgaven van het Brussels Hoofdstedelijk Gewest voor het begrotingsjaar 2014, Parl. Doc., sels Hoofdstedelijk Gewest, gewone zitting 2013-2014, nr. A-463/1, p. 142). 14 Nicolas BERNARD, Valérie LEMAIRE, Regionalisering van de woonbonus: naar een beleid op maat van het Brussels Gewest?, Brussels Studies, Nummer 83, 26 januari 2015, www.brusselsstudies.be Brussels Hoofdstedelijk Gewest 1990-2010, Brussels Hoofdstedelijk Gewest. Brussels Hoofdstedelijk Gewest 1990-2010, Brussels Hoofdstedelijk Gewest. Brussels Studies wordt gepubliceerd met de steun van: Brussels Studies wordt gepubliceerd met de steun van: MATHIEU, Agnès, 2014. Régionalisation du régime ﬁscal des déduc- tions/réductions d’impôt afférent à la maison d’habitation, Bruxelles, working paper. Innoviris, het Brussels Instituut voor Onderzoek en Innovatie MONNIER, Bernard et ZIMMER, Pol, 2008. Fiscalité immobilière: le coût de la brique dans le ventre. In: Les échos du logement. 2008. n°2, pp. 17-22. Universitaire Stichting OESO, 2013. Economische studies van de O.E.S.O. België 2013, Edi- ties OESO, 2013, pp. 1-121. Om deze tekst te citeren VALENDUC, Christian, 2008. Les incitations ﬁscales en faveur du lo- gement. In: Les échos du logement. 2008. n°2, pp. 1-16. BERNARD, Nicolas, LEMAIRE, Valérie, 2014. Regionalisering van de woonbonus: naar een beleid op maat van het Brussels Gewest?, In: Brussels Studies, Nummer 83, 26 januari 2015, www.brusselsstudies.be BERNARD, Nicolas, LEMAIRE, Valérie, 2014. Regionalisering van de woonbonus: naar een beleid op maat van het Brussels Gewest?, In: Brussels Studies, Nummer 83, 26 januari 2015, www.brusselsstudies.be VLAAMSE WOONRAAD, 2012. Advies nr. 2012/12 over de regionalise- ring van de woonbonus. Links Andere versies van deze tekst zijn beschikbaar ePub FR : http://tinyurl.com/BRUS83FREPUB ePub NL : http://tinyurl.com/BRUS83NLEPUB ePub EN : http://tinyurl.com/BRUS83ENEPUB pdf FR : http://tinyurl.com/BRUS83FRPDF pdf NL : http://tinyurl.com/BRUS83NLPDF pdf EN : http://tinyurl.com/BRUS83ENPDF De video's die verschenen zijn in Brussels Studies zijn te bezichtigen op het Vimeo kanaal van Brussels Studies op de volgende link: http://vimeo.com/channels/BruS De video's die verschenen zijn in Brussels Studies zijn te bezichtigen op het Vimeo kanaal van Brussels Studies op de volgende link: http://vimeo.com/channels/BruS
https://openalex.org/W2979798188	https://ruc.udc.es/dspace/bitstream/2183/25322/3/C.Gonzalez_Val_A_Convolutional_Approach_To_Qualit_2020.pdf	English	null	A convolutional approach to quality monitoring for laser manufacturing	Journal of intelligent manufacturing	2,019	cc-by	4,382	Abstract The extraction of meaningful features from the monitoring of laser processes is the foundation of new non-destructive quality inspection methods for the manufactured pieces, which has been and remains a growing interest in industry. We present ConvLBM, a novel approach to monitor Laser Based Manufacturing processes in real-time. ConvLBM uses a Convolutional Neural Network model to extract features and quality indicators from raw Medium Wavelength Infrared coaxial images. We demonstrate the ability of ConvLBM to represent process dynamics, and predict quality indicators in two scenarios: dilution estimation in Laser Metal Deposition, and location of defects in laser welding processes. Obtained results represent a breakthrough in the 3D printing of large metal parts, and in the quality control of welding processes. We are also releasing the ﬁrst large dataset of annotated images of laser manufacturing. Keywords Neural-networks · Convolutional-neural-networks · Quality-control · Laser-cladding · Laser-welding Keywords Neural-networks · Convolutional-neural-networks · Quality-control · Laser-cladding · Laser-welding Journal of Intelligent Manufacturing (2020) 31:789–795 https://doi.org/10.1007/s10845-019-01495-8 Journal of Intelligent Manufacturing (2020) 31:789–795 https://doi.org/10.1007/s10845-019-01495-8 A convolutional approach to quality monitoring for laser manufacturing Carlos Gonzalez-Val1 · Adrian Pallas1 · Veronica Panadeiro2 · Alvaro Rodriguez2 Received: 16 October 2018 / Accepted: 21 September 2019 / Published online: 9 October 2019 © The Author(s) 2019 B Carlos Gonzalez-Val carlos.gonzalez@aimen.es 1 Robotics and Control Unit, AIMEN Technology Centre, 36440 O Porriño, Spain B Carlos Gonzalez-Val carlos.gonzalez@aimen.es 1 Robotics and Control Unit, AIMEN Technology Centre, 36440 O Porriño, Spain 2 Department of Computer Science, University of A Coruña, Campus Elviña s/n, 15071 A Coruña, Spain Introduction fast manufacturing of larger and more complex parts (Hauser and De Weck 2007). Laser technologies play a key role in high precision processes in sectors such as automotive industry and aeronautics. In these sectors a continuous improvement towards the goal of “zero defects manufacturing” is essential to reduce costs and avoid exploitation problems. For this reason, new techniques are continuously developed for on-line quality monitoring and fast defect detection. We focus in two laser processes, Laser Metal Deposition (LMD), and laser welding. Laser welding industry is a mature technology imple- mented in most automotive industries. However, defects are a signiﬁcant issue in production lines and any deviation in the position of the work piece produces defects and non- compliance with quality standards. Process monitoring in real-timeanddetectinganydeviationfromthedesiredparam- eters will lead to a dramatic reduction in repairing costs and signiﬁcant increase in product quality. LMD is a manufacturing process based on the creation of a molten pool into which metal powder is injected as particles. This new additive technology is expected to enable 3D print- ing of net-shaped large metal components and is replacing previous techniques for coating and repairing critical parts in metallurgic industries. Currently, the biggest challenge for LMD is the lack of control in process dynamics at the high deposition rates required for large pieces (Song et al. 2012). Process monitoring in real time is critical for the efﬁcient and Automatic quality control in laser processes is challenging and stable online monitoring systems are still in early devel- opment stages. Nowadays, destructive analysis and visual inspection are the main techniques for defect detection. The automation of this process will suppose a great improvement in the production lines. In addition, many parameters such as track geometry or dilution can only be obtained through destructive testing, as metallographic analysis and 3D scan- ning. These procedures cannot be used for on-line quality controlsincetheyareeitherdestructiveortime-consuming.A vision system inspection allows the online monitoring show- ing key indicators of the studied processes. Monitoring and control of manufacturing processes have signiﬁcantly evolved, and recent research efforts rely on the use of non-intrusive sensors that can work with accuracy in a harsh environment, such as Charged Couple Device (CCD) 12 3 790 Journal of Intelligent Manufacturing (2020) 31:789–795 Fig. 1 Examples of MWIR images acquired during a Laser process or Complementary Metal–Oxide–Semiconductor (CMOS) cameras, photodiodes (Liu et al. 2019) and pyrometers (Song et al. 2012). Introduction Among these sensors, CCD/CMOS cameras provide the most comprehensive information from the process. These cameras are commonly used to obtain cladding height or geometry (Meriaudeau and Truchetet 1996; Iravani-Tabrizipour and Toyserkani 2007; Bi et al. 2007), particle-in-ﬂight velocity (Doubenskaia et al. 2004; Meriaudeau and Truchetet 1996), powder delivery distribu- tion (Doubenskaia et al. 2004), and melt pool features such as surface temperature (Meriaudeau and Truchetet 1996) and thermal images (Hu and Kovacevic 2003; Bi et al. 2007). Thermal imaging is an effective technique for defect detection (Jager and Hamprecht 2009; You et al. 2015) defect classiﬁcation (Lapido et al. 2015) and laser power control (Hofman et al. 2012; Rodriguez-Araujo et al. 2012). In par- ticular, high-speed Medium Wavelength Infrared (MWIR) imaging, has the beneﬁts of being low cost and providing a high thermal dynamic range (Rodríguez-Araújo et al. 2017), and has been successfully applied to the control of laser pro- cesses (Panadeiro-Castro et al. 2018; Garcia et al. 2018). Fig. 1 Examples of MWIR images acquired during a Laser process TheequipmentusedforlaserweldingandLMDisidealfor monitoring the molten pool with coaxial imaging. However, two major barriers limited the success of previous monitoring and control approaches. On one hand, images in the visible range from traditional sensors present problems of visual noise due to signal overﬂow and reﬂections exacerbated by dynamic range issues. These problems are solved with the appearance of low cost high-speed uncooled sensors in the MWIR range (Rodríguez-Araújo et al. 2017). On the other hand, obtaining measurements from images of the process is a challenging task due to the unintuitive nature of the images and the difﬁculty in obtaining annotated data with parameters of interest. These capabilities will lead to the development of new con- trol approaches for industrial laser manufacturing. Datasets ConvLBM is a CNN model that predicts laser power and speed from 28×28 raw MWIR coaxial images of a laser process. We implemented ConvLBM using Keras1 and Ten- sorFlow2 frameworks. We built three datasets of images for the different application scenarios and then, divided each one into a training and a val- idation set. The images were obtained with a Tachyon 1024 FPA camera at 1000 fps mounted coaxially to the laser head. The primary dataset is a large set of images annotated with the parameters of laser power and process speed. Then we created two smaller datasets; one with dilution information and one with labeled defects. Figure 2 details the architecture of ConvLBM. This design is a modiﬁcation of ResNet (He et al. 2016), a network that shows good results on the CIFAR-10 dataset, a well-known benchmark for image applications. This architecture, uses blocks made of four operators: The primary dataset consists of over 1600,000 images from 405 tracks of laser cladding, with 316L steel as powder and base material. Every track was cladded with different values of laser head speed and of laser power chosen from a range of values that delivered good quality tracks. We divided the dataset into a training and a validation set. In the training dataset,thedatasetwasresampledtoobtainasetofimagesfor every tested combination of laser power and speed, resulting in a total of 269,235 images. The validation dataset contains 268,470 images from 90 tracks (2983 images per track), and represents evenly distributed parameters over the range of tested laser power and speed. • Convolutional layers, which performs the feature calcula- tion on the image. They are a special type of perceptron with known performance in computer vision (Bengio 1997). • ReLu activations, that add non-linearities to the model (Ioffe and Szegedy 2015). • Batch normalization, a regularization technique to accel- erate training and to reduce overﬁtting (Ioffe and Szegedy 2015). • Shortcut connection, which performs an identity mapping carrying the input to the next block and skipping the convo- lutional layers. This approach tries to solve the saturation problem of deep networks, that results in higher training errors when adding more layers (He et al. 2016). The dilution dataset consists of LMD images with M2 tool steel as powder and base material and using the same camera conﬁguration as in the previous experiment. Materials and methods We assumed that high-speed MWIR images capture well the heat ﬂows that characterize the quasi-steady states of the melt pool, and that such steady states can be encoded in a CNN, providing an unequivocal representation of the process (Gar- cia et al. 2018). With this perspective, ConvLBM is trained using a two-step approach. The image processing ﬁeld is experiencing a huge trans- formation and many applications are meeting industry requirements with the use of Convolutional Neural Networks (CNN). A CNN is a type of feed-forward deep neural net- work based on a multilayer perceptron (Rosenblatt 1961), and designed to analyze visual information with minimal preprocessing. Schemes based on CNNs have a stunning ability to recognize images, objects, and image character- istics (Krizhevsky et al. 2012). These models use a labelled set of examples to train a model with convolutional elements that extract meaningful representations of the data without a handcrafted approach. First, we built a large dataset labelled with laser power and process speed. The selected variables are easy to control and good indicators of the energy and mass input. This is supported by recent efforts on modelling (Pinkerton 2015) and control (Tapia and Elwany 2014), where parameteriza- tion studies assume that two intrinsic magnitudes, enthalpy and mass input, drive the process and determine key quality indicators such as dilution, clad bead geometry and porosity (Zhong et al. 2015). To that end, 405 tests were recorded using different sets of parameters within acceptable ranges. Training ConvLBM on this dataset, the network captures the dynamic changes of the laser process from the raw images, without the need of manual feature extraction. CNNs approaches are capable of analysing MWIR ther- mal images (see Fig. 1) to extract parameters of laser processes (Kwon et al. 2018) and quality indicators (Amin- zadeh and Kurfess 2018; Zhang et al. 2019). We designed ConvLBM as a modular system that allows on-line quality control and defect detection in manufactured components. Then, we used a technique called transfer learning. This technique applies a second training to the model, using a smaller dataset speciﬁc for every process and laser conﬁg- uration. With transfer learning, ConvLBM can predict new 123 Journal of Intelligent Manufacturing (2020) 31:789–795 791 quality measurements while retaining the knowledge about the process gained from the initial training. Materials and methods To detect defects in images from laser welding, we replaced the output layer with a softmax layer that rep- resents the output as a category. The training mini- mizes the categorical cross entropy on the defects dataset, obtaining a classiﬁcation tool. This tool detects defects “segment-by-segment” in a component that is being man- ufactured. Transfer learning is used in two scenarios. In a ﬁrst exper- iment we used images from the LMD process to estimate dilution, quality indicator that can only be measured trough destructive testing. In a second experiment laser welding images are used to detect defects in the manufactured parts. 1 https://keras.io/. 2 https://www.tensorﬂow.org/. 1 https://keras.io/. 2 https://www.tensorﬂow.org/. Results and discussion Process representation for this experiment, with the objective of making a model capable of extracting meaningful features from raw images. Datasets M2 is known to be more challenging than 316L, and has signiﬁ- cant differences in process dynamics. The dataset contains over 8000 images from 20 clad tracks labeled with the dilu- tion value obtained from a manual inspection by experts. We used a strategy of leave-one-out cross validation, training the system with 7600 images from 19 clad tracks and using the remaining one for validation. The process was repeated 20 times using a different track for validation in every itera- tion. ConvLBM is made of 4 residual blocks followed by one fully connected layer. Residual blocks add the output of a shortcut convolutional layer to a stack of three convolutional layers with ﬁlters of different sizes. We use a ReLU activation and a batch normalization step after every convolution. We trained ConvLBM minimizing a mean squared error loss function on a ﬁrst dataset and used the resulting net as a base model to apply transfer learning in different qual- ity control applications. Thus, we replaced the last layer of ConvLBM and retrained it to measure dilution in LMD appli- cations and to detect defects in laser welding scenarios. The dataset of defects was acquired from a welding pro- cess with an overlap joint laser conﬁguration and DP600 as material. The camera conﬁguration is the same as in the para- metric dataset. We recorded 50 tracks with 24,444 images labeled with a binary classiﬁcation (good joint or defected joint), obtained from a metallographic inspection by experts. The datasets are divided in two sets of 5/6 and 1/6 for the training and validation sets respectively. To estimate the dilution, we added an additional neuron to the output layer in order to represent this variable, and trained the new model using the dilution dataset and minimizing mean squared error. 12 123 792 Journal of Intelligent Manufacturing (2020) 31:789–795 Journal of Intelligent Manufacturing (2020) 31:789 795 ConvLBM architecture lts and discussion ss representation for this experiment, with the objective of making a model capable of extracting meaningful features from raw images. The results prove that ConvLBM is able to capture the process dynamics using a regression analysis. A comparison Fig. 2 ConvLBM architecture Dilution estimation Dilution is a major quality indicator in LMD and cladding applications. During manufacturing, a low dilution may lead to insufﬁcient bound and produce warping, while a value too high indicates large heat affected zones and a high probability of defects due to thermal expansion. Dilution can only be measured with accuracy through metallographic inspection, a destructive procedure that requires cutting the sample. For the second experiment, we used the dilution dataset and applied transfer learning to the model of the previous experiment. The re-trained model was able of predicting dilu- tion measurements (Fig. 4) with a RMSE of 2.8%. Fig. 4 Estimation of dilution by ConvLBM. The magenta line corre- sponds to the mean of predicted values, the green represents 1 standard deviation (68.2% of values) and the blue area represents 3 standard deviations (99.7% of values) It is worth noting that in metallographic inspection, dilution is measured at a speciﬁc transversal cut, and the assumption is made that dilution is constant over the track. In practise, variations of dilution may occur on the track and affect the apparent performance of the model, especially if a peak value occurs in the position of the cut. processes generates residual stresses due to thermal expan- sion, which results in cracks or deformations that weakens the manufactured part. In order to detect these defects, the analysis and visual inspection of the piece is needed. Usu- ally a human operator checks the quality, weld by weld, and remakes the defective pieces so they are compliant with the quality standards. Most common defects are lack of fusion, undercuts, holes and pores. A signiﬁcant outcome of these results is that, despite the base model being built on LMD images and with a differ- ent material known to present a different thermal behaviour. ConvLBM achieved a good generalization from one material to the other. For the third experiment, we used the defects dataset and applied transfer learning to the model of the ﬁrst experiment, with the objective of creating a model able to detect defects during the laser welding process. Process representation The results prove that ConvLBM is able to capture the process dynamics using a regression analysis. A comparison between the true and predicted laser power and laser head speed is shown in Fig. 3. In the ﬁrst experiment, we trained ConvLBM to predict the known laser parameters power and speed, two parameters that drive heat and mass input in a laser process, and thus, are representative of its overall state. The ﬁrst dataset was used ConvLBM achieved an accuracy of about±0.05 kW for power and±1.75 mm/s for speed with over 99% conﬁdence. This accuracy is better than one step in the training stage 3 12 Journal of Intelligent Manufacturing (2020) 31:789–795 793 Fig. 3 Estimation of laser power (a) and laser head speed (b) using ConvLBM. The magenta line corresponds to the mean of predicted values, boxes represent the 50% conﬁdence interval, and bars represent the 99.3% conﬁdence interval Fig. 3 Estimation of laser power (a) and laser head speed (b) using ConvLBM. The magenta line corresponds to the mean of predicted values, boxes represent the 50% conﬁdence interval, and bars represent the 99.3% conﬁdence interval Fig. 4 Estimation of dilution by ConvLBM. The magenta line corre- sponds to the mean of predicted values, the green represents 1 standard deviation (68.2% of values) and the blue area represents 3 standard deviations (99.7% of values) for power (0.1 kW), and slightly higher in the case of speed (1 mm/s). Figure 3 shows the complete validation results. Conclusions Table 1 Confusion matrix for defects in laser welding in the validation We present a novel approach to monitor laser processes in real time based on MWIR imaging. The ConvLBM model is based on a residual CNN architecture trained with a novel dataset of images from LMD to capture the basic dynamics of laser processes and can be extended to different scenarios by modifying the output layer, and re-training the network. This training procedure ﬁts well to common calibration in industry practice. Table 2 Classiﬁcation metrics for the training set and the validation set Training set Validation set ConvLBM is able to estimate the dilution for LMD appli- cations and in the detection of defects for laser welding, obtaining very promising results. In LMD processes, ConvLBM can monitor accurately parametric indicators that can be used in real time appli- cations to deﬁne a control loop that keeps LMD processes in the desired window. For laser welding, we developed an on-line defect detec- tion tool capable of determining the position, size and length of defects. This tool can be easily integrated to increase the overall quality and reliability of the product. tively. Table 1 display the confusion matrices for the training and validation set, and Table 2 summarizes the most com- mon classiﬁcation metrics for both validation and training sets. These results show that ConvLBM can estimate laser parameters from high-speed thermal images and generalizes well to different processes and geometries. Furthermore, the good results of the model in three different materials (316L steel in the process dataset, M2 tool steel in the dilution dataset and DP600 dual phase steel in the defects dataset), proves its ﬂexibility and adaptability to new materials. The results indicate that the model is able to accurately identify defects in laser weld beads from raw IR images. Furthermore, this model was tested in an industrial scenario, detecting defects in real-time. Figure 5 shows an exam- ple of a successful defect detection in the laser welding case. In future work, we will validate ConvLBM with new indi- cators representative of cladding quality such as porosity or strength and more tests performed in a broader range of mate- rials. Monitoring and controlling these indicators is crucial in 3D applications of LMD, and better reference magnitudes for new control systems should be achieved. Detection of defects The detection of defects is a clear quality indicator in indus- trial laser welding scenarios. The thermal cycles in welding ConvLBM achieved in this experiment a F1-Score of 0.975 and 0.974 in the training and validation sets respec- 12 3 Journal of Intelligent Manufacturing (2020) 31:789–795 794 Table 1 Confusion matrix for defects in laser welding in the validation set True defective True non defective Predicted defective 2555 63 Predicted non defective 73 1383 Table 2 Classiﬁcation metrics for the training set and the validation set Training set Validation set Recall (TP rate) 0.973 0.972 Fallout (FP rate) 0.043 0.044 Speciﬁcity (TN rate) 0.957 0.956 Miss rate (FN rate) 0.027 0.028 Precision 0.976 0.976 Accuracy 0.968 0.967 F1 Score 0.975 0.974 References Aminzadeh, M., & Kurfess, T. R. (2018). Online quality inspection using bayesian classiﬁcation in powder-bed additive manufac- turing from high-resolution visual camera images. Journal of Intelligent Manufacturing, 30, 2505–2523. Rodriguez-Araujo, J., Rodríguez-Andina, J. J., Farina, J., Vidal, F., Mato, J. L., & Angeles Montealegre, M. (2012). Industrial laser cladding systems: FPGA-based adaptive control. IEEE Industrial Electronics Magazine, 6(4), 35–46. Bengio, Y. (1997). Convolutional networks for images, speech, and time-series MoDeep view project unsupervised learning of speech representations view project Rosenblatt, F. (1961). Principles of neurodynamics. Perceptrons and the theory of brain mechanisms. Cornell Aeronautical Lab Inc, Buffalo NY Bi, G., Schürmann, B., Gasser, A., Wissenbach, K., & Poprawe, R. (2007). Development and qualiﬁcation of a novel laser-cladding head with integrated sensors. International Journal of Machine Tools and Manufacture, 47(3–4), 555–561. Song, L., Bagavath-Singh, V., Dutta, B., & Mazumder, J. (2012). Con- trol of melt pool temperature and deposition height during direct metal deposition process. The International Journal of Advanced Manufacturing Technology, 58(1–4), 247–256. Doubenskaia,M.,Bertrand,P.,&Smurov,I.(2004).Opticalmonitoring of Nd: YAG laser cladding. Thin Solid Films, 453, 477–485. Tapia, G., & Elwany, A. (2014). A review on process monitoring and control in metal-based additive manufacturing. Journal of Manu- facturing Science and Engineering, 136(6), 60801. Garcia, A., Pallas, A., & Panadeiro, V. (2018). A modular approach to online monitoring for laser-based 3D printing using deep mwir features. Robotics and Computer Integrated Manufacturing. Hauser, D. P., & De Weck, O. L. (2007). Flexibility in compo- nent manufacturing systems: Evaluation framework and case study. Journal of Intelligent Manufacturing. https://doi.org/10. 1007/s10845-007-0033-9. You, D., Gao, X., & Katayama, S. (2015). WPD-PCA-based laser welding process monitoring and defects diagnosis by using FNN and SVM. IEEE Transactions on Industrial Electronics, 62(1), 628–636. He, K., Zhang, X., Ren, S., & Sun, J. (2016). Deep residual learning for image recognition. In Proceedings of the IEEE conference on computer vision and pattern recognition (pp. 770–78). Zhang, Y., You, D., Gao, X., Wang, C., Li, Y., & Gao, P. P. (2019). Real-time monitoring of high-power disk laser welding statuses based on deep learning framework. Journal of Intelligent Manu- facturing. https://doi.org/10.1007/s10845-019-01477-w. Hofman, J. T., Pathiraj, B., Van Dijk, J., de Lange, D. F., & Meijer, J. (2012). A camera based feedback control strategy for the laser cladding process. Journal of Materials Processing Technology, 212(11), 2455–2462. Zhong, C., Gasser, A., Kittel, J., Schopphoven, T., Pirch, N., Jinbao, F., et al. (2015). Compliance with ethical standards Conﬂicts of interest Carlos Gonzalez-Val, Adrián Pallas, Verónica Panadeiro and Alvaro Rodríguez declare that there is no conﬂict of interest regarding the publication of this paper. Liu, G., Gao, X., You, D., & Zhang, N. (2019). Prediction of high power laser welding status based on PCA and SVM classiﬁcation of multiple sensors. Journal of Intelligent Manufacturing, 30(2), 821–832. https://doi.org/10.1007/s10845-016-1286-y. Meriaudeau, F., & Truchetet, F. (1996). Control and optimization of the laser cladding process using matrix cameras and image pro- cessing. Journal of Laser Applications, 8(6), 317–324. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecomm ons.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. Panadeiro-Castro, V., Rodriguez-Araujo, J., Garcia-Diaz, A., & Ver- gara, G. (2018). Medium wavelength infrared (MWIR) imaging for high speed control of laser metal deposition (LMD). Lasers in Engineering (Old City Publishing) 39. Pinkerton, A. J. (2015). Advances in the modeling of laser direct metal deposition. Journal of Laser Applications, 27(S1), S15001. Rodríguez-Araújo, J., Garcia-Diaz, A., Panadeiro, V., & Knaak, C. (2017). Uncooled MWIR PbSe technology outperforms CMOS in RT closed-loop control and monitoring of laser processing. In Applied industrial optics: Spectroscopy, imaging and metrology, ATh2A–2. Optical Society of America. Conclusions It is important to note that, like in the dilution estimation, ConvLBM achieved a good generalization to the new mate- rial used in this experiment, which restates the generalization capabilities of the algorithm. Fig. 5 Real-time defect detection on a laser weld bead. a and c show two side views of the weld bead where the blue rectangles mark a defective section in the ﬁrst and ﬁnal segments due to undercuts and the yellow ellipses mark a region where some points have excessive porosity. c and d show the corresponding output of ConvLBM where red colour show frames labelled as defective and green show non defective frames 123 12 3 795 Journal of Intelligent Manufacturing (2020) 31:789–795 Acknowledgements This publication and research was partially funded by European Union’s Horizon 2020 research and innovation programme funds under GA ref. H2020-FoF-2015-680481 and GA ref. H2020–FoF-2016-723945. Iravani-Tabrizipour, M., & Toyserkani, E. (2007). An image-based fea- ture tracking algorithm for real-time measurement of clad height. Machine Vision and Applications, 18(6), 343–354. Jager, M., & Hamprecht, F. A. (2009). Principal component imagery for the quality monitoring of dynamic laser welding processes. IEEE Transactions on Industrial Electronics, 56(4), 1307–1313. Author contributions Carlos Gonzalez-Val wrote the main manuscript text and performed the experiments with the assistance of Verónica Panadeiro, Adrián Pallas implemented ConvLBM and Alvaro Rodríguez revised and edited the manuscript. Krizhevsky, A., Sutskever, I., & Hinton, G. E. (2012). Imagenet clas- siﬁcation with deep convolutional neural networks. In Advances in neural information processing systems (pp. 1097–1105). Kwon, O., Kim, H. G., Ham, M. J., Kim, W., Kim, G.-H., Cho, J.-H., et al. (2018). A deep neural network for classiﬁcation of melt-pool images in metal additive manufacturing. Journal of Intelligent Manufacturing,https://doi.org/10.1007/s10845-018-1451-6. Data availability All the data used to support the ﬁndings of this study have been deposited in a public repository and their DOI link will be provided with the ﬁnal version of this paper. Lapido, Y. L., Rodriguez-Araújo, J., García-Díaz, A., Castro, G., Vidal, F., Romero, P., & Vergara, G. (2015). Cognitive high speed defect detection and classiﬁcation in MWIR images of laser weld- ing. In Industrial laser applications symposium (ILAS 2015), 9657:96570B. References Study of process window development for high deposition-rate laser material deposition by using mixed process- ing parameters. Journal of Laser Applications, 27(3), 32008. Hu, D., & Kovacevic, R. (2003). Sensing, modeling and control for laser-based additive manufacturing. International Journal of Machine Tools and Manufacture, 43(1), 51–60. Publisher’s Note Springer Nature remains neutral with regard to juris- dictional claims in published maps and institutional afﬁliations. Ioffe, S., & Szegedy, C. (2015). Batch normalization: accelerating deep network training by reducing internal covariate shift. 12 3
https://openalex.org/W1829569078	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0141669&type=printable	English	null	Impact of Diverse Immune Evasion Mechanisms of Cancer Cells on T Cells Engaged by EpCAM/CD3-Bispecific Antibody Construct AMG 110	PloS one	2,015	cc-by	10,149	Background Citation: Deisting W, Raum T, Kufer P, Baeuerle PA, Münz M (2015) Impact of Diverse Immune Evasion Mechanisms of Cancer Cells on T Cells Engaged by EpCAM/CD3-Bispecific Antibody Construct AMG 110. PLoS ONE 10(10): e0141669. doi:10.1371/journal. pone.0141669 Bispecific T cell engager (BiTE1) are single-chain bispecific antibody constructs with dual specificity for CD3 on T cells and a surface antigen on target cells. They can elicit a poly- clonal cytotoxic T cell response that is not restricted by T cell receptor (TCR) specificity, and surface expression of MHC class I/peptide antigen complexes. Using human EpCAM/CD3- bispecific BiTE1 antibody construct AMG 110, we here assessed to what extent surface expression of PD-L1, cytoplasmic expression of indoleamine-2,3-deoxygenase type 1, Bcl- 2 and serpin PI-9, and the presence of transforming growth factor beta (TGF-β), interleukin- 10 (IL-10) and adenosine in culture medium can impact redirected lysis by AMG 110- engaged T cells. Editor: Lucienne Chatenoud, Université Paris Descartes, FRANCE Received: January 26, 2015 Accepted: October 12, 2015 Published: October 28, 2015 Copyright: © 2015 Deisting 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. Impact of Diverse Immune Evasion Mechanisms of Cancer Cells on T Cells Engaged by EpCAM/CD3-Bispecific Antibody Construct AMG 110 Wibke Deisting1, Tobias Raum1, Peter Kufer1, Patrick A. Baeuerle2, Markus Münz1* Wibke Deisting1, Tobias Raum1, Peter Kufer1, Patrick A. Baeuerle2, Markus Münz1* 1 Amgen Research (Munich) GmbH, Munich, Germany, 2 MPM Capital, Cambridge, Massachusetts, United States of America Wibke Deisting1, Tobias Raum1, Peter Kufer1, Patrick A. Baeuerle2, Markus Münz1* 1 Amgen Research (Munich) GmbH, Munich, Germany, 2 MPM Capital, Cambridge, Massachusetts, United States of America a1111 * mmuenz@amgen.com * mmuenz@amgen.com Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: Funding of this study has been supplied by AMGEN Inc., a publically listed company who provided support in the form of salaries for authors WD, TR, PK and MM, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. AMGEN supported this study, however, because of an interest in the results of this research. MPM Capital provided support in the form of a salary for author PAB, who was formerly employed by AMGEN RESEARCH ARTICLE Methods The seven factors, which are all involved in inhibiting T cell functions by cancer cells, were tested with human EpCAM-expressing Chinese hamster ovary (CHO) target cells at levels that in most cases exceeded those observed in a number of human cancer cell lines. Co- culture experiments were used to determine the impact of the evasion mechanisms on EC50 values and amplitude of redirected lysis by AMG 110, and on BiTE1-induced prolifera- tion of previously resting human peripheral T cells. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Introduction Therapies engaging a patient’s cytotoxic T cell response have proven to effectively treat and eventually cure cancer. For instance, adoptive transfer of ex-vivo expanded tumor-resident T cells [1], inhibition of immune escape by the PD-1/PD-L1 axis by monoclonal antibodies (mAbs) [2], intra-lesional injection of an oncolytic virus [3], or enhancing T cell differentiation and depleting regulatory T cells by CTLA-4-antagonistic mAbs [4] have all shown partial and complete responses in late-stage melanoma, a positive impact on progression-free or overall survival, and long-term remission if not cure in a small proportion of patients. Currently, response rates of these approaches are limited, which is why extensive biomarker programs aim at understanding resistance and multiple clinical programs search for combinations poten- tially increasing response rates and long-term benefit. All of the above approaches enable the generation, expansion and systemic spread of tumor-specific T cell clones that recognize cancer cells by their specific MHC class I/peptide complexes. BiTE1 antibody constructs engage cytotoxic T cells by a fundamentally different mecha- nism [5]. They use a soluble adapter to connect a surface target antigen on cancer cells—as is typically recognized by monoclonal antibody therapies—with the invariant CD3ε subunit of any T cell receptor (TCR) on T cells. Potentially all pre-existing cytotoxic T cells in a patient can be engaged by this approach, of which effector memory T cells seem to make the dominant contribution to anti-tumor activity [6]. With the BiTE1 technology, T cell recognition and activation is no longer dependent on T cell clones bearing a specific TCR, not on transport and expression of MHC I molecules to the cancer cell surface, or on the proteolytic generation, transport and surface display of peptide antigens [5]. The CD19/CD3-bispecific blinatumomab has provided clinical proof-of-concept that this non-natural engagement of T cells is highly effective and can elicit in a large proportion of ALL and NHL patients meaningful clinical responses [7–9]. Blinatumomab (Blincyto™) was recently approved by the FDA for treatment of patients with Philadelphia chromosome-negative relapsed/refractory B cell precursor ALL. Here, we used AMG 110, a well characterized EpCAM/CD3-bispecific BiTE1 antibody con- struct that is clinically being tested in late-stage cancer patients with different carcinomas [10, 11]. Cancer cells can be selected during tumor progression for numerous immune evasion mech- anisms, which for instance can impact MHC class I/peptide presentation [12, 13], or the gener- ation, differentiation, survival, migration and expansion of specific cytotoxic T cell clones. Impact of Immune Escape Mechanisms on BiTE Conclusions Research Munich, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of this author is articulated in the ‘author contributions’ section. Our data suggest that diverse mechanisms employed by cancer cells to fend off T cells can- not inactivate AMG 110-engaged T cells, and that inhibitory effects observed in vitro may be overcome by increased concentrations of the BiTE1 antibody construct. Competing Interests: Funding of this study has been supplied by AMGEN Inc., the employer of Wibke Deisting, Tobias Raum, Peter Kufer and Markus Münz and the former employer of Patrick A. Baeuerle who is now employed by MPM Capital. All authors have equity positions in the company. AMGEN is focused on the development of BiTE antibodies for the treatment of malignant diseases. AMGEN is the maker of AMG 110. Besides AMG 110 that was used for this study the BiTE platform holds other BiTE molecules in the pipeline and has one product marketed named BLINCYTO. The authors Tobias Raum, Peter Kufer, Markus Münz and Patrick A. Baeuerle hold various patents relevant for the BiTE platform, including a patent for AMG 110. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors. Findings An inhibitory effect on redirected lysis by AMG 110-engaged T cells was seen upon overex- pression of serpin PI-9, Bcl-2, TGF-βand PD-L1. An inhibitory effect on induction of T cell proliferation was only seen with CHO cells overexpressing IDO. In no case, a single evasion mechanism rendered target cells completely resistant to BiTE1-induced lysis, and even various combinations could not. 1 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0141669 October 28, 2015 NE \| DOI:10.1371/journal.pone.0141669 October 28, 20 PLOS ONE \| DOI:10.1371/journal.pone.0141669 October 28, 2015 Reagents AMG 110 was provided by Amgen Research (Munich) GmbH. It was produced by recombi- nant DNA technology and purified via a C-terminal hexa-histidine tag as previously described [24]. Impact of Immune Escape Mechanisms on BiTE T cell proliferation in response to AMG 110 were used as readouts for cytotoxic T cell performance. T cell proliferation in response to AMG 110 were used as readouts for cytotoxic T cell performance. T cell proliferation in response to AMG 110 were used as readouts for cytotoxic T cell performance. With PD-L1, adenosine, IL-10 and TGF-β we explored factors frequently produced by can- cer cells that bind negative regulatory surface receptors expressed on cytotoxic T cells. PD-L1 is expressed on the surface of tumor cells and prevention of binding its inhibitory receptor PD-1 on T cells by mAb monotherapies was shown to have pronounced anti-tumor activity in clini- cal trials [14, 15]. Adenosine can be synthesized by cancer cells and binds the A2a and A3 adenosine receptors on T cells, which can inhibit effector functions [16, 17]. IL-10 is considered an anti-inflammatory cytokine with broad function. Because cancer cells can secrete IL-10, a role in the escape of immunosurveillance has been ascribed to IL-10 in tumors [18]. By tran- scriptional repression, TGF-β secreted by cancer cells can broadly downmodulate effector T cell functions, for instance, by reducing expression of granzymes A and B, perforin and inter- feron-gamma (IFNγ) upon receptor binding [19]. The cytoplasmic protease inhibitor serpin PI-9 can directly block the enzymatic activity of granzyme B that is eventually delivered into the cytosol of cancer cells through a cytolytic synapse as formed by T cells [20]. With Bcl-2, we studied an anti-apoptotic cytosolic protein that can decrease the susceptibility of cancer cells to induction of apoptosis by granzyme B [21]. With IDO, we explored a catabolic enzyme that when overexpressed by cancer cells can starve the microenvironment of the essential amino acid tryptophane and release immunosuppressive metabolites, like l-kynurenine or 3-hydroxy- kynurenine, which can tolerize T cells [22, 23]. With the exception of IFNγ-induced IDO, all other evasion factors were studied at levels in transfected CHO cells that by far exceeded those found in six human cancer cell lines. Despite studying obviously high levels of evasion factors, we have observed only modest effects of some on redirected lysis and induction of T cell proliferation. In most cases, inhibitory effects could be compensated by higher dosing of BiTE1 antibody construct AMG 110, or be reversed by pharmacological means. The BiTE1-engaged T cells may have the potential to be active against cancer cells expressing a variety of frequent immune evasion mechanisms. Introduction In the present study, we investigated to what extent seven frequent evasion mechanisms impact the BiTE1 mode of action, which can potentially engage any pre-existing cytotoxic T cell in patients. We therefore focused on those mechanisms that can potentially impact cytotoxic T cell performance and left out those that, for instance, impair specific T cell recognition by MHC I/peptide complexes. To this end, we established rodent CHO cell lines expressing human EpCAM as surface target antigen that either overexpress proteins known to have immune evasion potential or where the immunosuppressive factor was added to the culture medium. Potency and amplitude of redirected CHO-EpCAM target cell lysis and induction of PLOS ONE \| DOI:10.1371/journal.pone.0141669 October 28, 2015 2 / 16 Antibodies The following antibodies were applied. For flow cytometry: mouse anti-human PD-L1-APC (M1H1, eBiosciences), mouse anti-human CD3-FITC (UCHT1; BD Biosciences), mouse anti- human CD25-APC (M-A251; BD Biosciences), mouse anti-human EpCAM (B302/323/A3; Abcam), goat anti-Mouse IgG (H+L)-APC (Jackson Immuno Research), mouse anti-human TGF-β-PE (9016; R&D Systems); for intracellular FACS or Western blot analysis: mouse anti- human PI-9 (7D8; Abcam), mouse anti-human GrB-APC (GB11; BD Biosciences), mouse anti-human Bcl-2 (100/D5; Thermo Scientific), mouse anti-human β-Actin (AC-15; Thermo Scientific), rabbit anti-human IDO (Abcam), goat anti-Mouse and anti-Rabbit HRP-coupled detection antibodies (Thermo Scientific); stimulating and neutralizing antibodies: mouse anti- human CD3 (OKT-3; Janssen-Cilag), mouse anti-human CD28 (L293; BD Biosciences), mouse anti-human TGF-β (1D11; R&D Systems) and mouse anti-human PD-L1 (M1H1; eBiosciences) 3 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0141669 October 28, 2015 Impact of Immune Escape Mechanisms on BiTE Human tumor cell lines and cell culture A549 [25], BxPC3 [26], KATOIII [27], SKBR3 [28] cells were all grown in RPMI1640 medium (Biochrom AG) supplemented with 10% fetal calf serum (Biochrom AG), 1% penicillin/ streptomycin (Biochrom AG), 50 μM 2-mercaptoethanol (Gibco), 1% nonessential amino acids (Biochrom AG), 1 mM sodium pyruvate (Biochrom AG) and 1 mM HEPES (Biochrom AG). SW480 [29] cells were grown in RPMI1640 medium supplemented with 10% fetal calf serum and 1% penicillin/streptomycin. A431 [25] cells were grown in DMEM (Biochrom AG) with 10% fetal calf serum and 1% penicillin/streptomycin. All cell lines were cultured according to standardized cell culture procedures. For induction of IDO and PD-L1 proteins, cells were treated for 48 h with 1000 U/ml IFNγ (Sigma-Aldrich). Construction, cultivation and testing of escape protein and of stably human EpCAM-expressing CHO cell lines Parental chinese hamster ovary (CHO) cells expressing full-length human EpCAM under the control of the EF1α promotor (provided by Amgen Research (Munich) GmbH) were stably transfected with a second expression plasmid coding for either human PI-9, IDO, Bcl-2, PD-L1, TGF-β1, IL-10 or the corresponding mock vector. Except for Bcl-2 (generated by gene synthesis; Geneart), all escape proteins were cloned from human cancer cell lines. Subsequent selection and amplification of protein expression were achieved by addition of methotrexate (Sigma-Aldrich), L-alanosine (TRC, Toronto) and dcF (Hospira). CHO EpCAM: Escape protein double-transfectants were grown in suspension in HyQ medium (HyClone) supplemented with 1% penicillin/streptomycin (Biochrom AG), 500 nM methotrexate, 10 mM HEPES (Biochrom AG), 1.1 mM adenosine (Sigma-Aldrich), 50 μM L-alanosine, 1 mM uri- dine (Sigma-Aldrich) and 0.1–1 μM dcF at 37°C in 5% CO2 humidified incubator. Expression of membrane-bound proteins EpCAM and PD-L1 and membrane-bound TGF- β was analyzed by flow cytometry, expression of PI-9 by intracellular FACS or Western blot analysis. For comparison of expression levels, the relative median (= median sample/median control) of the samples were calculated. Expression of intracellular IDO and Bcl-2 was deter- mined by Western blot analysis and quantified with Image J software. Expression levels of secreted IL-10 and TGF-β proteins were assessed in cell supernatant of 2.5x 105/ml after 48 h of culturing by ELISA using Quanti Glo IL-10 Elisa Kit (R&D Systems) and a Tecan Spectra- Fluor Plus (MTX Lab Systems) for detection or TGF beta Elisa Kit (Abcam) and an ELISA reader Power Wave X (BioTec Instruments) for detection. For functional analysis of TGF-β secreted from CHO transfectants, cell culture supernatant from 2.5x 105/ ml was collected after 48 h. One x106 CD3+ T cells were stimulated for 96 h with plate-bound CD3/CD28/IL-2 in the supernatant mixed with fresh medium (25% medium: 75% supernatant). Effects of TGF-β were analyzed by determining the expression of GrB by intra- cellular FACS analysis. The same assay was used for functional analysis of recombinant TGF-β and TGF-β neutralizing antibody. For functional analysis of adenosine effects, T cells were stimulated by plate-bound CD3/CD28/IL-2 with and without 1 mM adenosine. Effects of aden- osine were determined by measuring CD25 expression by FACS analysis after 24 or 96 h. FACS-based cytotoxicity assays Target cells were labeled with Vybrant DIO or DID (Life Technologies) fluorescent membrane dye, adjusted to 1.25x 105 cells/ml and co-incubated in 96-well plates with freshly isolated CD3+ T effector cells at an effector-to-target (E:T) ratio of 4:1 or with pre-stimulated CD8+ T effector cells at an E:T ratio of 1:1 in the presence of AMG 110 serial dilution. Further additions were added directly to the assay medium. Incubation was conducted at 37°C, in a 5% CO2 humidified atmosphere. After 24–72 h, living and dead cells were collected, resuspended in PBS/2% FCS with 1 μg/ml propidium iodide (PI) (Sigma-Aldrich), and analyzed in a FACS Canto™II flow cytometer (Becton Dickinson) equipped with FACSDiva software. Cytotoxicity was determined in duplicates by quantifying the labeled and PI positive non-viable target cells and the labeled PI negative target cells. Percentage of lysis was analyzed with respect to BiTE1 concentration using Prism 5 software (GraphPad). Analysis with Prism 5 included creation of sigmoidal dose-response curves, calculation of EC50 values as well as determination of top and bottom percentage of BiTE1-dependent lysis for calculation of lysis amplitude for a given time point. In order to compare the impact of escape proteins on BiTE1-induced redirected lysis relative change in EC50 was calculated by the formula: (EC50 values of escape transfectants)/ (EC50 values of parental control cells) and relative change in amplitude values by (amplitude of Escape transfectants)/(amplitude of parental control cells). Each assay was at least performed in duplicates. Impact of Immune Escape Mechanisms on BiTE was separated between different studies. Before each donation, volunteers provided a written consent for their participation in medical research. To protect the health of the donors, the blood pressure was measured and donors were asked about their current state of health before taking the blood. Moreover, blood values of each volunteer were checked quarterly by an exter- nal laboratory and strict intervals between two donations were kept. Each donor only supplied a small volume of blood not exceeding the range of blood samples drawn for minor investiga- tions during preventive medical checkups. Therefore, no permission of an ethic committee was sought. PBMCs were isolated from heparinized blood by Ficoll (Biochrom) density gradient centrifugation using standard procedures. For elimination of residual erythrocytes, cells were incubated with erythrocyte lysis buffer (155 mM NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA) for 5 min at 4°C. PBS Dulbecco (Biochrom) with 2% FCS was added to stop the reaction. After a centrifugation for 5 min at 600 x g, the supernatant with lyzed erythrocytes was discarded. CD3+ T cells were obtained by MACS magnetic bead cell separation using the pan T cell Isola- tion kit II (Miltenyi). CD8+ T cells were separated from freshly isolated or pre-stimulated PBMCs by either depletion of CD4+ and CD56+ cells using mouse anti-human CD4 (L200; BD, Heidelberg) and mouse anti-human CD56 antibodies (B159; BD), sheep anti-mouse IgG beads (Invitrogen) and Dynal magnets, or by using the MACS CD8+ Isolation Kit (Miltenyi). T cells were stimulated with plate-bound anti-CD3 (OKT-3; Janssen-Cilag) and anti-CD28 (BD) antibodies and 20 U/ml recombinant human interleukin-2 (IL-2, Chiron Corperation Ltd) in RPMI1640 medium (Biochrom AG), supplemented with the same ingredients as listed above. Supplemented RPMI was used for cultivation of T cell and all in vitro assays. Further additions as adenosine (Sigma-Aldrich), tryptophane (Sigma-Aldrich) recombinant cytokines (Miltenyi), or neutralizing antibodies were added directly into the culture medium at the beginning of the incubation period. T-cell isolation, stimulation and in vitro assays Human blood was donated by healthy volunteers, who were medical illuminated and registered in an internal blood bank of Amgen Research (Munich) GmbH. Each donor was assigned a number that was used for naming the blood samples. The blood was obtained directly before usage by a trained team not including the authors of this paper. In some cases the blood drawn 4 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0141669 October 28, 2015 Transfected CHO cell lines express high levels of human immune evasion factors Stably transfected, human EpCAM-expressing CHO cells were characterized for expression levels of human immune evasion proteins in comparison to natural levels found in six human cancer cell lines A549 (lung), BxPC3 (prostate), KATOIII (gastric), SKBR3 (breast), SW480 (colorectal), and A431 (skin). For IDO and PD-L1, cancer cells were in addition stimulated for 48 h with 1,000 U/ml IFNγ, which is known to induce the proteins [30, 31]. Various methods were employed to quantify and compare expression levels, including FACS (for serpin PI-9, PD-L1, EpCAM and TGF-β), ELISA (for TGF-β and IL-10) and Western blotting (for Bcl-2 and IDO; see analyses in S1 Fig). Western blot analyses confirmed the correct molecular size and the use of specific detection antibodies the identity of the respective proteins in cancer cell lines and transfected CHO cells. A summary of expression data shows that—apart from IDO after stimulation of A431, A549, BxPC3 and KATOIII cells with IFNγ—in all cases evasion proteins were expressed in stably transfected CHO cells to a much higher level than in the six cancer cell lines. The expression of the target antigen EpCAM in most cell lines (A431, BxPC3 and KATOIII, SkBR3 and SW480) was in the range of CHO transfectants (Factor 2–9 fold less) whereas A549 showed only moderate EpCAM expression (50x less than transfected CHO cells) (Table 1). CFSE-based proliferation assays To assess the proliferative potential of T cells in the presence of immune escape proteins, T cells were labeled with carboxyfluoresceine diacetate succinimidyl esther (CFSE) according to 5 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0141669 October 28, 2015 Impact of Immune Escape Mechanisms on BiTE the manufacturer`s instructions (Molecular Probes). Labeled T cells were co-incubated with CHO cell lines stably expressing EpCAM and one of the evasion proteins or control cells +/- adenosine in an incubator with 37°C, 5% CO2 atmosphere for 120 h in 48-well flat bottom plates (Nunc) in the presence or absence of 1 μg/ml AMG 110. The number of T cells was kept constant at 1x 104. CHO cells were washed twice to remove selection medium and were adjusted to E:T ratios of 1:8, 1:1 and 4:1. After the incubation period, proliferation of CD3+ T cells was determined by monitoring CFSE distribution by flow cytometry using a FACS Canto™ II flow cytometer and FACS DIVA™software. Subsequent analysis was performed using FlowJo 7.6.5 software. All assays were performed at least in duplicates. Statistical analysis Statistical analysis of EC50 values and amplitudes significant outliers were excluded with the Grubb0s test. Afterwards p values were calculated with Prism 5 (GraphPad Software) using unpaired t tests with Welch0s correction. Impact of Immune Escape Mechanisms on BiTE Table 1. Evasion protein expression in human cancer cell lines and stably expressing CHO transfectants. Rel. Expression Level [%] CHO transfectants A431 A549 BxPC3 KATOIII SKBR3 SW480 PI-9 100 9.5 20.1 11.1 7.1 18.5 13.2 Bcl-2 100 1 0 1.8 0.2 0.8 0 IDO 100 0 0 0 0 0 0 IDO + IFNγ 100 124.2 413.5 745.7 166.6 n.d.a 0 IL-10 100 0.9 0.7 0.3 1.2 0 18.7 TGF-β 100 0.03 0.2 0.03 0 0.06 0.2 PD-L1 100 2.4 0.08 0.4 0.08 0.3 0.08 PD-L1 + IFNγ 100 2.5 0.6 1.3 0.3 0.1 0.08 EpCAM 100 10.7 1.9 11.1 45.5 20.4 35.0 PI-9, PD-L1 and EpCAM expression was determined by FACS analysis, Bcl-2 and IDO expression was assessed by Western blot and subsequent analysis with Image J software, and TGF-β and IL-10 were detected with speciﬁc ELISA assays. The expression level observed in the CHO EpCAM transfectants was set to 100%. Stimulation of cells with IFNγ was at 1000 U/ml for 48 h. an.d. = not determined. ression in human cancer cell lines and stably expressing CHO transfectants. Table 1. Evasion protein expression in human cancer cell lines and stably expressing CHO transfectants. Table 1. Evasion protein expression in human cancer cell lines and stably exp PI-9, PD-L1 and EpCAM expression was determined by FACS analysis, Bcl-2 and IDO expression was assessed by Western blot and subsequent analysis with Image J software, and TGF-β and IL-10 were detected with speciﬁc ELISA assays. The expression level observed in the CHO EpCAM transfectants was set to 100%. Stimulation of cells with IFNγ was at 1000 U/ml for 48 h. an.d. = not determined. doi:10.1371/journal.pone.0141669.t001 factor was only noted for cells expressing IDO. At all three E:T ratios, AMG 110 could barely induce T cell proliferation in CHO-EpCAM cells stably transfected with human IDO cDNA. A missing impact of TGF-β and IL-10 was confirmed by addition of recombinant factors to the culture medium (see S2A Fig). For the experiments, three different human PBMC donors were used. one of them showed a weaker AMG 110 dependent proliferation (see Adenosine E. T = 1:1 and 4:1). IDO showed pronounced inhibitory effect on AMG 110-induced T cell proliferation AMG 110 and other BiTE1 antibody constructs have the capability to potently activate T cells, but only in the presence of target cells [32]. We here investigated the impact of seven immune escape factors on initiation of T cell proliferation by AMG 110 at three different effector to tar- get (E:T) ratios after co-culture for 120 h. In the absence of AMG 110, none of the transfected or control CHO cell lines elicited a proliferation of the CD3+ T cell population within added peripheral mononuclear cells (PBMCs) as monitored by FACS using CSFE staining (Fig 1). In the presence of 1 μg/ml AMG 110, control CHO-EpCAM cells (black) as well as CHO cells with evasion factors (red) showed strong T cell proliferation signals indicative of multiple rounds of cell cycling (Fig 1). Somewhat stronger signals were observed with increasing E:T cell ratios (compare E:T ratio of 1:8 with 4:1). A significant difference between control and evasion 6 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0141669 October 28, 2015 Serpin PI-9, TGF-β, Bcl-2 and PD-L1 reduce the potency of redirected target lysis by AMG 110 Using purified CD8+ T cells from healthy human PBMC donors, AMG 110 dose response anal- yses for redirected lysis compared EC50 values and the percentage of lysis between CHO-Ep- CAM control cells (black) and target cells in the presence of stably expressed or added evasion factors (red) (Fig 2). Co-culture was for 24 hours at an E:T ratio of 1:1. Depending on T cell donors, basal EC50 values for redirected lysis varied between 5 and 200 pg/ml AMG 110. The percentage of cell lysis ranged between 40 and 90%. Discrete shifts of dose response curves to higher EC50 values and lower percentages of lysis were noted for several evasion factors (Fig 2A). However, in no case, the presence of an evasion factor did abrogate redirected lysis. A quantitation of results is shown in Fig 2B and 2C. The strongest inhibitory effect on the potency of redirected lysis was seen with the granzyme B inhibitor serpin PI-9 showing a mean 18-fold increase in EC50 values (Fig 2A and 2B), with a trend toward statistical significance (p = 0.07; N = 5). Second strongest was the effect of surface expression of PD-L1 on CHO-EpCAM cells with a >5-fold reduction in potency reaching sta- tistical significance (p = 0.04; N = 7). Expression of TGF-β induced a 3-fold reduction in the potency of lysis with a strong trend (p = 0.06; N = 5). An inhibitory effect was confirmed by addition of recombinant TGF-β to the culture medium of CHO-EpCAM control cells (see S2B Fig). The >4-fold inhibitory effect of Bcl-2 on the EC50 value of lysis showed a weak trend (p = 0.09; N = 3), and a statistically significant albeit small reduction (see Fig 2C) in the per- centage of lysed cells (p = 0.01; N = 4). None of the other effects on the efficacy of lysis or per- centage of lysed cells described in Fig 2 reached statistical significance or showed a strong trend (S3A and S3B Fig). Similar results were obtained when CD3+ T cells were used instead of CD8+ T cells as effector cell population (see S4 Fig). 7 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0141669 October 28, 2015 Impact of Immune Escape Mechanisms on BiTE Fig 1. Impact of diverse immune evasion mechanisms on AMG 110-mediated T cell proliferation. Serpin PI-9, TGF-β, Bcl-2 and PD-L1 reduce the potency of redirected target lysis by AMG 110 Human CD3+ T cells were labeled with CFSE and co-cultured at effector to target (E:T) ratios of 1:8, 1:1 and 4:1 in 48-well plates with CHO cell lines expressing human EpCAM and one of the six stably transfected immune evasion proteins (PI-9, Bcl-2, IDO, IL-10, TGF-β or PD-L1) (red), or with parental EpCAM+ CHO cells in the presence of 1 mM adenosine (ADO) in the culture medium (black: parental cells). CFSE signals in cells were analyzed by flow cytometry. For each evasion protein one representative experiment is shown. Co- culture in the absence of AMG 110 did not gave CSFE signals, whereas co-culture in the presence of 1 μg/ml AMG 110 showed cycles of cell division affected by E:T ratio. Three different human PBMC were used. doi:10.1371/journal.pone.0141669.g001 Fig 1. Impact of diverse immune evasion mechanisms on AMG 110-mediated T cell proliferation. Human CD3+ T cells were labeled with CFSE and co-cultured at effector to target (E:T) ratios of 1:8, 1:1 and 4:1 in 48-well plates with CHO cell lines expressing human EpCAM and one of the six stably transfected immune evasion proteins (PI-9, Bcl-2, IDO, IL-10, TGF-β or PD-L1) (red), or with parental EpCAM+ CHO cells in the presence of 1 mM adenosine (ADO) in the culture medium (black: parental cells). CFSE signals in cells were analyzed by flow cytometry. For each evasion protein one representative experiment is shown. Co- culture in the absence of AMG 110 did not gave CSFE signals, whereas co-culture in the presence of 1 μg/ml AMG 110 showed cycles of cell division affected by E:T ratio. Three different human PBMC were used. doi:10.1371/journal.pone.0141669.g001 doi:10.1371/journal.pone.0141669.g001 doi:10.1371/journal.pone.0141669.g001 Impact of Immune Escape Mechanisms on BiTE Fig 2. Impact of diverse immune evasion mechanisms on AMG 110-mediated redirected lysis of target cell. AMG 110 dose-dependent lysis was compared between parental EpCAM+ CHO cells and EpCAM+ CHO cells stably transfected with one of six human evasions proteins or in the presence of 1 mM adenosine. Human CD8+ T cells were used as effector cells at an E:T ratio of 1:1. Percentage of target cell lysis after co-culture for 24 h and EC50 values from sigmoidal dose-response response curves were determined in a FACS-based cytotoxicity assay. (A) Representative dose response curves are shown for parental EpCAM+ CHO cells (black) and EpCAM+ CHO cells expressing evasion proteins or incubated in the presence of 1 mM adenosine (red). (B) Mean EC50 values for redirected lysis were calculated from the indicated number of independent experiments. The relative change in EC50 values for lysis was determined by dividing the mean of EC50 values from evasion conditions by the mean of EC50 values from control conditions. Error bars represent SEM values. (C) Relative changes in the percentage of target cell lysis after 24 h at the plateau of dose response curves. The mean amplitude of lysis under evasion conditions was divided by the mean amplitude of lysis under control conditions. Error bars represent SEM values. doi:10.1371/journal.pone.0141669.g002 Fig 2. Impact of diverse immune evasion mechanisms on AMG 110-mediated redirected lysis of target cell. AMG 110 dose-dependent lysis was compared between parental EpCAM+ CHO cells and EpCAM+ CHO cells stably transfected with one of six human evasions proteins or in the presence of 1 mM adenosine. Human CD8+ T cells were used as effector cells at an E:T ratio of 1:1. Percentage of target cell lysis after co-culture for 24 h and EC50 values from sigmoidal dose-response response curves were determined in a FACS-based cytotoxicity assay. (A) Representative dose response curves are shown for parental EpCAM+ CHO cells (black) and EpCAM+ CHO cells expressing evasion proteins or incubated in the presence of 1 mM adenosine (red). (B) Mean EC50 values for redirected lysis were calculated from the indicated number of independent experiments. The relative change in EC50 values for lysis was determined by dividing the mean of EC50 values from evasion conditions by the mean of EC50 values from control conditions. Error bars represent SEM values. The inhibitory activities of IDO, PD-L1, and TGF-β can be reversed by pharmacological means IDO was the only evasion factor in this study leading to strong inhibition of AMG 110-induced T cell proliferation (see Fig 1). To demonstrate that the inhibition was due to the enzymatic activity of IDO, we added 0.5 μM tryptophane to the cell culture medium and analyzed cell cycling by CFSE-based FACS. As shown in Fig 3A, replenishment with tryptophane completely reversed the inhibition of AMG 110-induced cell cycling in IDO-expressing CHO-EpCAM cells. The inhibitory effect seen upon expression of TGF-β on redirected lysis of target cells expressing and secreting human TGF-β could be reversed in the presence of 50 μg/ml of a neu- tralizing anti-TGF-β antibody (Fig 3B). The decreased lysis of PD-L1 expressing CHO-EpCAM 8 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0141669 October 28, 2015 Impact of Immune Escape Mechanisms on BiTE Fig 3. Reversal of inhibitory effects of PD-L1, IDO and TGF-β by pharmacological means. (A) Analysis of CD3+ T cell proliferation after 120 h of co- culture with control EpCAM+ CHO cells and IDO-expressing, EpCAM+ CHO cells in the presence or absence of 500 μM tryptophane (Trp) with and without 1 μg/ml AMG 110. (B) Dose-dependent, redirected target cell lysis of parental EpCAM+ CHO cells and EpCAM+ CHO TGF-β transfectants +/- 50 μg/ml TGF- β neutralizing anti-human TGF-β antibody in presence of AMG 110 and CD3+ T cells in an E:T ratio of 4:1 after 72 h incubation. (C) Dose-dependent redirected target cell lysis of control EpCAM+ CHO cells and EpCAM+ CHO PD-L1 transfectants with and without 5 μg/ml of an anti-human PD-L1-neutralizing antibody in the presence of AMG 110 and pre-stimulated CD8+ T cells. The E:T ratio was 1:1, the assay duration 24 h. Fig 3. Reversal of inhibitory effects of PD-L1, IDO and TGF-β by pharmacological means. (A) Analysis of CD3+ T cell proliferation after 120 h of co- Fig 3. Reversal of inhibitory effects of PD-L1, IDO and TGF-β by pharmacological means. (A) Analysis of CD3+ T cell proliferation after 120 h of co- culture with control EpCAM+ CHO cells and IDO-expressing, EpCAM+ CHO cells in the presence or absence of 500 μM tryptophane (Trp) with and without 1 μg/ml AMG 110. (B) Dose-dependent, redirected target cell lysis of parental EpCAM+ CHO cells and EpCAM+ CHO TGF-β transfectants +/- 50 μg/ml TGF- β neutralizing anti-human TGF-β antibody in presence of AMG 110 and CD3+ T cells in an E:T ratio of 4:1 after 72 h incubation. (C) Dose-dependent redirected target cell lysis of control EpCAM+ CHO cells and EpCAM+ CHO PD-L1 transfectants with and without 5 μg/ml of an anti-human PD-L1-neutralizing antibody in the presence of AMG 110 and pre-stimulated CD8+ T cells. The E:T ratio was 1:1, the assay duration 24 h. doi:10.1371/journal.pone.0141669.g003 Redirected target cell lysis is observed even if all seven evasion mechanisms are present in co-culture Several evasion factors investigated in this study can act in trans, such as TGF-β, IL-10, PD-L1, IDO and adenosine. Only Bcl-2 and serpin PI-9 proteins, which are confined to the cytoplasm of cancer cells, cannot. We here attempted to create in co-culture an immunosuppressive envi- ronment by having all seven factors present at the same time. In one condition, each of the six transfected CHO-EpCAM cell lines was present at the same number and 1 mM adenosine added to the culture medium. Even under this condition, redirected lysis was still observed (Fig 4). However, a substantial impact on the percentage of lysis was evident with an increasing number of factors combined. The impact on the EC50 value of lysis showed a trend and did not exceed a 7-fold reduction. (C) Relative changes in the percentage of target cell lysis after 24 h at the plateau of dose response curves. The mean amplitude of lysis under evasion conditions was divided by the mean amplitude of lysis under control conditions. Error bars represent SEM values. doi:10.1371/journal.pone.0141669.g002 cells by AMG 110 could be fully reversed in the presence of 5 μg/ml of an antagonistic anti- PD-L1 antibody (Fig 3C). Anti-TGF-β and anti-PD-L1 antibodies or PI-9 inhibition by shRNA had no effect on lysis of cancer cell lines expressing the respective evasion factors at low levels (see S4D, S4E and S4F Fig). 9 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0141669 October 28, 2015 Discussion This is the first study exploring the impact of diverse evasion mechanisms on the performance of cytotoxic T cells engaged by a BiTE1 antibody construct. Our experimental system used rodent target cells in co-culture with human T cells, which excludes mechanisms of regular T cell recognition based on matching MHC class I or co-stimulatory molecule interactions. T cell recognition was solely based on connecting human EpCAM on CHO-EpCAM cells with CD3ε 10 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0141669 October 28, 2015 Impact of Immune Escape Mechanisms on BiTE Fig 4. Combinatorial effect of evasion mechanisms on AMG 110-mediated redirected target cell lysis. Parental human EpCAM+ CHO cells and different combinations of EpCAM+ CHO cells expressing evasion proteins were co-cultured at an E:T ratio of 4:1 with CD3+ T cells and various AMG 110 concentrations for 72 h. For each curve an equal amount of control EpCAM+ CHO cells was replaced by an additional stable CHO cell line until the entire target cell population comprised evasion protein-expressing cells and contained 1 mM adenosine. (A) Target cell lysis was determined by a FACS-based cytotoxicity assays. Sigmoidal dose-response curves were created with GraphPad Prism software. Error bars represent SEM values. (B) Relative change in EC50 and (C) percentage of cell lysis after 72 h were calculated as described in Fig 3. Error bars represent SEM values from two independent experiments Fig 4. Combinatorial effect of evasion mechanisms on AMG 110-mediated redirected target cell lysis. Pa Fig 4. Combinatorial effect of evasion mechanisms on AMG 110-mediated redirected target cell lysis. Parental human EpCAM+ CHO cells and different combinations of EpCAM+ CHO cells expressing evasion proteins were co-cultured at an E:T ratio of 4:1 with CD3+ T cells and various AMG 110 concentrations for 72 h. For each curve an equal amount of control EpCAM+ CHO cells was replaced by an additional stable CHO cell line until the entire target cell population comprised evasion protein-expressing cells and contained 1 mM adenosine. (A) Target cell lysis was determined by a FACS-based cytotoxicity assays. Sigmoidal dose-response curves were created with GraphPad Prism software. Error bars represent SEM values. (B) Relative change in EC50 and (C) percentage of cell lysis after 72 h were calculated as described in Fig 3. Error bars represent SEM values from two independent experiments doi:10.1371/journal.pone.0141669.g004 on human T cells by the bispecific AMG 110 protein. Impact of Immune Escape Mechanisms on BiTE underestimate the impact of combinatorial effects of evasion factors. Heterogeneity of tumor cells has been comprehensively documented [35, 36] and may well extent to the number and combination of evasion factors selected during disease progression within tumor tissues. This is evident from the present study where more than one evasion protein was detectable in the six human cancer cell lines studied (see S1 Fig). In one experiment, we tried to address combi- nations of evasion factors by mixing all CHO cell clones in the presence of 1 mM adenosine. Even under this circumstance, redirected lysis was still detectable albeit at lower potency and reduced kinetics as reflected in a decrease of the amplitude of lysis. Another factor contributing to the insensitivity of AMG 110-engaged T cells could be the release of pro-inflammatory cyto- kines by BiTE1-activated T cells [32]. IFNγ, interleukins 2 and 6, TNF-α and many other immune-stimulatory cytokines were found to be released upon BiTE1 stimulation in vitro, in mouse studies and in patients [37, 38]. These could in part counteract the activity of evasion factors by induction of activating signal transduction pathways in T cells. For example, the addition of IL-2 has been shown to overcome PD-L1-dependent inhibition of proliferation [39]. Apart from that, the in-vitro assays investigating redirected lysis were executed with an E:T ratio of 1:1. In vivo, the number of T cells in the tumor might be less favorable. The ability of BiTE1 antibodies to engage all existing effector T cells in the body to eliminate mulitiple target cells might decrease the effects of inhibition of T cell proliferation (like IDO). Nevertheless, a surplus of tumor cells might require local t cell expansion. Another possibility is that evasion factors need to act for prolonged periods of time on cyto- toxic T cells to be effective. In our experiments, we studied redirected lysis for 24 or 72 hours and T cell proliferation for 120 hours. Pre-existing toxins in secretory granules of effector T cells and their discharge during synapse formation may be very difficult to intercept, for instance, by mechanisms that work in T cells by transcriptional repression (like TGF-β) or by inhibition of T cell proliferation (like IDO). In our experiments, we targeted CHO transfectants and tumor cells with high to moderate EpCAM expression. The expression of tumor antigen on tumor cells can be very heteroge- neous. PLOS ONE \| DOI:10.1371/journal.pone.0141669 October 28, 2015 Discussion Previous studies have shown that MHC I expression is dispensable for the activity of AMG 110 [33], and that T cell activation and ensu- ing target cell lysis is entirely dependent on proper recognition of the human EpCAM antigen [34]. By using stably transfected CHO-EpCAM cell clones, we established expression levels for six evasion proteins that, with the exception of IFNγ-induced IDO, exceeded by far those found in six human cancer lines. Our experimental system should therefore be suitable to faith- fully study the isolated activity of an evasion factor on BiTE1-induced T cell proliferation and redirected lysis with a suitable background and acceptable signal strength. Despite a high-level expression of evasion proteins or the abundance of soluble evasion fac- tors in culture medium, we were surprised to observe only subtle changes in the performance of AMG 110-activated T cells. Most substantial was an inhibition of T cell cycling by IDO, and shifts in EC50 values of redirected lysis compared to control CHO-EpCAM cells in cells expressing serpin PI-9, PD-L1, TGF-β and Bcl-2, of which only the effect of PD-L1 reached sta- tistical significance. The only statistically significant but small impact on the percentage of lysis was seen for Bcl-2. In all other cases, no significant inhibition was observed and higher concen- trations of AMG 110 could typically compensate for the reduced percentage of lysis during the 24-72-h co-culture experiments. Despite a high-level expression of evasion proteins or the abundance of soluble evasion fac- tors in culture medium, we were surprised to observe only subtle changes in the performance of AMG 110-activated T cells. Most substantial was an inhibition of T cell cycling by IDO, and shifts in EC50 values of redirected lysis compared to control CHO-EpCAM cells in cells What may explain the relative insensitivity of AMG 110-mediated T cell activation and lysis to most evasion factors? Firstly, our experimental system studying isolated factors may 11 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0141669 October 28, 2015 Supporting Information S1 Fig. Expression of escape proteins in CHO EpCAM transfectants and six human tumor cell lines. (A) Intracellular FACS analysis of PI-9 expression in fixed and permeabilized tumor cell lines and CHO EpCAM PI-9 transfectants. For comparison of expression levels the relative median (= median sample/median control) of the samples were compared with reference to the CHO EpCAM PI-9 cells. (B) FACS analysis of PD-L1 expression with unlabeled cells (open), untreated (black) and IFNγ pretreated (grey) cell lines labeled with anti-human PD-L1 antibody. For comparison of expression levels relative medians of all samples were calculated. (C) FACS analysis of EpCAM expression. For comparison of expression levels relative medians of all samples were calculated. (D) Western Blot Analysis of Bcl-2 expression and the corre- sponding loading control. 25 μg of total cell lysate were applied. Bcl-2 signals were analyzed with ImageJ software and evaluated with respect to loading control and CHO EpCAM Bcl-2 signal. (E) Western Blot analysis of IDO expression and the corresponding loading control. 100 μg protein of total cell lysate were applied. IDO signals were analyzed with ImageJ software and evaluated with respect to loading control and CHO EpCAM IDO signal. (F) ELISA analy- sis of IL-10 and (G) TGF-β expression in human cancer cell lines and CHO EpCAM tranfec- tants assessed in cell supernatant of 2,5x105 /ml after 48 h of culturing. Error bars represent SEM out of the two assays. (H) FACS analysis of extracellular TGF-β expression in CHO EpCAM transfectants with unlabeled cells (grey), parental CHO cells (blue) and CHO EpCAM TGF-β transfectants (closed) labeled with anti-human TGF-β antibody. (TIF) S2 Fig. Impact of rec. hum IL-10 and TGF-β on BiTE1-induced proliferation and target cell lysis. (A) Human CD3+ T cells were labeled with CFSE and co-cultured at effector to target (E:T) ratios of 1:8, 1:1 and 4:1 in 48-well plates in presence and absence of 1 μg/ml AMG 110 with CHO control cells, CHO EpCAM IL-10 cells or control cells in the presence of 10 ng/ml and 400 ng/ml hum IL-10 or with CHO control cells, CHO EpCAM TGF-β and control cells in the presence 100 ng/ml hum TGF-β. After 120 h, CFSE signals of CFSE-positive cells were ana- lyzed using a FACS Canto™II flow cytometer and FACS DIVA™software. It is possible, that only a small number of target antigens per cell could result in reduced t cell activation, leaving the t cells more susceptible to inhibitory signals. Follow up studies will have to address whether t cell inhibition increases with decreasing levels of target antigens. 1 The BiTE1 principle is not dependent on the generation, expansion, epitope spreading and migration of specific T cell clones. This in itself may circumvent a number of evasion mecha- nisms that impact the latter. How can the dose of AMG 110 eventually compensate for inhibitory effects of evasion fac- tors? AMG 110 was shown to induce formation of a normally structured, functional cytolytic synapse between T cells and EpCAM-expressing target cells [33]. It is conceivable that by increasing the concentration of AMG 110, this BiTE1-induced synapse is getting enlarged, which may allow for delivery of more granzymes and perforin across the synapse. 1 Future studies need to investigate whether the surface of BiTE1-induced synapses is scal- able and this way capable of delivering various doses of toxins in relationship to the BiTE1 concentration. Even if certain evasion mechanisms can impact BiTE1-engaged T cells in their proliferation or effector function, as here observed for IDO, PD-L1, serpin PI-9 or TGF-β, there may be means to enhance a reduced BiTE1 function by pharmacological intervention. We here show that antibodies neutralizing the PD-1/PD-L1 axis or TGF-β can relieve an inhibition of redi- rected lysis. Likewise, inhibitors of IDO may be able to restore BiTE1-induced T cell cycling. With the aggressive clinical development of antibodies blocking the PD-1/PD-L1 axis and 12 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0141669 October 28, 2015 Impact of Immune Escape Mechanisms on BiTE other checkpoint inhibitors, pharmacological tools will become available to clinically investi- gate whether BiTE1 antibody constructs can profit from a blockade of immune evasion. Supporting Information Dose-dependent redirected target cell lysis of human tumor cell lines with and without inhibition of endogenous (D) PI9 expression by shRNA, neutralization of endogenous (E) TGF-β or (F) endogenous PD-L1 by addition of neutralizing antibodies after (D-E) 48 h and (F) 24 h. (TIF) transfected with one of six human evasions proteins and the target antigen human EpCAM compared to parental EpCAM+ CHO cells or parental EpCAM+ CHO cells in presence or absence of evasion protein Adenosine using CD3+ T cells as effector cell population. Percentage of target cell lysis was assessed by a FACS-based cytotoxicity assay after 72 h of co-culture with CD3+ T cells at an E:T ratio of 4:1 using a FACS Canto™II flow cytometer. Mean EC50 values were calculated with GraphPad Prism software. Error bars represent SEM out of duplicates. For quantification of effects of immune escape mechanisms on BiTE1-mediated redirected target cell lysis. (B) Relative Change in EC50 and (C) relative change in amplitude were calcu- lated as described in Fig 2. Error bars represent SEM out of the assays performed for each dif- ferent cell line. The number of repetitions is indicated. Dose-dependent redirected target cell lysis of human tumor cell lines with and without inhibition of endogenous (D) PI9 expression by shRNA, neutralization of endogenous (E) TGF-β or (F) endogenous PD-L1 by addition of neutralizing antibodies after (D-E) 48 h and (F) 24 h. (TIF) S5 Fig. PD-1 increases upon stimulation. FACS analysis of PD-1 expression in CD3+T cells that were cultured with/without CD3/CD28/IL-2 96h after isolation. (TIF) S6 Fig. Adenosine decreases CD25 expression. FACS analysis of CD25 expression in CD3+T cells stimulated by CD3/CD28/IL-2 with/without 1 mM of Adenosine (ADO). (TIF) S6 Fig. Adenosine decreases CD25 expression. FACS analysis of CD25 expression in CD3+T cells stimulated by CD3/CD28/IL-2 with/without 1 mM of Adenosine (ADO). (TIF) S7 Fig. Functional analysis of rec. hum TGF-β, TGF-β from supernatant of CHO tranfec- tants and TGF-β neutralizing antibody. Intracellular FACS analysis of granzyme B (GrB) expression in CD3+ T cells (A) stimulated by CD3/CD28/IL-2 with/without 100 ng rec. hum TGF-β, (B) stimulated in CHO EpCAM control cell supernatant and (C) CHO EpCAM TGF-β supernatant +/- TGF-β neutralizing antibody. (TIF) Author Contributions Conceived and designed the experiments: TR MM WD. Performed the experiments: WD. Ana- lyzed the data: TR MM WD PK. Contributed reagents/materials/analysis tools: PK. Wrote the paper: MM WD PAB. Supporting Information (B) Dose-dependent redirected target cell lysis of CHO EpCAM control cells, CHO EpCAM-IL10 transfectans and control cells in presence of 10 ng/ml or 200 ng/ml hum IL-10 and dose-dependent redirected target cell lysis of CHO EpCAM control cells, CHO EpCAM TGF-β transfectans and control cells in presence of 80 ng/ml hum TGF-β. Percentage of target cell lysis was assessed by an FACS-based cytotoxicity assay after 72 h of co-culture with CD3+ T cells at an E:T ratio of 4:1 using a FACS Canto™II flow cytometer. Mean EC50 values were calculated with GraphPad Prism software. Error bars represent SEM out of duplicates. (TIF) S3 Fig. Statistical analysis of EC50 values and amplitudes of CHO escape transfectants and corresponding controls. (A) EC50 values and (B) amplitudes of all executed assays using CD8+ T cells as effector cell population were analyzed with the Grubb0s test to exclude significant out- liers. P values were calculated using unpaired t tests with welch0s correction with a significance level = p <0.05. (TIF) S4 Fig. Impact of diverse immune escape mechanisms on target cell lysis by redirected CD3+ T cells. Dose-dependent redirected target cell lysis of CHO cell lines (A) stably S4 Fig. Impact of diverse immune escape mechanisms on target cell lysis by redirected CD3+ T cells. Dose-dependent redirected target cell lysis of CHO cell lines (A) stably 13 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0141669 October 28, 2015 Impact of Immune Escape Mechanisms on BiTE transfected with one of six human evasions proteins and the target antigen human EpCAM compared to parental EpCAM+ CHO cells or parental EpCAM+ CHO cells in presence or absence of evasion protein Adenosine using CD3+ T cells as effector cell population. Percentage of target cell lysis was assessed by a FACS-based cytotoxicity assay after 72 h of co-culture with CD3+ T cells at an E:T ratio of 4:1 using a FACS Canto™II flow cytometer. Mean EC50 values were calculated with GraphPad Prism software. Error bars represent SEM out of duplicates. For quantification of effects of immune escape mechanisms on BiTE1-mediated redirected target cell lysis. (B) Relative Change in EC50 and (C) relative change in amplitude were calcu- lated as described in Fig 2. 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https://openalex.org/W4311169989	http://cds.cern.ch/record/2773697/files/ATL-ITK-PROC-2021-004.pdf	English	null	ATLAS ITk Pixel Detector Overview	Journal of physics. Conference series	2,022	cc-by	1,948	E-mail: fmunoz@cern.ch Abstract. In this work an overview of the layout and current status of development of the ITk Pixel detector is given. For the HL-LHC upgrade the current ATLAS Inner Detector is replaced by an all-silicon system. The Pixel Detector will consist of 5 barrel layers and a number of rings, resulting in about 14 m2 of instrumented area. Due to the huge non-ionising ﬂuence (1×1016 neq/cm2) and ionising dose (5 MGy), the two innermost layers, instrumented with 3D pixel sensors (L0) and 100 µm thin planar sensors (L1) will be replaced after about 5 years of operation. All hybrid detector modules will be read out by novel ASICs, implemented in 65 nm CMOS technology, with a bandwidth of up to 5 Gb/s. Data will be transmitted optically to the oﬀ-detector readout system. To save material in the servicing cables, the ASICs are serially powered. Large-scale prototyping programs are being carried out by all sub-systems. ATL-ITK-PROC-2021-004 22 June 2021 ATL-ITK-PROC-2021-004 22 June 2021 ATLAS ITk Pixel Detector Overview Francisca J. Mu˜noz S´anchez on behalf of the ATLAS collaboration Department of Physics and Astronomy, Faculty of Science and Engineering, University of Manchester, Oxford Road, M13 9PL United Kingdom. Francisca J. Mu˜noz S´anchez on behalf of the ATLAS collaboration Department of Physics and Astronomy, Faculty of Science and Engineering, University of Manchester, Oxford Road, M13 9PL United Kingdom. Francisca J. Mu˜noz S´anchez on behalf of the ATLAS collaboration Department of Physics and Astronomy, Faculty of Science and Engineering, University of Manchester, Oxford Road, M13 9PL United Kingdom. 1. Introduction The upgrade of the Large Hadron Collider (LHC) to the High Luminosity LHC (HL-LHC) will increase peak and integrated luminosities by factors 7.5 and 13 respectively [1]. The mean pile- up will average up to 200 events and the radiation ﬂuence reaches values up to 1×1016 neq/cm2 in some areas of the detector. The current inner detector cannot successfully perform under those conditions and it will be upgraded to an all-silicon tracker, the Inner Tracker (ITk). The ATLAS-ITk consists of a strip detector [2] in the outermost layers and a pixel detector [3] in those layers closer to the interaction point (shown in Figure 1 -left)). The pixel detector is divided in three diﬀerent sub-detectors’ covering up to η = 4, the Inner System (IS), the Outer Barrel (OB) and two Outer Endcaps (OEs), see Figure 1 -right). 2.2. Outer Barrel The OB layout has two diﬀerent sections, ﬂat and inclined. The local supports in the ﬂat section supports are called longerons while in the inclined, those in the inclined section are called inclined rings. In the OB, modules are loaded on module cells, which are screwed to base blocks on the local supports at a later integration stage, see details in Figure 4. Any of those cells can be integrated in both local support structures. Prototypes of layer-3 OB local supports 2. ITk Pixel layout In this section a brief description of the three sub-detectors mechanical designs is given. Some prototypes used to qualify the designs are also shown. 2.1. Inner System The IS is formed by a barrel and an endcap section and it will follow a quarter-shell integration structure. Each quarter shell has a barrel section where staves and rings are local support structures. It also has an endcap section where ﬂavours of rings will support the pixel modules. (see Figure 2). 2.1. Inner System The IS is formed by a barrel and an endcap section and it will follow a quarter-shell integration structure. Each quarter shell has a barrel section where staves and rings are local support structures. It also has an endcap section where ﬂavours of rings will support the pixel modules. (see Figure 2). ( g ) The IS has built several prototypes to qualify the mechanical design and to exercise the assembly of local supports. In Figure 3 diﬀerent prototypes are shown. ( g ) The IS has built several prototypes to qualify the mechanical design and to exercise the assembly of local supports. In Figure 3 diﬀerent prototypes are shown. Figure 1. ITk layout. Strip detector in blue and pixel detector inside the red rectangle on the left image [3], while the pixel detector quadrant coverage where the three sub-detectors are distinguishable is shown at the right [4]. Figure 1. ITk layout. Strip detector in blue and pixel detector inside the red rectangle on the left image [3], while the pixel detector quadrant coverage where the three sub-detectors are distinguishable is shown at the right [4]. Figure 2. Inner System layout. Left: Quarter shell layout with the barrel and endcap sections. Middle: stave local support composition. Right: ring local support composition. Figure 2. Inner System layout. Left: Quarter shell layout with the barrel and endcap sections. Middle: stave local support composition. Right: ring local support composition. Figure 3. IS prototypes. Left: ring prototype for thermal qualiﬁcation where some silicon heaters can be identiﬁed. Middle: Bare ring prototype. Right: Bare stave prototype. Figure 3. IS prototypes. Left: ring prototype for thermal qualiﬁcation where some silicon heaters can be identiﬁed. Middle: Bare ring prototype. Right: Bare stave prototype. Figure 4. OB mechanics. Top Left: OB layer layout. Top right: longeron breakdown. Bottom left: inclined ring drawing. Bottom right: longeron cross section and module cell. Figure 4. OB mechanics. 2. ITk Pixel layout Top Left: OB layer layout. Top right: longeron breakdown. Bottom left: inclined ring drawing. Bottom right: longeron cross section and module cell. Figure 5. Layer 3 OB local support prototypes. Figure 5. Layer 3 OB local support prototypes. are shown in Figure 5 together with a prototype of a single cell where modules will be loaded. These prototypes are used to test the production and assembling chains as well as to qualify their design. are shown in Figure 5 together with a prototype of a single cell where modules will be loaded. These prototypes are used to test the production and assembling chains as well as to qualify their design. 4. Power and data transmission The pixel system will be powered using a serial powering scheme [7] where up to thirteen pixel modules will be powered in the longest chain in the OEs. The use of the serial powering decreases signiﬁcantly the amount of material needed by reducing the number of required cables for powering. A Monitor Of the Pixel System (MOPS) chip will be used to monitor the temperature and the voltage drop in every module. Relative to the data transmission approach [8], the electrical signals will be transmitted at 1.28 Gbps from the module through twinax cables up to the opto-electrical conversion system outside of the pixel detector. This custom-designed system will equalise and aggregate the signals to 10.24 Gb/s and perform the conversion into optical signals. 3. Modules The pixel modules in the ITk pixel detectors are made using a hybrid technology, where silicon sensors are bump-bonded to the readout ASICs. The readout chip is based on the 65 nm technology developed by the RD53 collaboration [5]. The pixel modules in the ITk pixel detectors are made using a hybrid technology, where silicon sensors are bump-bonded to the readout ASICs. The readout chip is based on the 65 nm technology developed by the RD53 collaboration [5]. In order to optimise the performance of the ATLAS experiments an extensive study [6] was done by the collaboration to decide which sensor technology is used in diﬀerent parts of the detector. This study showed that best compromise between performance, yield and cost is achieved by having planar pixel sensors in layers 1 to 4, with 100 µm thick sensors in layer 1 and 150 µm thick sensors in layers 2, 3 and 4. In Layer 0, 3D silicon pixel sensors will be used due to their higher resistance to radiation. However, it was also found that diﬀerent pixel geometries in layer 0 provide a better resolution. Therefore, 50 × 50 µm2 pixels will be used on the rings while 25 × 100 µm2 will be used in the central staves. 2.3. Outer Endcap p In the case of the OE, the local supports are carbon-based half-ring structures where a titanium pipe is embedded. Pixel modules will be loaded on both sides of half-rings where a ﬂex PCB will also be loaded to route power and monitoring signals to each module. Each OE will consist of three layers of local supports, see Figure 6, which shows the complete layout of one endcap, together with the composition of a local support, and one of the prototypes used to qualify the design, test the production and assembly chains, and validate simulations. Though only local supports prototypes are shown in this proceeding, some prototypes of full-scale global support structures are already under test for the diﬀerent subsystems as well. In addition to the support structures, the services routing (comprising power and monitoring cables, cooling pipes and data lines) is currently in the latest stage of the design for all subsystems. Figure 6. OE mechanics layout. Left: one endcap layout. Centre: A half-ring local support breakdown. Right: Layer 2 half-ring prototype. Figure 6. OE mechanics layout. Left: one endcap layout. Centre: A half-ring local support breakdown. Right: Layer 2 half-ring prototype. Copyright 2021 CERN for the beneﬁt of the ATLAS Collaboration. CC-BY-4.0 license. Copyright 2021 CERN for the beneﬁt of the ATLAS Collaboration. CC-BY-4.0 license. 5. Demonstrators In order to test the performance of the pixel detectors and to investigate possible issues in the ﬁnal system, every sub-system is building realistic demonstrators from the local support up to patch panel 1, which is located at end of the pixel detector. These demonstrators will be real scale local supports prototypes populated with the nearly- ﬁnal modules, electronics, cooling and interfaces. Working with these prototypes will give the collaboration experience at operating a representative part of the detector in realistic conditions. Complete serial power chains will be tested while they are being cooled down to the operating temperatures and modules will be read out with full-length cables and realistic data acquisition systems. Demonstrators will provide the possibility to completely understand the system, validate simulations, and test the production plans. [ ] , p g f p , [8] L Franconi, The Opto-electrical conversion system for the data transmission chain of the ATLAS ITk Pixel detector upgrade for the HL-LHC, TIPP 2021 poster presentation, proceeding included in this conference publication. 6. Summary and outlook The ATLAS pixel community is facing the latest phase of R&D of the diﬀerent components and pre-production is expected to start in 2022-2023 (depending on the components). Intensive work is being carried out in order to understand complex systems in terms of cooling, power, data and supports that need to be integrated and fully operational in the ATLAS experiment in 2027. [7] L Gonella et al., A serial powering scheme for the ATLAS pixeldetector at sLHC, 2010 JINST5 C12002. [ ] References [1] High Luminosity LHC project, https://hilumilhc.web.cern.ch/. [2] ATLAS Collaboration, Technical Design Report for the ATLAS Inner Tracker Strip Detector, CERN-LHCC- 2017-005, 2017. [3] ATLAS Collaboration, Technical Design Report for the ATLAS Inner Tracker Pixel Detector, CERN-LHCC- 2017-021, 2017. [3] ATLAS Collaboration, Technical Design Report for the ATLAS Inner Tracker Pixel Detector, CERN-LHCC- 2017-021, 2017. , [4] ATLAS Collaboration, https://atlas.web.cern.ch/Atlas/GROUPS/PHYSICS/PLOTS/ITK-2020-002 [5] RD53 Collaboration, https://rd53.web.cern.ch/ [ ] // / [6] ATLAS Collaboration, Expected Tracking Performance of the ATLAS Inner Tracker at the HL-LHC, ATL- PHYS-PUB-2019-014, 2019. [7] L Gonella et al., A serial powering scheme for the ATLAS pixeldetector at sLHC, 2010 JINST5 C12002. [8] L Franconi, The Opto-electrical conversion system for the data transmission chain of the ATLAS ITk Pixel detector upgrade for the HL-LHC, TIPP 2021 poster presentation, proceeding included in this conference publication.
https://openalex.org/W4389787561	https://www.shs-conferences.org/articles/shsconf/pdf/2023/29/shsconf_icepcc2023_01016.pdf	English	null	The impact of American Philippine economic policy on Philippine modern economy during the Cold War	SHS web of conferences	2,023	cc-by	4,100	1 Introduction During the Cold War, in the 80 years since the end of World War II in the United States, the world pattern has gradually changed, from the bipolar pattern dominated by the United States and the Soviet Union to the obvious trend of multi-polarization. The relationship between countries has always been the embodiment of the change of the world pattern. After the 1950s, the cold war between the United States and the Soviet Union extended to Asia. With the improvement of its strategic position in Southeast Asia, the decolonization policy of the United States in Southeast Asia also reflects the characteristics of neocolonialism. As an important country in Southeast Asia, the Philippines is adjacent to the South China Sea in the West and the Pacific Ocean in the East. It is an important node of the first island chain, guarding the Bashi Strait, and has a very important strategic position. Therefore, during the Cold War, the United States' economic assistance policy to the Philippines on the one hand helped the economic development of the Philippines, on the other hand, strengthened the relationship between the United States and the Philippines, and infiltrated the forces into Philippine politics. As for the foreign aid of the United States, its foreign aid theory has made a lot of research results, among which the international interest theory is the most mainstream, and its main representatives are Morgenthau and Waltz. Morgenthau's theory is that no matter what form of foreign aid, its essence is political, and the main goal is to promote and protect national interests [1]. While Waltz believes that under the anarchy, the major world powers are competitors, they The impact of American Philippine economic policy on Philippine modern economy during the Cold War Yixuan Li1,* 1Beijing Huiwen Middle School, 100122 Beijing, China Yixuan Li1,* 1Beijing Huiwen Middle School, 100122 Beijing, China Abstract. As an important country in Southeast Asia, the Philippines has repeatedly occupied an important geographical position and had an important geographical and political position during the Cold War. Since the Cold War, the relationship between the United States and the Philippines has been very close, and the influence of the United States on the Philippines is self-evident. Therefore, studying the political influence of the United States on Philippine economic policy during the Cold War has important theoretical and practical significance. Starting from the economic policy adopted by the United States in the Philippines, this paper analyzes the economic assistance methods of the United States to the Philippines and its political influence on the Philippines and explains why the United States exercises neocolonial control over the Philippines from two aspects. This article argues that the United States has influenced the Philippine economy in a neocolonial way in an attempt to expand the political influence and sphere of influence of the United States itself. seek to establish hegemony and establish a security system, and foreign aid policy is a hegemonic tool [2]. Previous studies mainly focused on the changes in U.S. aid policy to the Philippines and the influence of U.S. colonial rule on the Philippines. The conclusion is that the American colonial rule has changed the direction of modern political development in the Philippines, making the Western colonial rule replace the political development with the traditional characteristics of the Philippines, or on the other hand, making the so-called oligarchic elite control the Philippine society in the special relationship of colonialism. That is to say, the political development of the Philippines after independence is a kind of inheritance relationship compared with the colonial period. The purpose of this study is to complement the economic impact of aid policy on politics, to highlight the importance of economic factors for social development, and to elaborate that this control method is different from the previous direct slavery of the suzerain power to the colonies, but to enhance its dependence on the United States with economic aid, to indirectly control. ICEPCC 2023 SHS Web of Conferences 180, 01016 (2023) ICEPCC 2023 SHS Web of Conferences 180, 01016 (2023) https://doi.org/10.1051/shsconf/202318001016 © 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/). s * Corresponding author: sking81477@student.napavalley.edu DP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 ses/by/4.0/). 2 The Philippines' strategic position towards the United States during the Cold War 3 The economic policy of the United St t t th Phili i d i th p To better explain the impact of the United States on the Philippines, the Neo-colonial view is very important. Neocolonialism is a more covert and indirect means of colonial aggression by Western powers against non- Western countries after the Second World War. The process of establishing the new colonial system is simply that, first of all, it was a colony of an imperialist country. Secondly, under the wave of the national liberation movement, the imperialist country (suzerain state) allowed its national independence and established an independent country. Finally, the imperialist country used its economic advantages to control the newly established colonial country politically, militarily, and culturally, Imperialist countries control and continue to extract wealth by fostering puppet regimes or using commerce (state power and transnational corporations are the two major ways of Neo colonialism infiltration). Under this scenario, the independent colonial countries will continue to be under control and serve as the commodity markets, raw material-producing areas, and investment sites of the imperialist countries. During the Cold War, the United States indirectly controlled the Philippines through foreign policy, which was in line with the control of imperialist international over the former colonial international under the Neo colonial model. While using the aid projects to the Philippines to enhance its influence on the Philippines, the United States also used this as a medium to skillfully infiltrate some regional organizations' activities related to this and take the opportunity to expand its influence in the political life of the Philippines. 2 The Philippines' strategic position towards the United States during the Cold War The Philippines has been a colony of the United States for half a century. The earliest can be traced back to 1898, when the United States won the Spanish-American war, The United States occupied the Philippines instead of Spain. Since then, the Philippines has become a ICEPCC 2023 SHS Web of Conferences 180, 01016 (2023) https://doi.org/10.1051/shsconf/202318001016 sphere of influence and a colony of the United States. But at this time, the Philippines has not yet become the focus of the United States. In 1899, the United States put forward the "open door" policy to carve up China with foreign powers. Southeast Asia is not its key area. But at this time, its impact on Philippine politics was obvious and profound. The later basic political systems introduced by the United States, such as the presidential system, the bicameral Parliament, and the two-party system, all began in this period. At that time, Taft stereotyped how the United States transplanted a democratic system for colonial rule in the Philippines. Taft's stereotype refers to cultivating a group of American Philippine upper-class elements by transplanting Western parliamentary democracy to facilitate the colonial rule of the United States. This foreshadowed the family elite politics in the Philippines. In 1946, the Macduffy Act was promulgated and the Philippines became independent. Since then, the world has entered the cold war. From 1947 to 1991, the North Atlantic Treaty Organization led by the United States, and the Warsaw Treaty led by the Soviet Union carried out political, military, and economic struggles between the two camps. The two superpowers of the United States and the Soviet Union competed for world hegemony. In 1948-1950, a large-scale Revolutionary Movement broke out in Southeast Asia. The United States believes that these revolutions were made by the Communist Party and the Workers' Party intelligence agency manipulated by the Soviet Union [3]. This is an indication that the Soviet Union wants to control Southeast Asia. The founding of the People's Republic of China in 1949 further expanded the power of the Soviet Union, which was also a communist country, in the Asia Pacific region, breaking the balance of power between the United States and the Soviet Union in East Asia. 3.1 Fourth point plan The fourth point plan, also known as the "plan for technical assistance to backward areas", is named because it is the fourth point of the aggression plan put forward by US President Truman in his inaugural address on January 20th, 1949. The target of the plan was developing countries, which was a supplement to the "Marshall Plan" implemented in Western Europe at that time. The fourth point is to provide economic and technical assistance to underdeveloped regions such as Asia, Africa, and Latin America. The goal of the fourth point plan is not to serve the economy of the recipient countries but to serve the United States to contain the Soviet Union The ultimate goal of United and communism. Therefore, the assistance of the United States is mostly given to countries that have important strategic significance in implementing the containment strategy. For example, India's Nehru government pursues a nonaligned foreign policy, while Pakistan is an ally of the United States, and the assistance received by the two 2 The Philippines' strategic position towards the United States during the Cold War In January 1950, Secretary of State Acheson delivered a speech, clearly announcing that the United States' defense line in the Asia Pacific region should be located in the Aleutian Islands in the north, through Japan and Ryukyu Islands, and extended to the Philippines [4]. At the same time, the strategic position of the Philippines has gradually improved. The documents nsc48/1 and nsc48/2 (the United States' position on Asia), nsc64 (the United States' position on Indochina), and nsc124/1 and nsc124/2 (the United States' policy statement on the objectives and course of action of the Communist Party of Southeast Asia's aggression in Southeast Asia) proposed by the Truman administration all believe that Southeast Asia has an important position in the Asian cold war [3]. Geographically, the Philippines is located in an important place, guarding the Bashi Strait and located in the first island chain. The island chain plan used it as a military base to blockade the Communist countries. Therefore, the Philippines has an outstanding strategic position for the United States to curb the Communist forces and expand its sphere of influence. Before and after World War II, the evolution of the United States' decolonization policy in East Asia was a process of opposing the old colonial order in Europe and establishing a new colonial order in the United States [5]. The United States uses economic aid and investment to control the economy of Southeast Asian countries and influence the politics of Southeast Asian countries. Before and after World War II, the evolution of the United States' decolonization policy in East Asia was a process of opposing the old colonial order in Europe and establishing a new colonial order in the United States [5]. The United States uses economic aid and investment to control the economy of Southeast Asian countries and influence the politics of Southeast Asian countries. sphere of influence and a colony of the United States. But at this time, the Philippines has not yet become the focus of the United States. In 1899, the United States put forward the "open door" policy to carve up China with foreign powers. Southeast Asia is not its key area. But at this time, its impact on Philippine politics was obvious and profound. The later basic political systems introduced by the United States, such as the presidential system, the bicameral Parliament, and the two-party system, all began in this period. 2 The Philippines' strategic position towards the United States during the Cold War At that time, Taft stereotyped how the United States transplanted a democratic system for colonial rule in the Philippines. Taft's stereotype refers to cultivating a group of American Philippine upper-class elements by transplanting Western parliamentary democracy to facilitate the colonial rule of the United States. This foreshadowed the family elite politics in the Philippines. In 1946, the Macduffy Act was promulgated and the Philippines became independent. Since then, the world has entered the cold war. From 1947 to 1991, the North Atlantic Treaty Organization led by the United States, and the Warsaw Treaty led by the Soviet Union carried out political, military, and economic struggles between the two camps. The two superpowers of the United States and the Soviet Union competed for world hegemony. In 1948-1950, a large-scale Revolutionary Movement broke out in Southeast Asia. The United States believes that these revolutions were made by the Communist Party and the Workers' Party intelligence agency manipulated by the Soviet Union [3]. This is an indication that the Soviet Union wants to control Southeast Asia. The founding of the People's Republic of China in 1949 further expanded the power of the Soviet Union, which was also a communist country, in the Asia Pacific region, breaking the balance of power between the United States and the Soviet Union in East Asia. In January 1950, Secretary of State Acheson delivered a speech, clearly announcing that the United States' defense line in the Asia Pacific region should be located in the Aleutian Islands in the north, through Japan and Ryukyu Islands, and extended to the Philippines [4]. At the same time, the strategic position of the Philippines has gradually improved. The documents nsc48/1 and nsc48/2 (the United States' position on Asia), nsc64 (the United States' position on Indochina), and nsc124/1 and nsc124/2 (the United States' policy statement on the objectives and course of action of the Communist Party of Southeast Asia's aggression in Southeast Asia) proposed by the Truman administration all believe that Southeast Asia has an important position in the Asian cold war [3]. Geographically, the Philippines is located in an important place, guarding the Bashi Strait and located in the first island chain. The island chain plan used it as a military base to blockade the Communist countries. Therefore, the Philippines has an outstanding strategic position for the United States to curb the Communist forces and expand its sphere of influence. 3.3 USAID assistance to the Philippines The aid activities of the International Development Agency in the Philippines have become an important part of the alliance between the two countries. It is a strategic means for the United States to consolidate and deepen the relationship between the United States and the Philippines. Based on the content of the assistance, the International Development Agency also has the Food Security Bureau, the Economic Growth and Agriculture and Trade Bureau, the Democracy and Conflict Management and Humanitarian Relief Bureau, and the Global Health Bureau. In addition to the above departments, the International Development Agency also has institutions such as policy planning and training, legislation and public affairs, and the inspector general's office to coordinate and supervise the operation and management of administrative and operational departments [7]. UNDP is an assistance system covering politics, economy and trade, social development, and other aspects, and can realize the connection and supervision of all links from policy planning to project implementation. The Philippines is a deeply involved country, and its influence on the Philippines has penetrated all aspects of political and economic activities. For example, according to the statistical prediction, by the end of the UNDP assistance project in 2009-2013, 90% of the poor people in the Philippines will enjoy the financial assistance provided by social medical insurance [8]. It can be seen that the United States' assistance to the Philippines plays a key role in the economic development and stability of the Philippines. So its action decision is enough to influence and make the United States indirectly control the political economy of the Philippines. y From the economic perspective, the fourth plan is to a large extent the extension and continuation of the traditional U.S. foreign economic policy in the new situation, that is, the foreign policy serves the U.S. economy, especially helping U.S. enterprises to develop commodity markets and raw material supply channels overseas, to ensure the prosperity of the U.S. domestic economy. To carry out the fourth point plan, the United States sent a large number of technical personnel abroad for technical cooperation and received personnel from many countries to the United States for training. The cooperation projects mainly involve agriculture, raw material development, transportation, vocational education, and health. By the end of 1951, the "fourth point plan" had been extended to 33 countries. 4 Impact of the above measures To help the Philippines improve its economic problems, the United States provided economic and technical assistance to the Philippines. According to the documents, the total amount of economic and technical assistance of the United States to the Philippines in 1954 was US $ 220 million, specifically including the first, agricultural assistance projects: designed to help the 3.3 USAID assistance to the Philippines From July 1st, 1945 to June 30th, 1967, more than 30% of the cumulative US $117.2 billion in foreign economic and military assistance was given to non-Communist underdeveloped countries. 3 The economic policy of the United States to the Philippines during the Cold War 3 The economic policy of the United States to the Philippines during the Cold War 2 https://doi.org/10.1051/shsconf/202318001016 ICEPCC 2023 SHS Web of Conferences 180, 01016 (2023) https://doi.org/10.1051/shsconf/202318001016 countries is very different. India, with a population of 400 million during the Nehru administration, was the most populous noncommunist country in the world at that time, receiving only US $ 45 million (the US foreign aid budget was US $6.5 billion in 1952). As a reward for its political and military alliance with the US, Pakistan received on average twice as much economic aid as India in addition to military aid [6]. Philippines increase food production. The second is the public health project: help the Philippines control the spread of malaria and establish regional health units. Education project: assist the Philippines in establishing professional and professional teacher training. Transportation projects: Assist in the construction of roads and ports, and carry out extensive and comprehensive transportation surveys. Mineral resources project: assist the Philippines in developing its mineral resources and provide technical guidance to government officials. Public administration project: It aims to improve the efficiency of the government, and the labor- management relations center has been established by the government's School of Public Management. The plan aims to control these areas politically. Truman also hoped that through the economic and technical assistance provided by the Fourth Point Plan, the economic situation of some third-world countries would be improved, so that these countries could achieve political stability, and then eliminate the soil of communism infiltration. In June 1949, Truman pointed out in his State of the Parliament address that underdeveloped regions must have a "solid economic foundation ". Without such an economic foundation, it is impossible to meet the needs of the people in these regions. "If they are frustrated and disillusioned, they will turn to pursue those heresies advocating development through autocracy. Therefore, the free country, that is, the United States, must help these regions establish a solid economic foundation. At this time, it believes that the United States is the only country capable of providing such assistance. Therefore, the United States, through economic and technical assistance, helps the economically underdeveloped countries and regions in Asia, Africa, and Latin America to develop their national economies, consolidate the economic foundation of governance, and resist the influence of "heresy", that is, communism. 5 Conclusion In fiscal year 2008-2009, the specific measures of the United States in the field of energy and environmental protection are as follows: first, addressing climate change. First, develop alternative energy and clean energy. Help promote alternative energy. About 180 million liters of coconut biogasoline were produced and sold. 31 million liters of ethanol were sold. This saved us $ 270 million in foreign exchange. Second, establish modern energy facilities. More than 30,000 people have access to electricity. The Renewable Energy Act was passed. Increased efficiency in the energy sector. More than $4 million was invested in energy projects. Second, strengthen environmental supervision. More than 270000 hectares of forests and coastal ecosystems have been better managed and maintained. More than 60 local government departments have funded a variety of environmental protection projects. Illegally cut timber valued at about US $180000 was confiscated. Third, improve the urban environment. About 600000 people have access to potable water. Attracted more than $16.5 billion in private funds. 18 local governments recycled more than 25% of local waste products [10]. The economic growth theory proposed by Rostow, a famous American economist, believes that a country's economic growth needs to go through six stages, namely, the traditional social stage, the stage of preparing to take off, the stage of taking off, the stage of advancing towards maturity, the stage of high mass consumption and the stage of pursuing quality of life. Rostow suggested that the United States provide economic assistance to developing countries according to their stage so that developing countries can choose the Western development model, implement Western democracy politically, promote market economy economically, achieve political stability and economic development, and completely get rid of the influence of communism and the Eastern European model of the Soviet Union. It can be seen that this theory explains the main purpose of the United States' economic assistance to the Philippines during the Cold War, that is, to deal with communism. After the Cold War, terrorism soon became a major threat to the national security of the United States and became one of the main purposes of the United States' assistance to the Philippines. H h h f i h l h i The resource exchange relationship between the United States and the Philippines has strong common interests. 5 Conclusion The United States has provided a large amount of assistance to the Philippines to obtain some special strategic interests from the Philippines. This strategic interest acted as a deterrent during the Cold War to prevent the "expansion" of communism in Southeast Asia; After 9.11 after the end of the Cold War, strategic interests focused on the prevention of terrorism and the rise of China. From the perspective of the Philippines in general, the economic assistance of the United States is very important. This is conducive to its economic development. For the Philippines, which has a large population and an underdeveloped economy, this is undoubtedly a very valuable resource. 4.1 Philippine economic situation In the 1950s, the Philippines was considered one of the most promising countries in Asia. The level of economic development is second only to Japan. In 1950, Japan's per capita GDP was 454 US dollars, the Philippines 293 US dollars, Taiwan 237 US dollars, South Korea 157 US 3 ICEPCC 2023 SHS Web of Conferences 180, 01016 (2023) https://doi.org/10.1051/shsconf/202318001016 dollars, Thailand 129 US dollars, and Indonesia 98 US dollars [9]. and pays attention to the dependence of donor countries on recipient countries [11]. References 1. H. Morgenthau, X. Xu, et al, translated, Interstate politics: power struggle and peace, Beijing: Peking University Press. 2. K. Waltz, Q. Xin, translated, International political theory, Shanghai: Shanghai People's Publishing House. 3. J. Cady, N. Yao, et al, translated, Postwar history of Southeast Asia, Shanghai: Shanghai People's Publishing House. 4. D. Acheson, L. L. Wu, et al, translated, Memoirs of Acheson: Volume II, Shanghai: Shanghai People's Publishing House. 5. L. F. Liu, J Guizhou Norm Univ (Soc Sci Edit), 5, 69-73 (2006). 6. W. J. Zhang, J Three Gorg Univ (Hum soc sci ed), 39, 183-185 (2017). 7. C. M. Zhang, Sci econ soc, 2, 65 (2002). 8. N. Han, Se Asia Sou Asia stud, 2, 46-51+93 (2012). 9. USAID, Search for Country asistancess trategy philipines (2009), Available online at: https://www.usaid.gov/search/site-search? keys=Country+asistancess+trategy+philipines. 10. C. P. Goldberg, Economic development, Shanghai: Shanghai Translation Publishing House. However, the theory of economic growth also has its shortcomings, that is, it overemphasizes the purpose and interests of the donor countries and belittles the status and interests of the recipient countries. Social exchange theory can make up for this defect to a great extent. Dingshaobin, a well-known domestic expert on aid, pointed out that the theory overcomes the one-sidedness of the theory of the interests of donors and the theory of the needs of recipient countries. It inspires researchers to observe and understand the relationship between aid and aid from the perspective of reciprocity, attaches importance to the power of 'weak' recipient countries, 11. USAID, Energy and environment: acquisitions (2011), Available online at: http://philippines Usaidgov/programs/energy environment ent/accomplishments. 4
https://openalex.org/W3130664472	https://www.researchsquare.com/article/rs-229945/latest.pdf	English	null	Classification of Colorectal Carcinoma Based on Ferroptosis-related Gene Signature	Research Square (Research Square)	2,021	cc-by	6,489	Xianglong Duan (  duanxl_spph@163.com ) Xianglong Duan (  duanxl_spph@163.com ) Xizang Mingzu University, Xianyang, Shaanxi, P.R. China Department of General Surgery, Shaanxi Provincial People's Hospital, Affiliated Hospital of Northwestern Polytechnical University, Xi'an, Shaanxi, P.R. China. Xizang Mingzu University, Xianyang, Shaanxi, P.R. China Department of General Surgery, Shaanxi Provincial People's Hospital, Affiliated Hospital of Northwestern Polytechnical University, Xi'an, Shaanxi, P.R. China. Jun Bai Department of Medical Oncology, Shaanxi Provincial People's Hospital, Affiliated Hospital of Northwestern Polytechnical University, Xi'an, Shaanxi, P.R. China Classification of Colorectal Carcinoma Based on Ferroptosis-related Gene Signature QingFang Yue Shaanxi Provincial People's Hospital https://orcid.org/0000-0003-4664-0305 Yuan Zhang Department of Medical Oncology, Shaanxi Provincial People's Hospital, Affiliated Hospital of Northwestern Polytechnical University, Xi'an, Shaanxi, P.R. China Fei Wang Department of gynecology, Shaanxi Provincial People's Hospital, Affiliated Hospital of Northwestern Polytechnical University, Xi'an, Shaanxi, P.R. China Fei Cao Department of Medical Oncology, Shaanxi Provincial People's Hospital, Affiliated Hospital of Northwestern Polytechnical University, Xi'an, Shaanxi, P.R. China Xianglong Duan (  duanxl_spph@163.com ) Xizang Mingzu University, Xianyang, Shaanxi, P.R. China Department of General Surgery, Shaanxi Provincial People's Hospital, Affiliated Hospital of Northwestern Polytechnical University, Xi'an, Shaanxi, P.R. China. Jun Bai Department of Medical Oncology, Shaanxi Provincial People's Hospital, Affiliated Hospital of Northwestern Polytechnical University, Xi'an, Shaanxi, P.R. China Research Keywords: Colorectal cancer, ferroptosis related-genes, classification Posted Date: February 23rd, 2021 DOI: https://doi.org/10.21203/rs.3.rs-229945/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License QingFang Yue Shaanxi Provincial People's Hospital https://orcid.org/0000-0003-4664-0305 Yuan Zhang xi Provincial People's Hospital https://orcid.org/0000-0003-4664-0305 Department of Medical Oncology, Shaanxi Provincial People's Hospital, Affiliated Hospital of Northwestern Polytechnical University, Xi'an, Shaanxi, P.R. China Fei Wang Fei Wang Department of gynecology, Shaanxi Provincial People's Hospital, Affiliated Hospital of Northwestern Polytechnical University, Xi'an, Shaanxi, P.R. China Fei Cao Department of Medical Oncology, Shaanxi Provincial People's Hospital, Affiliated Hospital of Northwestern Polytechnical University, Xi'an, Shaanxi, P.R. China Department of Medical Oncology, Shaanxi Provincial People's Hospital, Affiliated Hospital of Northwestern Polytechnical University, Xi'an, Shaanxi, P.R. China Research Keywords: Colorectal cancer, ferroptosis related-genes, classification Posted Date: February 23rd, 2021 DOI: https://doi.org/10.21203/rs.3.rs-229945/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. R d F ll Li Page 1/21 Abstract Background: Ferroptosis is an iron-dependent form of programmed cell death, involves in the development of many cancers. However, systematic analysis of ferroptosis related-genes in colorectal carcinoma (CRC) remains to be clarified. We herein analyzed the public databases to identify the molecular features of CRC by the development of a classification based on the gene expression profile of ferroptosis-related genes. Methods: We collected the gene expression data and clinical information from The Cancer Atlas and Gene Expression Omnibus to explore the correlation of ferroptosis-related gene expression of CRC. Consensus clustering was performed to determine the clusters of colorectal cancer patients, then we analyzed the prognostic value, transcriptome features, immune microenvironment, drug sensitivity, gene mutations differences of the subclasses. Results: Four subclasses of CRC (C1, C2, C3 and C4) were identified. There were significant differences in the prognosis of patients between the four subtypes. Functional enrichment suggested that these ferroptosis related-genes were associated with biological processes such as metabolic processes and oxidative stress, and the KEGG pathway suggested that it was closely related to ferroptosis and glutathione metabolic pathway. There were significant differences in immune cell infiltrations, immune score, stromal score and tumor purity among four subclasses. The expression of PD-L1 was differentially expressed in four subclasses and PD-L1 was significantly correlated with the expression of several ferroptosis-related genes in the differentially expressed subtypes. There were significant differences in stemness indices and drug sensitivity in four subclasses, and gene mutations analysis showed that ferroptosis-related genes such as TP53 had high mutations in different subtypes, and there were significant differences in mutation frequencies in the subclasses. Conclusion: This study established a new CRC classification based on ferroptosis-related genes expression profiles, and different subgroups may have their unique gene expression patterns, indicating that the heterogeneity within CRC and the classification might provide valuable stratification for the design of future treatment. Data collection Colon carcinoma(n=461) and rectal carcinoma(n=172) samples with mRNA sequencing data and clinical properties were downloaded from The Cancer Genome Atlas (TCGA, http://cancergenome.nih.gov/). In addition, gene expression data together with clinical properties of 114 CRC samples in the GSE152430 data set was downloaded from Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/). Thus, in this study three datasets were merged into a metadata set of 747 CRC samples and the combat function in the SVA R package was applied to remove the batch effects[13]. The data from TCGA and GEO are both publicly available. The current research follows the TCGA and GEO data access policies and publication guidelines. Background Colorectal cancer (CRC) is the third most commonly diagnosed cancer and accounts for the second leading cause of cancer death globally[1, 2]. Although the current new treatments such as biotargeted therapy, immunotherapy, and precise treatment have applied for CRC treatment[3, 4], the clinical outcomes are still disappointing. CRC is a heterogeneous disease[5], it is critical to uncover the underlying molecular mechanisms for the development of novel targeted therapies. Ferroptosis is a recently defined regulated cell death characterized by the iron-dependent accumulation of lipid peroxidation[6]. A plethora of researches have implicated that ferroptosis play an important role in Page 2/21 Page 2/21 carcinogenesis and that ferroptosis promises to be a novel option for the cancer treatment, especially for tumors which are resistant to conventional treatments[7, 8]. carcinogenesis and that ferroptosis promises to be a novel option for the cancer treatment, especially for tumors which are resistant to conventional treatments[7, 8]. In recent years, the role of ferroptosis in CRC has attracted much attention. For example, the research by Xie et al. demonstrated that ferroptosis related gene TP53 could inhibit dipeptidyl peptidase 4 activity in an independent way of transcription, which can restrain erastin-induced ferroptosis in CRC[9]. Park et al. demonstrated that bromelain effectively causes ferroptosis in Kras mutant cell lines compared to in Kras wild-type cells by modulating ACSL-4 levels[10]. Sui et al. revealed that GPX4 inhibition was a key determinant in RSL3-induced ferroptosis in CRC[11]. Xu et al reported that targeting SLC7A11 might selectively kill colorectal cancer stem cells by inducing ferroptosis and attenuate chemoresistance in CRC[12]. Although previous studies have identified several gene markers associated with ferroptosis in CRC. However, the relationship between ferroptosis-related genes (FRGs) and CRC treatment and prognosis is still unclear. In the present study, we firstly collected the mRNA expression profiles and corresponding clinical data of CRC patients from public databases. Then, three cohorts were merged into a metadata set of 747 patients. We classified CRC into four distinct subtypes based on consensus clustering of FRGs profiles. Furthermore, we evaluated the prognosis value, transcriptome features, immune microenvironment, drug sensitivity as well as gene mutation alterations of the four subclasses, and analyzed the correlation with the conventional clinical features. The identification of ferroptosis-related subclasses may can provide an important prognostic and therapeutic value for CRC. In the present study, we firstly collected the mRNA expression profiles and corresponding clinical data of CRC patients from public databases. Background Then, three cohorts were merged into a metadata set of 747 patients. We classified CRC into four distinct subtypes based on consensus clustering of FRGs profiles. Furthermore, we evaluated the prognosis value, transcriptome features, immune microenvironment, drug sensitivity as well as gene mutation alterations of the four subclasses, and analyzed the correlation with the conventional clinical features. The identification of ferroptosis-related subclasses may can provide an important prognostic and therapeutic value for CRC. Calculation of stemness index We calculate the stemness index using an innovative one-class logistic regression (OCLR) machine learning algorithm as previously described[25]. By using a linear transformation, the stemness index is mapped to the range [0,1] by subtracting the minimum value and dividing it by the maximum value. Functional enrichment analysis The "clusterProfiler" R package was applied to conduct Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses based on the FRGs in CRC[22]. P values were adjusted with the BH method. The thresholds for analyses were determined by a p-value < 0.05 indicating significantly enriched functional annotations Consensus cluster analysis to identify ferroptosis-related subtypes Initially, in order to perform consensus classification of CRC for metadate set, we used the R package “ConsensusClusterPlus”, which offers stable quantitative and visual evidence for estimating the number of unsupervised clusters in a dataset[19]. In each algorithm, tumors were sampled 100 times, and the k- means algorithm with the Euclidean distance metric was carried out. The clustering number was assessed according to the area under the cumulative distribution function (CDF) curve[20]. The R package survminer is then used to evaluate the prognostic value of subtypes [21] and a heat map was draw according to the tumor/node/metastasis (TNM) stage of the samples. In addition, the analysis of variance (ANOVA) test was utilized to screen the differentially expressed ferroptosis-related genes (DEGs) among different subtypes. and the P values were adjusted by Benjamini & Hochberg (BH) correction (P <0.05). Then, correlation analysis and principal component analysis based on DEGs were carried out. Immune characterizations analysis The R package "CIBERSORT" was used to evaluate the infiltration of immune cells, among ferroptosis subclasses[23]. The immune score, matrix score, and tumor purity of the sample were calculated by the R package "ESTIMATE"[24]. In recent years, cancer immunotherapy based on immune checkpoint inhibitors (ICIs) has achieved considerable success in the clinic. We extracted the expression profile of immune checkpoint molecular (PD-L1) and FRGs in metadate set, and evaluated the differential expression of immune checkpoint molecular (PD-L1) in subclasses, then the R package "ggplot2" was used to analyze the correlation between PD-L1 and FRGs. Construction of ferroptosis subclasses in CRC. The expression of 60 FRGs were obtained from the previous literatures[14-17] and which are provided in Supplementary Table S1, next, we extracted gene expression profiles of ferroptosis-related genes in CRC samples and the candidate genes were used for clustering. The R package “Rcircos”[18] was applied to characterize the location distribution of FRGs in CRC samples. Page 3/21 Page 3/21 Correlation analysis of drug sensitivity among subclasses To identify which target compounds might be useful in the subtypes, the R package "pRRophetic" was used to predict the drug sensitivity in subclasses[26]. Ferroptosis subclasses in CRC The flow chart of this study is shown in Figure 1A. A total of 461 colon adenocarcinoma (COAD) and 172 rectum adenocarcinomas (READ) patients from TCGA database and 114 CRC patients from the GEO database were finally enrolled. We merged TCGA and GEO data and adopted the ComBat method to remove the batch effect. Next, we extracted ferroptosis expression gene profiles and found that there were 59 ferroptosis-related genes expressed in CRC tissues. Distribution of FRGs by circosplot were shown in Figure 1B. By applying consensus clustering on the gene expression profile of the above mentioned 59 FRGs on metadata set 747 CRC samples, four resulting clusters were defined as C1-C4(Figure2A, B). In addition, a high similarity of gene expression pattern was observed within each subgroup based on consensus matrix k=4(Figure2C). Survival analysis indicated a significant prognostic difference between four subgroups in metadate set (Figure 2D, p=0.034). Next, we developed a heatmap demonstrating the relationship between stage and 59 genes (Figure 2E). To validate the subclasses’ assignments, we performed the correlation analysis of 59 genes, the results showed that there were co-expression correlations among some FRGs (Figure 3A). Principal component analysis (PCA) was applied to decrease the dimension of features and we found the subtype designations were largely concordant with two- dimensional PCA distribution patterns (Figure3B). Hence, it was reasonable to classify FRGs into four subclasses. Independence of the FRGs subclass from CRC patients’ clinical characteristics In order to evaluate whether the FRGs subclass was independent variable when considering other conventional clinical features (age, gender, TNM stage, tissue or organ of origin, and body mass index (BMI)) in CRC patients. The correlation analysis by using RCIRCOS were performed. Statistical analysis The Kaplan–Meier with log-rank test was used to assess survival difference between groups. All statistical analyses were conducted using R software. If not specified above, the P value less than 0.05 was considered statistically significant. The analysis of genomic alterations difference Page 4/21 Page 4/21 According to the gistic score by GenePattern(http://cloud.genepattern.org/), and the R package "Maftools" was used to characterize the difference of mutations and copy number variations of different subclasses[27]. Tumor immune microenvironment characteristics among four subclasses To uncover the immune heterogeneity among the four subtypes, we resorted to immune-related tools. Through the CIBERSORT method, we evaluated the differences in the immune infiltration of 22 immune cell types between four subclasses in CRC patients. As illustrated in Figure 6, the infiltration of 18 immune cells were different between the four subclasses. Compared with the other three subclasses, tumor in C3 had higher abundance of 7 immune cell populations (B cells, M2 macrophages, resting mast cells, monocytes, NK cells, plasma cells, CD8+T cells). In addition, C3 also exhibited low infiltration of M1 macrophages, activated mast cells, neutrophils, Tfh cells and Treg cells (Figure 6). We furtherly investigated stromal and immune scores based on the ESTIMATE method. The results indicated that tumor in C3 had higher immune and stromal scores but lower tumor purity (Figure7). Transcriptomes of the CRC subclasses To further characterize the differentially expressed FRGs (DEGs)in different subtypes, ANOVA differential expression analysis were performed. Gene expression differences were considered significant if the Page 5/21 Page 5/21 adjusted P value was < 0.05('BH' correction), the results indicated that all 59 FRGs were differentially expressed in all of these subtypes (Figure4). To elucidate the biological functions and pathways of the 59 DEGs in CRC. Gene Oncology enrichment analysis of the signature genes was conducted using the CLUSTERPROFILER package, and significantly enriched biological processes are shown in Figure 5. GO enrichment analysis showed that these FRGs were significantly associated with biological processes such as metabolic processes and oxidative stress, and KEGG pathway analysis revealed that they were associated with metabolic pathways such as iron- dependent cell death and glutathione metabolism. Assessment of immunotherapeutic response in subclasses Immunotherapy have emerged as a promising strategy for the treatment of many cancers. We subsequently investigated the expression of PD-L1 which is a pivotal immunomodulator. As shown in Figure 8A, the expression of PD-L1 were different between the four subclasses, and tumor in C3 had higher expression of PD-L1 expression than other subclasses. In addition, we investigated the correlation between PD-L1 expression and FRGs in CRC patients. The results revealed that the expression of PD-L1 was significantly correlated with multiple FRGs such as ACSL4, ALOX5, HOMX1 and IREB2(Figure8B, FigureS1). Cancer stem cells are known to play a critical role in the growth, metastasis, and recurrence of tumors. Recent studies have reported that higher stemness indices could limit the antitumor immune responses and be associated with decreased PD-L1 expression[28]. We herein applied an innovative one- class logistic regression (OCLR) machine-learning algorithm to calculate stemness indices of subclasses. As shown in Figure9, there was a significant difference in the stemness index among the subtypes, and we observed that patients in C3 had lower stemness indices value than other subtypes. Thus, these data support our analysis that patients in C3 may have better prognosis, and might be more promising in response to immunotherapy. Difference sensitivity to antitumor drugs for CRC subclasses Correlation of the CRC subclasses with mutations and CNV differences The tumoral genomic landscape has been proven to be associated with the application and efficacy of targeted therapies and immunotherapy. We further evaluated the difference of gene mutations and copy number variation (CNV) among the four subtypes. The results revealed that distinct subtypes had different subclasses tend to have different mutational characteristics. For example,49% samples in C1 had TP53 mutation while 62% in C2 and 71% in C4(Figure11A). What’s more, we further obtained gistic scores of different subtypes according to the genepattern website, and the results indicated that there was a significant difference in mutation frequency between the four subtypes. These data could protect the samples in different clusters from the option of individualized therapeutic agents. Correlation of CRC subclasses with clinical parameters In order to elucidate whether the ferroptosis-related subclasses for CRC is independent indicator when considering other conventional clinical parameters. Based on the integrated clinical information downloaded from COAD and READ in TCGA with the clinical information obtained from GEO, we obtained the clinical information of the metadate dataset. By applying the RCIRCOS method, we compared the distribution of ferroptosis subclasses with the patients’ stage, age, gender, tissue or organ of origin, and BMI. As shown in Figure 10, ferroptosis subclasses do not show significant crossover with other clinical parameters, and have significantly difference with the parameters. Hence, the subclass may serve as a potential molecular subtype which can provide an important prognostic and therapeutic value for CRC. Difference sensitivity to antitumor drugs for CRC subclasses Page 6/21 By applying the R package pRRophetic, we further explored the association between the CRC subclasses and sensitivity toward antitumor drugs. For gemcitabine, there were significant differences between the four subtypes(p<0.0001). For eriotinib, C3 exhibited significant association of eriotinib-resistant group. For bortezomib, the results showed significant differences between some subtypes (Figure10). In addition, other drugs such as cytarabine, dasatinib and sorafenib were explored, and significant differences were observed between some subtypes (Supplementary Figure). These data demonstrated the CRC subclasses might provide precise therapeutic approaches in the further. Discussion Ferroptosis is a new form of programmed cell death which is different from other types of cell death, the correlation between ferroptosis and cancer is extremely complicated and inducing ferroptosis is considered a novel approach for cancer therapy[29–31]. However, the systematic analysis of CRC has yet to be elucidated. In this study, to obtain the classification of ferroptosis-related genes profile of CRC samples, we analyzed gene expression profile of samples in TCGA and GEO datasets, and the batch effect from different platforms were successfully removed. The consensus clustering based on our large sample size indicated that our transcriptome classification was robust. Then we screened 59 FRGs that could be used to cluster patients with CRC into four groups, with significant differences in clinicopathology and prognosis. Next, transcriptome features, immune makers and drug sensitivity of the subclasses were explored. clinicopathology and prognosis. Next, transcriptome features, immune makers and drug sensitivity of the subclasses were explored. Page 7/21 Page 7/21 Recently, a few studies have indicated that several genes could regulate drug-induced ferroptosis in CRC, however, their relationship with CRC patient’s clinical outcome remains unknown. In this study, we found most (59/60) of were differently expressed in CRC tissues, and each subclass with different prognosis. These results significantly demonstrated the potential role of ferroptosis in CRC. Tumor microenvironment and immune cell infiltration are reported to be correlated with cancer prognosis[32]. And some studies conducted over the past five years revealed that the ferroptosis are tightly associated with antitumor immunity[33]. We investigated the relationship between subclasses and immune markers including immune score, tumor purity, and infiltrating immune cells. As we expected, the ferroptosis subclasses could successfully distinguished the CRC patients based on the above makers, which implied the credibility of the classification in evaluating the immune response. Moreover, the relevant estimation demonstrated that C3 had the higher immune score, stromal score and the lowest tumor purity. These data suggested that C3 subclass was of high heterogeneity and might be refractory. In recent years, there has been great success in applying immune checkpoint inhibitors for the treatment of various cancers, the PD-L1 level is a commonly used maker for predicting the efficacy of Tumor microenvironment and immune cell infiltration are reported to be correlated with cancer prognosis[32]. And some studies conducted over the past five years revealed that the ferroptosis are tightly associated with antitumor immunity[33]. Discussion We investigated the relationship between subclasses and immune markers including immune score, tumor purity, and infiltrating immune cells. As we expected, the ferroptosis subclasses could successfully distinguished the CRC patients based on the above makers, which implied the credibility of the classification in evaluating the immune response. Moreover, the relevant estimation demonstrated that C3 had the higher immune score, stromal score and the lowest tumor purity. These data suggested that C3 subclass was of high heterogeneity and might be refractory. In recent years, there has been great success in applying immune checkpoint inhibitors for the treatment of various cancers, the PD-L1 level is a commonly used maker for predicting the efficacy of immunotherapy. Patients with high levels of PD-L1 in tumor tissue who received PD-1/PD-L1 inhibitors had better survival outcomes compared to patients who did not receive this treatment[34, 35]. Thus, we investigated the expression of immune checkpoint molecules PD-L1 and the correlation between PD-L1 and ferroptosis-related genes, the results showed that there were significant differences in the expression of PD-L1 among the four subgroups, and there was a significant correlation between PD-L1 and several genes, which further indicated that ferroptosis was significantly correlated with immunotherapy. Cancer stemness had been reported to be associated with suppressed immune responses across cancers. As expected in our study, we found that there were significant differences between subgroups. Based on the above evidence, we hypothesize that the subclass based on ferroptosis-related genes may act as reliable immune-related biomarker for cancer therapy. As we were known, gene mutation may induce treatment resistance. In our study, genomic analysis revealed a distinct mutation and copy number change landscape among subclasses. Results indicated that distinct subclasses tended to have different mutation proportion of each gene. These data might demonstrate that our identified ferroptosis subtypes attached importance to gene alterations. however, the correlations between ferroptosis and somatic mutations need to be further studied. Next, utilizing the R package pRRophetic, we tested the sensitivity of antitumor drugs, the results showed significant differences in drug sensitivity among the four subtypes, which suggests that there are different mechanisms involved in CRC among the four subtypes. Then, we compared the classification with distribution of the conventical clinical parameters. As a result, it revealed that molecular classification of FRGs has no significant overlap with the classification of other clinical parameters in CRC. Funding This study was supported by Natural Science Foundation of Shaanxi Province (No. 2019JM-563), National Natural Science Foundation of China (No.81760441), Key Research and Development Program Projects of Shaanxi Province (No.2019ZDLSF02-09-01) and Scientific Research Project of Shaanxi Education Department (No.18JC027). Conclusion In conclusion, in this study we established a new CRC classification based on ferroptosis-related genes expression profiles, and different subgroups may have their unique gene expression patterns, and our work contributes to the insights of ferroptosis signatures in CRC and provides valuable information for personalized treatment and prognosis prediction. Discussion Page 8/21 The present study was a pioneering work for the classification of CRC based on the molecular signature of ferroptosis. However, we have to acknowledge some flaws of our present work. First, a larger sample size dataset is critically needed to validate our classification. Second, it will be necessary to validate our classification in clinical samples. In addition, further basic experiments are important to understand the difference in the mechanisms of the four subtypes in CRC. size dataset is critically needed to validate our classification. Second, it will be necessary to validate our classification in clinical samples. In addition, further basic experiments are important to understand the difference in the mechanisms of the four subtypes in CRC. Abbreviations CRC: Colorectal carcinoma; FRGs: Ferroptosis-related genes; GEO: Gene Expression Omnibus; TCGA: The Cancer Genome Atlas; TNM: tumor/node/metastasis; DEGs: differentially expressed ferroptosis-related genes; ANOVA: analysis of variance; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; PCA: Principal component analysis; BMI: body mass index; COAD: Colon adenocarcinoma; READ: Rectum adenocarcinomas; OCLR: one-class logistic regression; CNV: Copy number variation; Authors’ contributions The corresponding authors are responsible for the whole article design, Qingfang Yue is responsible for the manuscript writing and bioinformatics analysis, Yuan Zhang, Fei Cao and Fei Wang are responsible for the proofreading and partial statistics. Consent for publication Not applicable Competing interests The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. 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Herbst RS, Baas P, Kim DW, Felip E, Perez-Gracia JL, Han JY, Molina J, Kim JH, Arvis CD, Ahn MJ, et al: Pembrolizumab versus docetaxel for previously treated, PD-L1-positive, advanced non-small-cell lung cancer (KEYNOTE-010): a randomised controlled trial. Lancet 2016, 387:1540-1550. 35. Borghaei H, Paz-Ares L, Horn L, Spigel DR, Steins M, Ready NE, Chow LQ, Vokes EE, Felip E, Holgado E, et al: Nivolumab versus Docetaxel in Advanced Nonsquamous Non-Small-Cell Lung Cancer. N Engl J Med 2015, 373:1627-1639. 35. Borghaei H, Paz-Ares L, Horn L, Spigel DR, Steins M, Ready NE, Chow LQ, Vokes EE, Felip E, Holgado E, et al: Nivolumab versus Docetaxel in Advanced Nonsquamous Non-Small-Cell Lung Cancer. N Engl J Med 2015, 373:1627-1639. Figures Page 12/21 Figure 1 Figure 1 Page 12/21 (A) The flow chart of this study. (B) Circos plot showed the distribution of ferroptosis-related genes in human chromosome. (A) The flow chart of this study. (B) Circos plot showed the dist human chromosome. Figure 2 (A) The consensus among clusters for each category number k clustering. (C) Consensus clustering of CRC samples with k = 4 Page 13/21 Figure 2 (A) The consensus among clusters for each category number k. (B) Delta area curve of consensus clustering. (C) Consensus clustering of CRC samples with k = 4. (D) Kaplan-Meier survival curves of CRC in each ferroptosis subclass. (E) Heatmap shows the correlation between the expression of ferroptosis- Figure 2 Figure 2 (A) The consensus among clusters for each category number k. (B) Delta area curve of consensus clustering. (C) Consensus clustering of CRC samples with k = 4. (D) Kaplan-Meier survival curves of CRC in each ferroptosis subclass. (E) Heatmap shows the correlation between the expression of ferroptosis- (A) The consensus among clusters for each category number k. (B) Delta area curve of consensus clustering. (C) Consensus clustering of CRC samples with k = 4. (D) Kaplan-Meier survival curves of CRC in each ferroptosis subclass. (E) Heatmap shows the correlation between the expression of ferroptosis- Page 13/21 Page 13/21 related genes and the stage of the four patient classes. The color codes for different stage are as indicated. The expression of ferroptosis-related genes is also indicated by a color code bar, where red refers to high expression or upregulation and green refers to low expression or downregulation. Figure 3 (A) Heatmap describing co-expression of ferroptosis-related genes; (B) Principal component analysis (PCA) of ferroptosis-related genes expression profiles of the metadata set. Figure 3 (A) Heatmap describing co-expression of ferroptosis-related genes; (B) Principal component analysis (PCA) of ferroptosis-related genes expression profiles of the metadata set. Page 14/21 Figure 4 59 ferroptosis-related genes differentially expressed in different subclasses. 59 ferroptosis-related genes differentially expressed in different subclasses. Page 15/21 Page 15/21 Figure 5 Functional enrichment analysis of differentially expressed ferroptosis-related genes in subtypes. Figure 5 Functional enrichment analysis of differentially expressed ferroptosis-related genes in subtypes. Functional enrichment analysis of differentially expressed ferroptosis-related genes in subtypes. Page 16/21 Page 16/21 Page 17/21 Figure 6 The difference of immune cell infiltration abundances CRC subtypes. Figure 7 Figure 6 The difference of immune cell infiltration abundances CRC subtypes. The difference of immune cell infiltration abundances CRC subtypes. Page 17/21 The difference of immune cell infiltration abundances CRC subtypes. Figure 7 The difference of immune cell infiltration abundances CRC subtypes. Figure 7 Page 17/21 Immune scores, stromal scores and tumor purity in CRC subclasses. u e sco es, st o a sco es a d tu o pu ty C C subc asses. Figure 8 Figure 8 (A) The PD-L1 mRNA expression in ferroptosis subclasses of CRC patients. (B) The correlation between PD-L1 with the expression of ACSL4, ALOX5, HMOX1 and IREB2. Figure 8 (A) The PD-L1 mRNA expression in ferroptosis subclasses of CRC patients. (B) The correlation between PD-L1 with the expression of ACSL4, ALOX5, HMOX1 and IREB2. Page 18/21 Page 18/21 Page 18/21 gure 9 fference of stemness indices among four ferroptosis subtypes. Figure 9 Difference of stemness indices among four ferroptosis subtypes. Page 19/21 Page 19/21 Figure 10 Differences in antitumor drug sensitivity in ferroptosis subclasses of CRC patients. Figure 10 Differences in antitumor drug sensitivity in ferroptosis subclasses of CRC patients. Figure 12 Circos plot describing the comparison of ferroptosis subclass with the distribution of conventional clinical parameters. Figure 10 Figure 10 Differences in antitumor drug sensitivity in ferroptosis subclasses of CRC patients. Differences in antitumor drug sensitivity in ferroptosis subclasses of CRC patients. Page 20/21 Page 20/21 Page 20/21 Figure 11 Figure 11 (A) OncoPrint of mutation status of genes in ferroptosis subclasses. (B) CNV mutation map among ferroptosis subclasses. (C) The violin plot of mutation frequency in ferroptosis subclasses. (A) OncoPrint of mutation status of genes in ferroptosis subclasses. (B) CNV mutation map among ferroptosis subclasses. (C) The violin plot of mutation frequency in ferroptosis subclasses. Figure 12 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. TableS1.xls Supplementaryfigures.pdf Page 21/21
https://openalex.org/W4302760198	http://journals.krc.karelia.ru/index.php/precambrian/article/download/1656/1211	Russian	null	RELICTS OF DESTROYED OCEANIC ISLAND ARCS IN THE PALEOPROTEROZOIC VYATKA OROGEN (EAST-EUROPEAN CRATON)	Trudy Karelʹskogo naučnogo centra Rossijskoj akademii nauk	2,022	cc-by	2,495	Труды Карельского научного центра РАН. 2022. № 5. С. 34–37 Transactions of the Karelian Research Centre RAS. 2022. No. 5. P. 34–37 DOI: 10.17076/geo1656 КРАТКИЕ СООБЩЕНИЯ Short communications УДК 550.4; 550.93 РЕЛИКТЫ РАЗРУШЕННЫХ ОКЕАНИЧЕСКИХ ОСТРОВНЫХ ДУГ В ПАЛЕОПРОТЕРОЗОЙСКОМ ВЯТСКОМ ОРОГЕНЕ (ВОСТОЧНО-ЕВРОПЕЙСКИЙ КРАТОН) К. Г. Ерофеева1,2, А. В. Самсонов1, Т. А. Пилицына1, А. Н. Ларионов3, В. А. Спиридонов4, А. В. Постников5, А. Ю. Вовшина3, А. С. Дубенский2 1 Институт геологии рудных месторождений, петрографии, минералогии и геохимии РАН (Старомонетный пер., 35, Москва, Россия, 119017), xenin.erofeeva@ya.ru 2 Геологический институт РАН (Пыжевский пер., 7, Москва, Россия, 119017) 3 Всероссийский научно-исследовательский геологический институт имени А. П. Карпинского (Средний проспект, 74, Санкт-Петербург, Россия, 199106) 4 ВНИИ Геосистем (Варшавское шоссе, 8, Москва, Россия, 117105) 5 РГУ нефти и газа им. И. М. Губкина (Ленинский просп., 63/2, Москва, Россия, 119991) Труды Карельского научного центра РАН. 2022. № 5. С. 34–37 Transactions of the Karelian Research Centre RAS. 2022. No. 5. P. 34–37 DOI: 10.17076/geo1656 КРАТКИЕ СООБЩЕНИЯ Short communications УДК 550.4; 550.93 К. Г. Ерофеева1,2, А. В. Самсонов1, Т. А. Пилицына1, А. Н. Ларионов3, В. А. Спиридонов4, А. В. Постников5, А. Ю. Вовшина3, А. С. Дубенский2 1 Институт геологии рудных месторождений, петрографии, минералогии и геохимии РАН (Старомонетный пер., 35, Москва, Россия, 119017), xenin.erofeeva@ya.ru 2 Геологический институт РАН (Пыжевский пер., 7, Москва, Россия, 119017) 3 Всероссийский научно-исследовательский геологический институт имени А. П. Карпинского (Средний проспект, 74, Санкт-Петербург, Россия, 199106) 4 ВНИИ Геосистем (Варшавское шоссе, 8, Москва, Россия, 117105) 5 РГУ нефти и газа им. И. М. Губкина (Ленинский просп., 63/2, Москва, Россия, 119991) На основе изучения раннедокембрийских метаморфизованных вулканогенно- осадочных пород Вятского пояса (Волго-Уральский сегмент Восточно-Европей- ского кратона) рассмотрены вопросы тектонических обстановок его формирова- ния и места в палеоконтинентальных корреляциях 2,2–2,0 млрд лет назад. К лю че вые сло в а: Восточно-Европейский кратон; Волго-Уралия; орогены; островные дуги Для цитирования: Ерофеева К. Г., Самсонов А. В., Пилицына Т. А., Ларионов А. Н., Спиридонов В. А., Постников А. В., Вовшина А. Ю., Дубенский А. С. Реликты раз- рушенных океанических островных дуг в палеопротерозойском Вятском орогене (Восточно-Европейский кратон) // Труды Карельского научного центра РАН. 2022. № 5. С. 34–37. doi: 10.17076/geo1656 Финансирование. Исследование выполнено при финансовой поддержке в рамках госзадания лаборатории петрографии ИГЕМ РАН. K. G. Erofeeva1,2, A. V. Samsonov1, T. A. Pilitsyna1, A. N. Larionov3, V. A. Spiridonov4, A. V. Postnikov5, A. Yu. Vovshina3, A. S. Dubenskiy2. RELICTS OF DESTROYED OCEANIC ISLAND ARCS IN THE PALEOPROTEROZOIC VYATKA OROGEN (EAST-EUROPEAN CRATON) 34 Transactions of the Karelian Research Centre of the Russian Academy of Sciences. 2022. No. 5 2 Geological Institute, Russian Academy of Sciences (7 Pyrzhevsky Per., 119017 Moscow, Russia) 2 Geological Institute, Russian Academy of Sciences (7 Pyrzhevsky Per., 119017 Moscow, Russia) 3 Karpinsky Russian Geological Research Institute (74 Sredny Prospect, 199106 St. Petersburg, Russia) 4 VNIIGeosystem (8 Varshavskoe Shosse, 117105 Moscow, Russia) 5 Gubkin State University of Oil and Gas (63/2 Leninsky Ave., 119991 Moscow, Russia) 2 Geological Institute, Russian Academy of Sciences (7 Pyrzhevsky Per., 119017 Moscow, Russia) 3 Karpinsky Russian Geological Research Institute (74 Sredny Prospect, 199106 St. Petersburg, Russia) 4 VNIIGeosystem (8 Varshavskoe Shosse, 117105 Moscow, Russia) 5 Gubkin State University of Oil and Gas (63/2 Leninsky Ave., 119991 Moscow, Russia) 4 VNIIGeosystem (8 Varshavskoe Shosse, 117105 Moscow, Russia) y ( , , ) 5 Gubkin State University of Oil and Gas (63/2 Leninsky Ave., 119991 Moscow, Russia) y 5 Gubkin State University of Oil and Gas (63/2 Leninsky Ave., 119991 Moscow, Ru On the basis of the study of Early Precambrian metamorphosed volcanogenic-sedi- mentary rocks of the Vyatka belt of the northeastern Volga-Ural megablock, East Euro- pean Craton, the paper will consider the tectonic settings for the formation of this belt and its position in the paleocontinental correlations 2.2-2.0 Ga ago. Ke ywords: East European Craton; Volgo-Uralia; orogens; oceanic island arcs For cit a t ion: Erofeeva K. G., Samsonov A. V., Pilitsyna T. A., Larionov A. N., Spiri- donov V. A., Postnikov A. V., Vovshina A. Yu., Dubenskiy A. S. Relicts of destroyed oce- anic island arcs in the Paleoproterozoic Vyatka Orogen (East-European Craton). Trudy Karel’skogo nauchnogo tsentra RAN = Transactions of the Karelian Research Centre RAS. 2022;5:34–37. doi: 10.17076/geo1656 Funding. The study was funded under state assignment to the Petrography Laboratory IGEM RAS. Вятский пояс выделен по результатам буре- ния и оконтурен по геофизическим данным как крупная вулканогенно-осадочная структура, зажатая между архейскими гранито-гнейсовы- ми блоками [Bogdanova et al., 2016]. Вятский пояс слагают Hbl-Bt-гнейсы, сохранившие Pl- порфировую структуру и образованные за счет вулканогенного протолита, и Crd-Sil-Grt-Bt- гнейсы первично осадочного происхождения. Эти вулканогенно-осадочные породы, объе- диняемые в унийскую свиту, прорваны жилами гнейсовидных Ms-Bt-трондьемитов талицкого комплекса мощностью от первых сантиметров до 5 метров. что не обнаруженного, источника палеопроте- розойского магматического циркона. Труды Карельского научного центра Российской академии наук. 2022. № 5 Литература Бибикова Е. В., Богданова С. В., Постников А. В., Попова Л. П., Кирнозова Т. И., Фугзан М. М., Глущен- ко В. В. Зона сочленения Сарматии и Волго-Уралии: изотопно-геохронологическая характеристика су- пракрустальных пород и гранитоидов // Стратигра- фия и геологическая корреляция. 2009. № 6. С. 3–16. Bogdanova S. V., Gorbatschev R., Garetsky R. G. EUROPE \| East European Craton. Reference Module in Earth Systems and Environmental Sciences. Elsevier; 2016. doi: 10.1016/B978-0-12-409548-9.10020-X Condie K. C., Pisarevsky S. A., Puetz S. J. LIPs, orogens and supercontinents: The ongoing saga. Gondwana Res. 2021;96:105–121. doi: 10.1016/j. gr.2021.05.002 Гурьянов В. А., Диденко А. Н., Песков А. Ю., Рога- нов Г. В., Дымович В. А. Раннедокембрийские грани- тоиды Батомгского выступа фундамента Юго-Вос- тока сибирской платформы: возраст и геодинами- ческая обстановка формирования // Тихоокеанская геология. 2016. Т. 35, № 3. С. 23–44. Elming S.-Å., Salminen J., Pesonen L. J. Chapter 16: Paleo-Mesoproterozoic Nuna supercycle. Peso- nen L. J., Salminen J., Evans D. A. D., Elming S.-Å., Veikkolainen T. (eds.). Ancient Supercontinents and the Paleogeography of the Earth. Elsevier; 2021. P. 499– 548. doi: 10.1016/B978-0-12-818533-9.00001-1 Котов А. Б. Граничные условия геодинамических моделей формирования континентальной коры Ал- данского щита: Автореф. дис. … д-ра геол.-минер. наук. СПб.: Изд-во СПбГУ, 2003. 78 с. Guryanov V. A., Didenko A. N., Peskov A. Y. Roganov G. V., Dymovich V. A. Early Precambrian grani- toids of the Batomga inlier of the southeastern Siberian Platform basement: Age and geodynamic formation settings. Russ. J. of Pac. Geol. 2016;10:168–188. doi: 10.1134/S1819714016030039 Кузьмин В. К., Богомолов Е. С., Глебовицкий В. А. Раннепротерозойский габбро-диорит-гранитный магматизм Батомгского поднятия (Северо-Восток Алданского щита): Sm-Nd-изотопно-геохимические свидетельства // ДАН. 2016. Т. 466, № 6. С. 707. doi: 10.7868/S0869565216060177 Kotov A. B. Boundary conditions of geodynamic models of the formation of the continental crust of the Aldan Shield: Summary of DSc (Dr. of Geol.-Miner.) the- sis. St. Petersburg: SPbGU; 2003. (In Russ.) Мишкин М. А., Ленников А. М., Баянова Т. Б., Вовна Г. М., Сахно В. Г., Октябрьский Р. А., Бадреди- нов З. Г. Первые результаты U-Pb геохронологиче- ских исследований докембрийских гранитоидов Ба- томгского блока Алданского щита // Тихоокеанская геология. 2010. Т. 29, № 3. С. 45–49. Kuzmin V. K., Bogomolov E. S., Glebovitskii V. A. Paleoproterozoic gabbro-diorite-granite magmatism of the Batomga Rise (NE Aldan Shield): Sm-Nd isotope geochemical evidence. Dokl. Earth Sci. 2016;466:199– 205. doi: 10.1134/S1028334X16020203 Bogdanova S. V., Gorbatschev R., Garetsky R. G. K. G. Erofeeva1,2, A. V. Samsonov1, T. A. Pilitsyna1, A. N. Larionov3, V. A. Spiridonov4, A. V. Postnikov5, A. Yu. Vovshina3, A. S. Dubenskiy2. RELICTS OF DESTROYED OCEANIC ISLAND ARCS IN THE PALEOPROTEROZOIC VYATKA OROGEN (EAST-EUROPEAN CRATON) Получен- ные данные расширяют список палеопроте- розойских орогенов с возрастом 2,2–2,0 млрд лет, в которых начало амальгамации отдельных архейских блоков в суперконтинент Нуна про- исходило на фоне главного этапа раскола ар- хейских суперкратонов 2,2–2,0 млрд лет назад [Condie et al., 2021]. [Котов, 2003; Мишкин и др., 2010; Гурьянов и др., 2016; Кузьмин и др., 2016] и существую- щими палеоконтинентальными корреляциями [Elming et al., 2021 и ссылки в ней]. Получен- ные данные расширяют список палеопроте- розойских орогенов с возрастом 2,2–2,0 млрд лет, в которых начало амальгамации отдельных архейских блоков в суперконтинент Нуна про- исходило на фоне главного этапа раскола ар- хейских суперкратонов 2,2–2,0 млрд лет назад [Condie et al., 2021]. Terentiev R. A., Santosh M. Detrital zircon geochro- nology and geochemistry of metasediments from the Vorontsovka terrane: implications for microcontinent tectonics // Int. Geol. Rev. 2016. Vol. 58, no. 9. P. 1108– 1126. doi: 10.1080/00206814.2016.1147386 References Bibikova E. V., Bogdanova S. V., Postnikov A. V., Popova L. P., Kirnozova T. I., Fugzan M. M., Glushchen- ko V. V. Sarmatia-Volgo-Uralia junction zone: Isotopic- geochronologic characteristic of supracrustal rocks and granitoids. Stratigr. Geol. Correl. 2009;17(6):561–573. doi: 10.1134/S086959380906001X Поступила в редакцию / received: 19.08.2022; принята к публикации / accepted: 29.08.2022. Авторы заявляют об отсутствии конфликта интересов / The authors declare no conflict of interest. K. G. Erofeeva1,2*, A. V. Samsonov1, T. A. Pilitsyna1, A. N. Larionov3, V. A. Spiridonov4, A. V. Postnikov5, A. Yu. Vovshina3, A. S. Dubenskiy2. RELICTS OF DESTROYED OCEANIC ISLAND ARCS IN THE PALEOPROTEROZOIC VYATKA OROGEN (EAST-EUROPEAN CRATON) Циркон с возрастом 2,05 млрд лет, найденный во всех породах в виде каем обрастания и мелких кри- сталлов, мог образоваться при внедрении Ms- Bt-трондьемитов, возраст которых оценен в 2,05 млрд лет. Радиогенный изотопный состав Nd в метавулканитах (εNd(2100) = 3,2 ± 0,2) ука- зывает на ювенильный источник магм. Более низкие величины εNd(2100) = 2,5 ± 0,3 в метао- садочных породах свидетельствуют о неболь- шом вкладе более древнего материала, что со- гласуется с находками в них единичных архей- ских зерен циркона. По составу метавулканиты отвечают известко- во-щелочным высокоглиноземистым андезитам и дацитам калинатрового ряда (Na2O/K2O > 1), имеют сильно обогащенные спектры легких РЗЭ, умеренно фракционированные спектры тя- желых РЗЭ и отрицательные аномалии Sr, Eu, Ti и всех HFSE. Метаосадочные породы отвечают грауваккам и по содержанию большинства глав- ных и редких элементов близки к метавулкани- там, которые могли служить их источником. Весь комплекс данных дает убедительные свидетельства об образовании пород унийской свиты Вятского пояса на удалении от архейских блоков, во внутриокеанической надсубдукци- онной обстановке, при формировании двух разновозрастных островных дуг 2,10 и 2,14 млрд лет, разрушение которых с образованием граувакковых отложений могло происходить за счет субдукционной эрозии [например, Straub et al., 2020]. U-Pb-изотопное датирование циркона из метавулканитов и метаосадков в разных ча- стях пояса дает близкие спектры возрастов с тремя главными пиками. Циркон с возрастом 2,10 млрд лет доминирует в метавулканитах (> 90 %) и составляет до 70 % популяции в ме- таосадочных породах, что служит еще одним аргументом в пользу образования осадков за счет разрушения смежных вулканических по- строек. Циркон с возрастом 2,14 млрд лет со- ставляет до 15 % популяций во всех пробах метаосадочных пород, что указывает на при- сутствие в Вятском поясе еще одного, пока Близкие по геохимии флишоидные осадки с ювенильными островодужными источниками и с возрастом циркона 2,1–2,2 млрд лет слага- ют Волго-Донской пояс [например, Бибикова и др., 2009; Terentiev, Santosh, 2016], который располагается между Волго-Уральским и Сар- матским мегаблоками [Bogdanova et al., 2016]. Восточный фланг Вятского пояса, возможно, был частью обширного палеопротерозойско- го орогена вместе с Батомгским поясом юго- восточной части Сибирского кратона. Такая корреляция обоснована сходством возраста корообразующих процессов этих двух поясов 35 P. 574–589. do osh M. Detrital zirc f di Environ. 2020. Vol. 1. P. 574–589. doi: 10.1038/ s43017-020-0095-1 [Котов, 2003; Мишкин и др., 2010; Гурьянов и др., 2016; Кузьмин и др., 2016] и существую- щими палеоконтинентальными корреляциями [Elming et al., 2021 и ссылки в ней]. Transactions of the Karelian Research Centre of the Russian Academy of Sciences. 2022. No. 5 Труды Карельского научного центра Российской академии наук. 2022. № 5 СВЕДЕНИЯ ОБ АВТОРАХ: Ерофеева Ксения Геннадьевна канд. геол.-мин. наук, научный сотрудник e-mail: xenin.erofeeva@ya.ru Самсонов Александр Владимирович член-корр. РАН, д-р геол.-мин. наук, главный научный сотрудник e-mail: samsonovigem@mail.ru Пилицына Татьяна Андреевна младший научный сотрудник e-mail: allafia@yandex.ru Ларионов Александр Николаевич д-р геол.-мин. наук e-mail: alexander_larionov@vsegei.ru Спиридонов Виктор Альбертович ведущий специалист e-mail: vik-spiridonov@yandex.ru Постников Александр Васильевич д-р геол.-мин. наук, зав. кафедрой литологии e-mail: postnikov.a@gubkin.ru Вовшина Анна Юрьевна ведущий геолог e-mail: anna_vovshina@vsegei.ru Дубенский Александр Сергеевич канд. хим. наук, старший научный сотрудник e-mail: alexchem206@gmail.com СВЕДЕНИЯ ОБ АВТОРАХ: Ерофеева Ксения Геннадьевна канд. геол.-мин. наук, научный сотрудник e-mail: xenin.erofeeva@ya.ru Самсонов Александр Владимирович член-корр. РАН, д-р геол.-мин. наук, главный научный сотрудник e-mail: samsonovigem@mail.ru Пилицына Татьяна Андреевна младший научный сотрудник e-mail: allafia@yandex.ru Ларионов Александр Николаевич д-р геол.-мин. наук e-mail: alexander_larionov@vsegei.ru Спиридонов Виктор Альбертович ведущий специалист e-mail: vik-spiridonov@yandex.ru Постников Александр Васильевич д-р геол.-мин. наук, зав. кафедрой литологии e-mail: postnikov.a@gubkin.ru Вовшина Анна Юрьевна ведущий геолог e-mail: anna_vovshina@vsegei.ru Дубенский Александр Сергеевич канд. хим. наук, старший научный сотрудник e-mail: alexchem206@gmail.com Литература EUROPE \| East European Craton // Reference Module in Earth Systems and Environmental Sciences, Elsevier, 2016. doi: 10.1016/B978-0-12-409548-9.10020-X Mishkin M. A., Lennikov A. M., Bayanova T. B., Vovna G. M., Sakhno V. G., Oktyabr’skii R. A., Badredi- nov Z. G. First results of U-Pb geochronological studies of the Precambrian granitoids of the Batomga block of the Aldan Shield. Russ. J. of Pac. Geol. 2010;4:223– 227. doi: 10.1134/S1819714010030048 Condie K. C., Pisarevsky S. A., Puetz S. J. LIPs, oro- gens and supercontinents: The ongoing saga // Gond- wana Res. 2021. Vol. 96. P. 105–121. doi: 10.1016/j. gr.2021.05.002 Straub S. M., Gómez-Tuena A., Vannucchi P. Sub- duction erosion and arc volcanism. Nat. Rev. Earth Envi- ron. 2020;1:574–589. doi: 10.1038/s43017-020-0095-1 Elming S.-Å., Salminen J., Pesonen L. J. Chapter 16: Paleo-Mesoproterozoic Nuna supercycle // Peso- nen L. J., Salminen J., Evans D. A. D., Elming S.-Å., Veikkolainen T. (eds.). Ancient Supercontinents and the Paleogeography of the Earth. Elsevier, 2021. P. 499–548. doi: 10.1016/B978-0-12-818533-9.00001-1 Terentiev R. A., Santosh M. Detrital zircon geochro- nology and geochemistry of metasediments from the Vorontsovka terrane: implications for microcontinent tectonics. Int. Geol. Rev. 2016;58(9):1108–1126. doi: 10.1080/00206814.2016.1147386 Terentiev R. A., Santosh M. Detrital zircon geochro- nology and geochemistry of metasediments from the Vorontsovka terrane: implications for microcontinent tectonics. Int. Geol. Rev. 2016;58(9):1108–1126. doi: 10.1080/00206814.2016.1147386 Terentiev R. A., Santosh M. Detrital zircon geochro- nology and geochemistry of metasediments from the Vorontsovka terrane: implications for microcontinent tectonics. Int. Geol. Rev. 2016;58(9):1108–1126. doi: 10.1080/00206814.2016.1147386 Straub S. M., Gómez-Tuena A., Vannucchi P. Sub- duction erosion and arc volcanism // Nat. Rev. Earth Поступила в редакцию / received: 19.08.2022; принята к публикации / accepted: 29.08.2022. Авторы заявляют об отсутствии конфликта интересов / The authors declare no conflict of interest. Поступила в редакцию / received: 19.08.2022; принята к публикации / accepted: 29.08.2022. Авторы заявляют об отсутствии конфликта интересов / The authors declare no conflict of interest 36 37 Труды Карельского научного центра Российской академии наук. 2022. № 5 канд. геол.-мин. наук, научный сотрудник Cand. Sci. (Geol.-Miner.), Researcher e-mail: xenin.erofeeva@ya.ru Самсонов Александр Владимирович член-корр. РАН, д-р геол.-мин. наук, главный научный сотрудник Samsonov, Alexander RAS Corr. Fellow, Dr. Sci. (Geol.-Miner.), Chief Researcher e-mail: samsonovigem@mail.ru Пилицына Татьяна Андреевна младший научный сотрудник Pilitsyna, Tatiana Junior Researcher e-mail: allafia@yandex.ru Ларионов Александр Николаевич д-р геол.-мин. наук Larionov, Alexander Dr. Sci. (Geol.-Miner.) e-mail: alexander_larionov@vsegei.ru Спиридонов Виктор Альбертович ведущий специалист Spiridonov, Viktor Leading Specialist e-mail: vik-spiridonov@yandex.ru Постников Александр Васильевич д-р геол.-мин. наук, зав. кафедрой литологии Postnikov, Alexander Dr. Sci. (Geol.-Miner.), Head of Lithology Department e-mail: postnikov.a@gubkin.ru Вовшина Анна Юрьевна ведущий геолог Vovshina, Anna Leading Geologist e-mail: anna_vovshina@vsegei.ru Дубенский Александр Сергеевич канд. хим. наук, старший научный сотрудник Dubenskiy, Alexander Cand. Sci. (Chem.), Senior Researche e-mail: alexchem206@gmail.com Samsonov, Alexander RAS Corr. Fellow, Dr. Sci. (Geol.-Miner.), Chief Researcher Самсонов Александр Владимирович член-корр. РАН, д-р геол.-мин. наук, главный научный сотрудник e-mail: samsonovigem@mail.ru 37 37
https://openalex.org/W3011336384	https://www.econjournals.com/index.php/ijeep/article/download/9066/5010	English	null	FEASIBILITY ANALYSIS OF A RENEWABLE AUTONOMOUS POWER SUPPLY SYSTEM AT A COASTAL AREA IN INDONESIA	International journal of energy economics and policy	2,020	cc-by	5,661	1. INTRODUCTION reduce people’s dependence on the use of fossil fuels through the Self-Sufficient Energy Village program (energypedia.info, 2019). Increasing population will increase the demand for electrical energy. The need for electrical energy now has shifted from secondary needs to primary needs, the need for equal distribution of electrical energy so that every layer of society in both urban and rural areas can enjoy the utilization of electrical energy. The expansion of electricity grids by electricity service providers in solving the problem of electricity distribution in isolated settlements is often hampered by geographic conditions to get to the area. The main roads that are difficult to pass vehicles cause new electrical grid material is difficult to reach the area. In 2010, the government provided solar panels in isolated areas to meet the needs of electrical energy by optimizing available renewable energy to be able to Development of hybrid power plants by many countries continues to increase in number, ranging from power generation to tens of watts to tens of kilowatts. The increasingly expensive use of fossil fuels can be reduced by applying power generation from renewable energy sources that generate clean and environmentally friendly electrical energy with very minimal levels of carbon dioxide gas. Accepted: 18 February 2020 DOI: https://doi.org/10.32479/ijeep.9066 Received: 04 December 2019 ABSTRACT The Indonesian government program in providing solutions of electrical energy distribution problems to get to remote or isolated areas is to optimize the potential of renewable energy in an area. The combination of conventional power plants (diesel generators) with renewable energy (photovoltaic and wind turbine) power plants is expected to solve the problem of electricity service in isolated areas in southern Tulungagung regency, namely residential area in Brumbun Beach. The existence of government assistance in the form of solar panels distributed to each family head still can not optimize the utilization of electrical energy for 24 h in a day, this is because the generation of diesel generators and solar panels are done separately. This research focuses on the design and analysis of renewable autonomous power supply system which consists of centralized solar-powered diesel generator (solar panel – wind turbine – diesel generator) systems using HOMER software. This software is in addition to being used to create designs, is also capable of performing the most optimal system design evaluation by sorting based on overall cost, basic electricity tariff, and carbon dioxide gas emissions. The research from the design of four power plant configurations shows that the use of 10 kW diesel generators, 8 kW solar panels, and 6 kW wind turbines is the best solution, from the combination of the three energy sources shows the net present cost value of US $ 44,680, Cost of Energy of 0.268 kWh/$, CO2 emissions of 1,077 kg/year, and diesel generator use only 54 min a day. Keywords: Cost of Energy, Hybrid Power Generation, HOMER, Net Present Cost, Renewable Energy JEL Classifications: C63, C88, Q42 Keywords: Cost of Energy, Hybrid Power Generation, HOMER, Net Present Cost, Renewable Energy JEL Classifications: C63, C88, Q42 This Journal is licensed under a Creative Commons Attribution 4.0 International License Department of Electrical Engineering, State Polytechnic of Malang, Soekarno-Hatta Street No. 9, Malang 65141, Indonesia. E il h @ li id Department of Electrical Engineering, State Polytechnic of Malang, Soekarno-Hatta Street No. 9, Malang 65141, Indonesia. Email: moh.noor@polinema.ac.id Accepted: 18 February 2020 2. SYSTEM DESCRIPTION From the design and simulation of planned generator system to get optimal result with configuration of hybrid power plant system between diesel generator, PV panel, and wind turbine. Power plants with centralized system configuration have many advantages over dispersed systems. The most optimal system configuration of power plant design simulated by HOMER software is the use of 10 kW diesel generator, 8 kW capacity PV panel, 6 kW wind turbine, 24 V busbar with 8000 Ah capacity and 5 kW inverter capacity. Based on the technical review, the operation of the generator for 1 year is 341 h or 0.9 h/day, resulting in less fuel use and longer diesel generator life. While based on economic reviews, the value of net present cost (NPC) of US $ 44,680, the value of cost of energy (COE) of US $ 0.268 $/kWh, and CO2 gas emissions of 1,077 kg/year. This research designs and simulates hybrid power plants suitable for community settlements in Brumbun Beach where they have not enjoyed the utilization of electrical energy due to remote locations of urban and hilly road access that are difficult to carry out network expansion. From the design and simulation of planned generator system to get optimal result with configuration of hybrid power plant system between diesel generator, PV panel, and wind turbine. Power plants with centralized system configuration have many advantages over dispersed systems. i The configuration of the hybrid power plant system is based on the working principle (Madziga et al., 2018, Nayar et al., 1993), among others: (1) Series hybrid system, (2) hybrid system switch, and (3) parallel hybrid system. In serial system configurations, all power plants (generators, PV panels, wind turbines) are converted in DC form and equipped with a charge controller to charge the battery charge, to serve the load with AC voltage specifications required by the inverter. In this system, the electrical energy generated is paralleled by the battery, so the battery life becomes less durable and reduces the efficiency of the system, then the generator occurs voltage losses because of the AC voltage to DC to supply the load is converted again into AC voltage using the inverter. 2. SYSTEM DESCRIPTION Hybrid power plant system is a power plant consisting of two or more renewable energy sources that are operated together to obtain his Journal is licensed under a Creative Commons Attribution 4.0 International License International Journal of Energy Economics and Policy \| Vol 10 • Issue 3 • 2020 Hidayat, et al.: Feasibility Analysis of a Renewable Autonomous Power Supply System at a Coastal Area in Indonesia an efficient and optimal system in supplying electrical energy in accordance with the load demand. The combination of various renewable energy sources can provide the balance and stability of a power plant system (Magarappanavar and Koti, 2016). The advantages of this system (Sopian et al., 2005, Nayar et al., 1993), among others: (1) Increasing the reliability of the system in serving the load demand, (2) reducing the use of fossil fuels, (3) utilization of electricity within 24 h, (4) system or component of the plant, and (5) the use of electrical energy effectively and efficiently. serves to bridge between components that produce DC voltage source and AC voltage source generated from diesel generator, BDI can charge battery from diesel generator while operating as rectifier (AC-DC Converter) or from renewable energy source (PV panels and wind turbines), BDI can also operate as an inverter (DC-AC Converter) while serving AC-powered AC loads. The advantages of parallel hybrid configuration (Nayar et al., 1993), among others: (1) Optimal in the supply of electrical energy at the load, (2) efficient in the operation and maintenance of electrical energy generating equipment, and (3) minimization of components so as to reduce initial investment costs. Figure 1 is a hybrid power plant system configuration (Madziga et al., 2018, Nayar et al., 1993). Component required in hybrid power generation (Sopian et al., 2005), among others: (1) An inverter with a power rating of 60% greater than the loaded power required, (2) one or two generator diesel generators with a larger or minimum capacity (3) electrical energy storage using lead-acid batteries; (4) use of solar panels equipped with regulators and controllers; and (5) microprocessor-based or system-based controls microcontroller for monitoring and system automation. 2.1. Renewable Energy Resource This research designs and simulates hybrid power plants suitable for community settlements in Brumbun Beach where they have not enjoyed the utilization of electrical energy due to remote locations of urban and hilly road access that are difficult to carry out network expansion. 2. SYSTEM DESCRIPTION In a switched switch system, the diesel and the inverter can be operated as an AC voltage source capable of directly supplying the voltage during the average load and peak load conditions thus increasing the efficiency of the system, the excess energy generation of the diesel generator can be allocated to charge the battery charge. At low loads, the diesel generator becomes inactive and the electrical load requirements are only supplied by renewable energy and battery by converting DC voltage into AC voltage using inverter. In parallel configuration systems, the need for electrical loads can be served in parallel from both the diesel generator and from the inverter. Bi-Directional Inverter (BDI) 2.2. System Configuration The isolated problem of power grids in remote areas and islands with small population communities can be solved by implementing the construction of Stand-Alone or off-grid hybrid power plants, apart from costly and costly network expansion as well as to minimize fossil fuels in energy generation electricity on diesel generators. The components used in the design of power plant system configuration in this study are as follows: 2.2.1. Diesel generator or genset A diesel generator is a power plant that is used to supply electrical loads on a small power capacity and to increase electrical power during peak loads. Optimization of diesel generator use is done in isolated area and not yet reached by power grid. For selection of capacity of diesel generator/generator to be used by using equation 1 (Alayan, 2016; Okwu et al., 2017). Monocrystalline type because it has the highest efficiency level with 24.1% (GSES, 2016). Modeling Solar panels in HOMER software model the arrangement of PV as a device that produces DC electricity in proportion to the incidence of solar radiation globally present, regardless of the temperature and the affected voltage (Lambert et al., 2006), to determine the capacity of the PV integrated with the battery, solar charge controller, and inverter by using equation 2 (Alayan, 2016; Okwu et al., 2017). ( ) 130% diesel diesel diesel E t P η = × (1) (1) Where: Estimated capacity increase of 30% Pdiesel: Output power of diesel generator (KW) Pdiesel: Output power of diesel generator (KW) Ediesel: Electric load to be met per hour (kWh)i Ediesel: Electric load to be met per hour (kWh)i ηdiesel: Diesel efficiency (assuming=0.9). ηdiesel: Diesel efficiency (assuming=0.9). P E I k H PV load b inv m bcu = × × × × × × 0 0 η η η η (2) (2) 2.1.3. Load profilei Figure 3: Load profile data Load profile is a characteristic of the use of electrical energy for 24 h on the consumer of electrical energy. Some facilities, facilities, and infrastructure that require electrical energy, among others: residential residents, places of worship, schools, and shops. Figure 3 shows the peak load profile at 17.00 - 19.00 WIB of 30.480 kW. 2.1.2. Wind speed p Electric energy can be generated from wind gusts to wind turbines, so wind speed parameters are required in mechanical design of wind turbines. The wind speed data is equipped with a high anemometer where wind speed is measured (HOMER Manual Book, 2017). Figure 2 shows the largest wind velocity (6.1 m/s) obtained in August and minimum wind speed (2.95 m/s) in December, while the mean annual wind velocity at Brumbun Beach is 4.43 m/s. 2.2.2. Solar panels Commonly known as photovoltaic panels (PV) are modern equipment capable of generating electrical energy by converting photon energy from sunlight into electrical energy (Quaschning, 2016). Based on the manufacture technology (Handbook for Solar Photovoltaic (PV) Systems, 2017) solar cells are divided into 3 kinds, namely: (a). Monocrystalline Solar Cell; (b). Polycrystalline Solar Cell; and (c). Thin Film Solar Cell (TSFC). In this study the solar panel used is Where: 2.1.1. Solar radiation The characteristics and potential of solar energy in selected locations are analyzed based on global solar radiation with monthly averages as well as monthly brightness (Olatomiwa, 2015). Global Horizontal Irradiance (GHI) is the total solar radiation that occurs on the horizontal surface (HOMER Manual Book, 2017). Figure 1 shows the minimum value of solar radiation in January at 4,850 kWh/m2/day with a brightness index of 0.448 and in International Journal of Energy Economics and Policy \| Vol 10 • Issue 3 • 2020 176 Figure 1: Hybrid power generation system configuration (Madziga et al., 2018, Nayar et al., 1993) International Journal of Energy Economics and Policy \| Vol 10 • Issue 3 • 2020 Hidayat, et al.: Feasibility Analysis of a Renewable Autonomous Power Supply System at a Coastal Area in Indonesia Monocrystalline type because it has the highest efficiency level with 24.1% (GSES, 2016). Modeling Solar panels in Figure 2: Renewable energy potential data at Brumbun beach (NASA, 2016) Figure 3: Load profile data Figure 2: Renewable energy potential data at Brumbun beach (NASA, 2016) Figure 2: Renewable energy potential data at Brumbun beach (NASA, 2016) September is a maximum value of 6.220 kWh/m2/day with a brightness index of 0.613, while the average annual solar radiation is 5,37 kWh/m2/day. Dimana: Ppeak load: Peak load daily Pinv: Inverter produced the power ηinv: Inverter efficiency PV Pmax−out: The load energy that the generator must meet (kWh). It refers to the turbine’s power curve to calculate its power output at that wind speed assuming standard air density, (d). It multiplies that power output value by the air density ratio, which is the ratio of actual water density to the standard water density. To determine the capacity of wind turbine generator (PWG) using equation 5 and to determine the energy generated using equation 6 (Balachander et al., 2012) Dimana: Where: Where: PPv: Solar panel power capacity (kWp) Eload: Electric load to be supplied I0: Standard radiation k: Coefficient H0: Average radiation ηb: Battery efficiency ηinv: Inverter efficiency PPv: Solar panel power capacity (kWp) Pv Eload: Electric load to be supplied International Journal of Energy Economics and Policy \| Vol 10 • Issue 3 • 2020 Hidayat, et al.: Feasibility Analysis of a Renewable Autonomous Power Supply System at a Coastal Area in Indonesia shortage factor, energy storage is required. The required battery capacity (Ah) can be determined using equation 6 (Ishaq and Ibrahim, 2013) ηm: Maching efficiencyi m ηbuc: Efficiency of unit control batteries Or to determine the amount of energy produced Eel PV panel used, can be determined using equation 3 (Alayan, 2016; Okwu et al., 2017) ( ) Ah load bat max bat bat nom E Dayof Automous C DOD V η − × = × × (7) (7) Dimana: Dimana: 0 0 max out b inv m bcu el P H E k I η η η η − × × × × × = × (3) Cbat: Batteray capacity (Ah) Eload: Electric load to be supplied (kWh) Day of Autonomous: DODmax: Deep of discharge ηbat: Battery Efficiency Vbat−nom: Battery nominal voltase (3) Cbat: Batteray capacity (Ah) Cbat: Batteray capacity (Ah) Eload: Electric load to be supplied (kWh) Day of Autonomous: DODmax: Deep of discharge ηbat: Battery Efficiency Vbat−nom: Battery nominal voltase As for determining the number of PV panels (NPV) used can be determined using equation 4 (Alayan 2016, Okwu et al., 2017). N P P PV PV max out = − (4) : Battery nominal voltase (4) And to determine number of battery (Nbat) used equation 7. And to determine number of battery (Nbat) used equation 7. Where: N C V bat bat bat nom = − (8) NPV: Number of PV panels (8) PV PPV: The energy from PV panels (kWh) PV Pmax−out: The load energy that the generator must meet (kWh). 2.3. Cost Optimization The economic evaluation of the entire hybrid system is achieved by optimizing the total life cycle cost of the system configurations. The NPC (or life-cycle cost) of a component is the present value of all the costs of installing and operating that components over the project lifetime, minus the present value of all revenues that it earns over the project lifetime. To determine the value of NPC is shown in equation 9 (HOMER Manual Book, 2017). P A C V WG r P W = × × × × 1 2 3 ρ (5) 3 1 2 P W C V ρ π = × × × × E t P t WG WG ( ) = × (6) (5) (6) ( ) ann tot NPC C C CRF i N = (10) (10) Dimana: PWG: Output power of wind turbine generator EWG: Energy produced wind turbine ρ: Air density Ar: The area of the rotor Cp: Power coefficient VW: Wind velocity t: Time Dimana: PWG: Output power of wind turbine generator EWG: Energy produced wind turbine ρ: Air density Ar: The area of the rotor Cp: Power coefficient VW: Wind velocity t: Time ( ) ann tot NPC C CRF i N = Dimana: CNPC: Overall cost over lifetime Cann tot: Total annualized cost CRF: Capital recovery factor I: Interest rate Rproj: Project period. PWG: Output power of wind turbine generator Dimana: CNPC: Overall cost over lifetime Cann tot: Total annualized cost CRF: Capital recovery factor I: Interest rate Rproj: Project period. PV Pmax−out: The load energy that the generator must meet (kWh). 2.2.5. Bidirectional converter 2.2.5. Bidirectional converter Bidirectional Converter is a tool used to convert DC-voltage power sources from 12, 24, 48 Volts generated from electrical sources such as solar panels, wind turbines and batteries, into AC-inverter power sources that can be used to power equipment electronics as per specification (120 or 240 V AC, 50 or 60 Hz) or equivalent to PLN electricity voltage and vice versa (rectifier) (Purwadi et al., 2012). The efficiency of a converter is better when the output load approaches the rated work of the converter, and vice versa. In equation 8 is shown to determine the working capacity of the inverter (Purwadi et al., 2012). 2.2.3. Wind turbine Wind turbine is a device used to convert wind energy into kinetic energy connected to the generator to generate electrical energy. The voltage generated by the wind turbine can be either DC or AC voltage seseuai with the type of generator used. With HOMER software can be determined the use of the optimum amount of wind turbines in serving the load. Determination of output power from wind turbine in software HOMER can be done with four stages (Lambert et al., 2006), namely: (a). It determines the average wind speed for the hour at the anemometer height by referring to the wind resource data, (b). It calculates the corresponding wind speed at the turbine’s hub using the logarithmic law or the power law, (c). It refers to the turbine’s power curve to calculate its power output at that wind speed assuming standard air density, (d). It multiplies that power output value by the air density ratio, which is the ratio of actual water density to the standard water density. To determine the capacity of wind turbine generator (PWG) using equation 5 and to determine the energy generated using equation 6 (Balachander et al., 2012) Wind turbine is a device used to convert wind energy into kinetic energy connected to the generator to generate electrical energy. The voltage generated by the wind turbine can be either DC or AC voltage seseuai with the type of generator used. With HOMER software can be determined the use of the optimum amount of wind turbines in serving the load. P P inv peakload inv = ×1 15 , η (9) (9) (b). It calculates the corresponding wind speed at the turbine’s hub using the logarithmic law or the power law, (c). 3. DISCUSSION In this research, the design and simulation of hybrid power plant configuration is solved using HOMER software. HOMER is a simulation tool developed by NREL that is used for the design, simulation, and evaluation of hybrid power generation systems (Lambert et al., 2006). HOMER simulates to obtain an optimized off-grid and grid-connected power grid system (Sopian et al., 2005, Purwadi et al., 2012, Olatomiwa, 2015), which consists of a combination of solar panels, wind turbines, microhydro, biomass, diesel generators, micro turbines, fuel-cells, battery, and hydrogen storage, also serves AC and DC power loads, as well as thermal loads. The optimization algorithm and HOMER software sensitivity analysis facilitate the evaluation of various possible system configurations (HOMER Manual Book, 2017). Many references suggest the use of HOMER software to make the design of optimal hybrid power generation systems (Magarappanavar and Koti, 2016; Sopian et al., 2005; Lambert et al., 2006; NASA, 2016; Handbook for Solar Photovoltaic (PV) Systems, 2016). Source: Author’s Computation using HOMER, 2017) 179 Table 1: Component data input Panel PV Technology Monocrystal Capital cost $7,800 Replacement $7,800 Lifetime 15 years Derating faktor 80% Wind turbin generator Technology Low speed Capital cost $ 3,000 Replacement $ 3,000 O & M cost $ 100 Lifetime 15 years Converter Capital cost $ 2,000 Replacement cost $ 2,000 Efficiency 95% Lifetime 15 years Diesel generator Capital Cost $ 5,575 Replacement $ 5,575 O & M cost $ 0.7 Lifetime 15.000 h Battery Technology GEL Deep Cycle Capital cost $ 4,000 Replacement cost $ 4,000 O & M cost $ 10 Battery busbar voltage 24 Volt Lifetime 10 years Source: Author’s computation using HOMER, 2017 Table 1: Component data input In this research the hybrid power plant design uses a parallel configuration with the advantages shown in Figure 4. Consists of 100 Wp photovoltaic panel, 1kW wind turbine generator, 5 kW converter, 10 kW diesel generator, and GEL Deep Cycle battery. To optimize the power output of the wind turbine and the resulting power is fluctuating, it must be connected to the DC generator. Diesel generator operates at peak load and as back-up voltage source when power capacity of PV panel output, wind turbine, battery in minimum condition. With a decent hybrid system diesel generator configuration can be active maximum for 1 h/day to keep the diesel generator is always in good condition also keep the voltage stability. 2.2.4. Battery The capital recovery factor is a ratio used to calculate the present value of an annuity (a series of equal annual cash flows) (HOMER Manual Book, 2017). y Battery is an equipment used to store unidirectional DC (DC) charge. To improve the efficiency of the system by reducing International Journal of Energy Economics and Policy \| Vol 10 • Issue 3 • 2020 Hidayat, et al.: Feasibility Analysis of a Renewable Autonomous Power Supply System at a Coastal Area in Indonesia Figure 4: Hybrid Power Plant Configuration Purposed HOMER defines the COE as the average cost per kWh of useful electrical energy produced by the system (HOMER Manual Book, 2017). To determine the value of COE, HOMER compares the value of the total annual cost (NPC) with the actual electrical load of the serve by the hybrid system (kWh/year). To determine the value of COE is shown in equation 10 (Laksmhi et al., 2012) Figure 4: Hybrid Power Plant Configuration Purposed Source: Author’s Computation using HOMER, 2017) , , ann tot load AC load DC def gridsales C COE E E E E = + + + (11) (11) Where: COE: Cost of energy Eload,AC: The load is served AC voltage Eload,DC: The load is served DC voltage Edef: Deferable load Egrid sales: Total sales of power grids. 7. Battery capacity 30.480 Wh 3 6.350 Ah 0,75 0,80 24 Volt bat C × = = × × 8. Number of battery 8. Number of battery 8. Number of battery 6.350 Ah 64 unit 100 Ah bat N = ≈ al of Energy Economics and Policy \| Vol 10 • Issue 3 • 2020 Source: Author’s computation using HOMER, 2017 Source: Author’s computation using HOMER, 2017 Figure 6: Comparison of net present cost value Source: Author’s computation using HOMER, 2017 Figure 7: Comparison of cost of energy value Source: Author’s computation using HOMER, 2017 9. Power capacity of inverter ( ) .1 25% 3,220 0,40 .1 ,25 0,96 peak load inv inv P P η + = = Pinv=4,72 kW≈5 kW Source: Author’s computation using HOMER, 2017 Figure 6: Comparison of net present cost value Source: Author’s computation using HOMER, 2017 Figure 6: Comparison of net present cost value Figure 6: Comparison of net present cost value To maintain the safety and stability of the inverter should be considered with a size 25-30% greater than the total load. The inverter that should be installed with a minimum specification is 5 kW. 5. Power output of wind turbine 1 .1 ,225 . 1 ,07 . 2,293 . 0,59 4,64 kW 2 WT P = = Source: Author’s computation using HOMER, 2017 Figure 5: Net present cost, cost of energy, and CO2 system values based on HOMER software Source: Author’s computation using HOMER, 2017 Figure 5: Net present cost, cost of energy, and CO2 system values based on HOMER software Figure 5: Net present cost, cost of energy, and CO2 system values based on HOMER software 6. Number of power turbine 6. Number of power turbine 30480 6,57 7unit 464 0 WT N = = ≈ 7. Battery capacity 4. Solar panel capacity ( ) 27 1 1,1 4.850 0,85 0,95 1 0,95 peak P × × = × × × × Ppeak=7,96 kW≈8 kW ( ) 27 1 1,1 4.850 0,85 0,95 1 0,95 peak P × × = × × × × Ppeak=7,96 kW≈8 kW Figure 8 illustrates that the selling price of electricity in the hybrid configuration system (Diesel - WT) is 0.556 $/kWh or 34% cheaper than the Diesel configuration system as the main supply. 5. Power output of wind turbine 3. DISCUSSION Table 1 is shown in detail rather than hybrid power plant components to be planned. Sizing of Design Hybrid Power Generation: 1. Load demand Total energy usage=30,480 kWh/day 2. Capacity of diesel generator P t diesel ( ) = + × = 5 34 0 4 0 9 130 8 29 , , , % , kW 1. Load demand Total energy usage=30,480 kWh/day 2. Capacity of diesel generator Hidayat, et al.: Feasibility Analysis of a Renewable Autonomous Power Supply System at a Coastal Area in Indonesia And in the HOMER software provided generator capacity is close to 8.29 kW is a generator with a capacity of 10 kW. configuration as the main supply in the fulfillment of electrical loads with a value of US $ 267,023 and the lowest value is the configuration of a hybrid power plant (Diesel - PV - WT). The total NPC value includes all costs used during the project, consisting of component procurement costs, replacement costs, operations and maintenance costs (O & M), fuel costs, emission fine costs (if any), and interest rates. 3. Solar energy potential ( ) ( ) ( ) 1 4,850 0,85 0,95 1 0,95 1,1 1 el E × × × × × = × Eel = 3,213 kWh (Energy produced 1 h) Figure 7 shows a bar chart of the COE value or the price of electricity per kWh of each planned system configuration. The largest electricity price in Diesel configuration as the main supply in the fulfillment of electrical loads with a value of US $ 1.60/kWh and the lowest value is the configuration of a hybrid power plant (Diesel - PV - WT). 4. Solar panel capacity REFERENCES Alayan, S. (2016), Design of a PV-Diesel Hybrid System with Unreliable Grid Connection in Lebanon Thesis. Balachander, K., Kuppusamy, S., Dan Vijayakumar, P. (2012), Comparative Study of Hybrid Photovoltaic-fuel Cell System/Hybrid Wind-Fuel Cell System for Smart Grid Distributed Generation System. India: International Conference on Emerging Trends in Science, Engineering and Technology. GSES. (2016), A Practical Guide to PV Efficiency. GSES Technical Paper. Source: Author’s computation using HOMER, 2017 ( )i y p Handbook for Solar Photovoltaic (PV) Systems. (2011), Singapore: Grenzone Pte Ltd, Phoenix Solar Pte Ltd, Singapore Polytechnic, Solar Energy, Research Institute of Singapore. SP PowerGrid, Urban Redevelopment Authority. p4-15. Table 2: Tariff of electricity power of four PLTH configuration systems Configurations COE (Rp/kWh) Diesel 1.600 Diesel–PV 0.370 Diesel–WT 0.556 Diesel–PV–WT 0.268 Source: Author’s computation using HOMER, 2017. COE: Cost of energy Table 2: Tariff of electricity power of four PLTH configuration systems Configurations COE (Rp/kWh) Diesel 1.600 Diesel–PV 0.370 Diesel–WT 0.556 Diesel–PV–WT 0.268 Source: Author’s computation using HOMER, 2017. COE: Cost of energy HOMER HOMER® Pro Version 3.7 User Manual. Available from: https://www.homerenergy.com/pdf/HOMERHelpManual.pdf. [Last accessed on 2017 Aug 08]. Indonesia Energy Situation. Available from: https://www.energypedia.info/ wiki/Indonesia_Energy_Situation. [Last accessed on 2019 Sep 21]. Source: Author’s computation using HOMER, 2017. COE: Cost of ener Ishaq, M., Ibrahim, U.H. (2013), Design of an off grid photovoltaic system: A case study of government technical college, Wudil, Kano State. International Journal of Scientific and Technology Research, 2(12), 175-181. The hybrid configuration system (Diesel - PV) is 0.370 $/kWh or i The hybrid configuration system (Diesel - PV) is 0.370 $/kWh or 67% cheaper than the hybrid configuration system (Diesel - WT). Among the four planned configuration systems, the selling price of the hybrid configuration system (Diesel - PV - WT) is 0.268 $/kWh. Table 2 shows the Electricity Tariff (TDL) of several Hybrid Power Generation configuration systems when changed according to the electricity tariff in the State of Indonesia. Laksmhi, M.V.S., Babu, C.S. dan Prasad, S. (2012), Design of Off-grid Homes with Renewable Energy Sources”, Third International Conference on Sustainable Energy and Intelligent System, Chennai and Vivekanandha College of Technology for Women, Dept. of EEE. India: Regency Institute of Technology. Lambert, T., Gilman, P., Lilienthal, P. (2006), Micropower system modeling with HOMER. Integration of Alternative Sources of Energy, 1(15), 379-418. Figure 8 shows the gas emission stem diagram (CO2) produced by each planned system configuration. 4. RESULT To get optimal system configuration of Hybrid Power Generation (PLTH) in Brumbun Beach by modeling electrical condition and availability of renewable energy potency, so there are 4 models of system design configuration which will be simulated using Software HOMER, that is: (1). Disel Diesel Generator Configuration; (2). Diesel Generator + Panel + PV + Battery Configuration; (3). Diesel Generator Configuration + Wind Turbine + Battery; and (4). Diesel Generator + PV Panel + Wind Turbine + Battery Configuration. The feasibility of a power plant is reviewed on the technical and economic side. Based on the design and simulation using HOMER software obtained comparison of several output parameters (NPC, COE, and CO2) of the four system configuration of PLH shown in Figure 5. Source: Author’s computation using HOMER, 2017 Figure 7: Comparison of cost of energy value Figure 7: Comparison of cost of energy value Figure 6 shows a bar chart of the NPC value value or the total total cost of each planned system configuration. The largest NPC value in Diesel Hidayat, et al.: Feasibility Analysis of a Renewable Autonomous Power Supply System at a Coastal Area in Indonesia Hidayat, et al.: Feasibility Analysis of a Renewable Autonomous Power Supply System at a Coastal Area in Indonesia Source: Author’s computation using HOMER, 2017 Figure 8: Comparison of CO2 emission Source: Author’s computation using HOMER, 2017 Figure 8: Comparison of CO2 emission the value of COE of US $ 0.268 $/kWh, and CO2 gas emissions of 1,077 kg/year. REFERENCES The largest gas emission value in Diesel configuration as the main supply in the fulfillment of electrical load is 28,462 kg/year and the lowest value is hybrid power plant configuration (Diesel - PV - WT). For 1 year the generator continues to operate, but every configuration system with the addition of a generator from renewable energy occurs a reduction in operating time. Madziga, M., Rahil, A., Mansoor, R. (2018), Comparison between three off-grid hybrid systems (solar photovoltaic, diesel generator and battery storage system) for electrification for Gwakwani village, South Africa. Environments, 5(5), 57. Magarappanavar, U.S., Koti, S. (2016), Optimization of wind-solar- diesel generator hybrid power system using HOMER. International Research Journal of Engineering and Technology, 3(6), 522-526. NASA Surface Meteorology and Solar Energy. Available from: https://www. eosweb.larc.nasa.gov/cgi-bin/sse/grid.cgi?&email=&step=1&p=&lat =8.18&submit=Submit&lon=111.94. [Last accessed on 2016 Dec 28]. 5. CONCLUSIONS Nayar, C.V., Phillips, S.J., James, W.L., Pryor, T.L., Remmer, D. (1993), Novel wind/diesel/battery hybrid energy system. Solar Energy, 51(1), 65-78. This research designs and simulates hybrid power plants suitable for community settlements in Brumbun Beach where they have not enjoyed the utilization of electrical energy due to remote locations of urban and hilly road access that are difficult to carry out network expansion. From the design and simulation of planned generator system to get optimal result with configuration of hybrid power plant system between diesel generator, PV panel, and wind turbine. Power plants with centralized system configuration have many advantages over dispersed systems. The most optimal system configuration of power plant design simulated by HOMER software is the use of 10 kW diesel generator, 8 kW capacity PV panel, 6 kW wind turbine, 24 V busbar with 8000 Ah capacity and 5 kW inverter capacity. Based on the technical review, the operation of the generator for 1 year is 341 h or 0.9 h/day, resulting in less fuel use and longer diesel generator life. While based on economic reviews, the value of NPC of US $ 44,680, Okwu, P.I., Chidiebere, C.E.O., Obinna, E.O., Uche, E.A. (2017), Details of photovoltaic solar system design alculations and the accessories. International Journal for Research in Applied Science and Engineering Technology, 5(6), 2933-2938.i Olatomiwa, L. (2016), Optimal configuration assessments of hybrid renewable power supply for rural healthcare facilities. Energy Reports, 2, 141-146. Purwadi, A., Haroen, Y., Zamroni, M., Heryana, N., dan Suryanto, A. (2012), Study of Hybrid PV-Diesel Power Generation System at Sebira IslandKepulauan Seribu. Conference on Power Engineering and Renewable Energy. Bali, Indonesia: IEEE. and Renewable Energy. Bali, Indonesia: IEEE. Quaschning, V. (2016), Understanding Renewable Energy Systems. 2nd ed. New York: Routledge, Taylor and Francis Group. Sopian, K., dan Othman, M.Y. (2005), Performance of a photovoltaic diesel hybrid system in Malaysia. Isesco Science and Technology Vision, 96, 37-39. 181 International Journal of Energy Economics and Policy \| Vol 10 • Issue 3 • 2020
https://openalex.org/W1592352382	https://dash.harvard.edu/bitstream/1/41384847/1/111836%2012545.full.pdf	English	null	How Visual Is the Visual Cortex? Comparing Connectional and Functional Fingerprints between Congenitally Blind and Sighted Individuals	The journal of neuroscience/The Journal of neuroscience	2,015	cc-by	15,592	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 Permanent link http://nrs.harvard.edu/urn-3:HUL.InstRepos:41384847 http://nrs.harvard.edu/urn-3:HUL.InstRepos:41384847 Citation Wang, X., M. V. Peelen, Z. Han, C. He, A. Caramazza, and Y. Bi. 2015. “How Visual Is the Visual Cortex? Comparing Connectional and Functional Fingerprints between Congenitally Blind and Sighted Individuals.” Journal of Neuroscience 35 (36): 12545–59. https://doi.org/10.1523/ jneurosci.3914-14.2015. Received Sept. 18, 2014; revised July 16, 2015; accepted July 22, 2015. Authorcontributions:M.V.PandY.B.designedresearch;X.W.,Z.H.,andC.H.performedresearch;X.W.analyzed data; X.W., M.V.P., A.C., and Y.B. wrote the paper. This study was supported by National Key Basic Research Program of China (2013CB837300, 2014CB846100), National Natural Science Foundation of China (31222024, 31171073, 31271115, 81030028, 31221003), and Pro- gram for New Century Excellent Talents in University (12-0055, 12-0065). A.C. was supported by the Fondazione CassadiRisparmiodiTrentoeRovereto.WethankDr.YongHeforhelpfulcomments;Drs.XuemingLuandYujunMa for help with data analyses; and Beijing Normal University-CNLab members for assistance in data collection. The authors declare no competing financial interests. Correspondence should be addressed to Dr. Yanchao Bi, State Key Laboratory of Cognitive Neuroscience and Learning & IDG/McGovern Institute for Brain Research, Beijing Normal University, Beijing, China 100875. E-mail: ybi@bnu.edu.cn. DOI:10.1523/JNEUROSCI.3914-14.2015 Copyright © 2015 the authors 0270-6474/15/3512545-15$15.00/0 How Visual Is the Visual Cortex? Comparing Connectional and Functional Fingerprints between Congenitally Blind and Sighted Individuals Xiaoying Wang,1 XMarius V. Peelen,2 Zaizhu Han,1 Chenxi He,1 Alfonso Caramazza,2,3 and Yanchao Bi1 1State Key Laboratory of Cognitive Neuroscience and Learning and IDG/McGovern Institute for Brain Research, Beijing Normal University, Beijing 100875, China, 2Center for Mind/Brain Sciences, University of Trento, 38068 Rovereto, Italy, and 3Department of Psychology, Harvard University, Cambridge, Massachusetts 02138 Classical animal visual deprivation studies and human neuroimaging studies have shown that visual experience plays a critical role in shaping the functionality and connectivity of the visual cortex. Interestingly, recent studies have additionally reported circumscribed regionsinthevisualcortexinwhichfunctionalselectivitywasremarkablysimilarinindividualswithandwithoutvisualexperience.Here, by directly comparing resting-state and task-based fMRI data in congenitally blind and sighted human subjects, we obtained large-scale continuous maps of the degree to which connectional and functional “fingerprints” of ventral visual cortex depend on visual experience. We found a close agreement between connectional and functional maps, pointing to a strong interdependence of connectivity and function. Visual experience (or the absence thereof) had a pronounced effect on the resting-state connectivity and functional response profile of occipital cortex and the posterior lateral fusiform gyrus. By contrast, connectional and functional fingerprints in the anterior medial and posterior lateral parts of the ventral visual cortex were statistically indistinguishable between blind and sighted individuals. These results provide a large-scale mapping of the influence of visual experience on the development of both functional and connectivity properties of visual cortex, which serves as a basis for the formulation of new hypotheses regarding the functionality and plasticity of specific subregions. Key words: blind; connectional fingerprints; functional fingerprints; resting-state functional connectivity; ventral visual cortex; visual experience Significance Statement Howisthefunctionalityandconnectivityofthevisualcortexshapedbyvisualexperience?Bydirectlycomparingresting-stateand task-based fMRI data in congenitally blind and sighted subjects, we obtained large-scale continuous maps of the degree to which connectional and functional “fingerprints” of ventral visual cortex depend on visual experience. In addition to revealing regions that are strongly dependent on visual experience (early visual cortex and posterior fusiform gyrus), our results showed regions in which connectional and functional patterns are highly similar in blind and sighted individuals (anterior medial and posterior lateral ventral occipital temporal cortex). These results serve as a basis for the formulation of new hypotheses regarding the functionality and plasticity of specific subregions of the visual cortex. 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 The Journal of Neuroscience, September 9, 2015 • 35(36):12545–12559 • 12545 Behavioral/Cognitive Introduction hierarchical (interactive) process starting from V1, processing simple visual features, such as line orientation, to higher-level visual regions in inferior temporal cortex processing more com- plex visual properties (Felleman and Van Essen, 1991). Although the role of VOTC in vision is well characterized, its involvement The ventral occipital-temporal cortex (VOTC) is primarily im- plicated in visual object recognition. This is achieved through a 12546 • J. Neurosci., September 9, 2015 • 35(36):12545–12559 Wang et al. • Function and Connection of Blind Visual Cortex Table 1. Background information of congenitally blind subjects Subject Age (yr), gender Years of education Cause of blindness Light perception Reported age at blindness onset Experiments participated 1 21, M 12 Congenital microphthalmia None 0 Task-fMRI 2 18, M 12 Congenital glaucoma and cataracts None 0 Task-fMRI 3 23, M 0 Congenital anophthalmos None 0 Task-fMRI 4 30, M 12 Congenital microphthalmia and microcornea None 0 Task-fMRI 5 38, F 12 Congenital glaucoma None 0 Both 6 45, M 12 Congenital microphthalmia; cataracts; leukoma None 0 Both 7 44, F 9 Congenital glaucoma None 0 Both 8 46, F 12 Cataracts; congenital eyeball dysplasia Faint 0 Both 9 53, M 12 Congenital eyeball dysplasia None 0 Both 10 36, M 12 Congenital leukoma Faint 0 Both 11 36, F 12 Congenital optic nerve atrophy Faint 0 Both 12 58, M 9 Congenital glaucoma and leukoma None 0 Both 13 41, M 12 Congenital glaucoma None 0 Both 14 28, F 15 Congenital microphthalmia; microcornea; leukoma Faint 0 Resting-state fMRI 15 37, F 12 No professional medical establishment of cause of blindness Faint 0 Resting-state fMRI 16 60, M 12 No professional medical establishment of cause of blindness Faint 0 Resting-state fMRI 17 46, M 12 No professional medical establishment of cause of blindness Faint 0 Resting-state fMRI 18 54, F 9 No professional medical establishment of cause of blindness Faint 0 Resting-state fMRI Table 1. Background information of congenitally blind subjects rest in terms of vectors of correlations between resting-state time- series) (Biswal et al., 1995). We characterized the functional and connectional profiles of regions within VOTC that were strongly influenced by visual experience, showing divergent functional and connectivity patterns between blind and sighted, as well as regions within VOTC that appeared polymodal, showing highly similar patterns in the blind and sighted groups. Participants Thi p Thirteen congenitally blind (4 females) and 16 sighted controls (7 fe- males) participated in the object category experiments. All blind subjects reported that they had been blind since birth. Because medical records of onset of blindness were not available for most subjects, it cannot be ruled out that some of the subjects may have had vision very early in life. None of the subjects remembered to have ever been able to visually recognize shapes. All blind subjects were examined by an ophthalmologist to con- firm their blindness and to establish the causes if possible. Five blind subjects reported to have had faint light perception in the past. Three blind subjects had faint light perception at the time of testing. Detailed diagnoses for all blind subjects are listed in Table 1. The blind and sighted groups were matched on handedness (all right-handed), age (blind: mean SD 38 12 years, range 18–58 years; sighted: mean SD 43 11 years, range 26–59 years; t(27) 1), and years of education (blind: mean SD 11 3 years, range 0–12 years; sighted: mean SD 12 2 years, range 9–15 years; t(27) 1). Intriguingly, a set of recent findings showed that the categorical response preferences of other parts of VOTC are surprisingly inde- pendent of visual experience (for review, see Ricciardi et al., 2014). For example, several category-preferring regions in VOTC respond similarly (in nonvisual tasks) in the congenitally blind and the sighted, including selectivity for scenes and large objects in the para- hippocampal place area (Wolbers et al., 2011; He et al., 2013), for toolsinleftlateraloccipital-temporalcortex(Peelenetal.,2013),and for bodies in the right extrastriate body area (Kitada et al., 2014; using a training procedure: Striem-Amit and Amedi, 2014). These results suggest that some parts of VOTC are relatively polymodal, or metamodal (Pascual-Leone and Hamilton, 2001) in nature, exhibit- ing similar functional profiles in terms of category preference in the absence of visual input/experience. The resting-state fMRI scanning was performed on 14 congenitally blind (7 females; mean age 45 years, range 26–60 years), 9 of whom participated in our object category experiment (Table 1), and 34 right- handed sighted subjects (20 females; age: mean SD 22.5 1.3 years, range 20–26 years) from separate studies (Wei et al., 2012; Peelen et al., 2013). Introduction in processing input from other modalities is less clear (Morrell, 1972; Gibson and Maunsell, 1997; for a review, see Ghazanfar and Schroeder, 2006). In the present study, we provide large-scale maps of the degree to which the functionality and connectivity of VOTC are shaped by visual experience, revealing both visual and nonvisual parts of VOTC. p Animal visual deprivation studies (Hubel et al., 1977; Heil et al., 1991)andhumanbrainimagingstudieswithcongenitallyblindsub- jects(Sadatoetal.,1996,1998;Cohenetal.,1997;Bu¨cheletal.,1998; Burton et al., 2002, Burton, 2003; Amedi et al., 2003; Lewis et al., 2010;forreviews,seeMerabetetal.,2005;Pascual-Leoneetal.,2005) haveshownthatvisualinputhasacriticalroleinshapingearlyvisual corticalfunction.Absenceofvisualexperienceearlyinlifealsoaffects the functionality of VOTC regions beyond early visual cortex, such as regions selective for faces (Goyal et al., 2006) or motion (Saenz et al., 2008; Bedny et al., 2010). These functional changes are reflected in altered anatomical and functional connectivity patterns of visual cortex in early blind adults (Liu et al., 2007; Bedny et al., 2010; Wat- kins et al., 2012; Butt et al., 2013; Burton et al., 2014; Wang et al., 2014; for reviews, see Bock and Fine, 2014; Ricciardi et al., 2014). Stimuli Image acquisition All fMRI and structural MRI data were collected using a 3T Siemens Trio Tim Scanner at the Beijing Normal University MRI center. The stimuli consisted of 18 categories, each containing 18 items. The 18 categories included human face parts, human body parts, daily scenes, tools, mammals, reptiles, birds, fishes, bugs, fruits and vegetables, flowers, pre- servedfood,clothes,musicalinstruments,vehicles,furniture,celebrities,and famous places (for exemplars for each category, see Table 2). Our analyses here focused on the first 16 common objects. Celebrities (presented as faces in visual experiment) and famous places might be differently represented in the brain (e.g., Ross and Olson, 2012) and were not analyzed in this paper. This set of categories was chosen: (1) to cover a range of common object categories people know about and interact with in daily life; and (2) to in- clude categories that have previously been shown to evoke relatively distinct response patterns in VOTC (e.g., tools, animals, bodies, faces, furniture, scenes). Our set was a subset of the categories studied in an earlier study in sighted subjects (Downing et al., 2006). For the object category fMRI experiments, high-resolution anatomical 3D MP-RAGE images were collected in the sagittal plane (144 slices, TR 2530 ms, TE 3.39 ms, flip angle 7°, matrix size 256 256, voxel size 1.33 1 1.33 mm). BOLD activity was measured with an EPI sequence covering the whole cerebral cortex and most of the cerebel- lum (33 axial slices, TR 2000 ms, TE 30 ms, flip angle 90°, matrix size 64 64, voxel size 3 3 3.5 mm with gap of 0.7 mm). Fourteen congenitally blind and 34 sighted subjects underwent a resting-state scan in separate studies. Subjects were instructed to close their eyes and to not fall asleep. The resting-state scan lasted 6 min 40 s for the congenitally blind group and 8 min for the sighted group. A T1-weighted MP-RAGE structural image with the same scanning parameters as reported above and 200 volumes of resting-state functional images using an EPI sequence (32 axial slices, TR 2000 ms, TE 33 ms, flip angle 73°, matrix size 64 64, voxel size 3.125 3.125 4 mm with gap of 0.6 mm) were acquired for each of the 14 blind subjects. Participants Thi The potential confound of the age difference between the two groups of subjects was examined in a control analysis where a separate group of 7 sighted adults (2 females; age: mean SD 42 10 years, range 26–54 years; years of education: mean SD 12 2 years, range 9–15 years; all right-handed; 2 of them also took part in the object category experiment) that were age-matched with the congenitally blind group were used. In sum, previous studies with blind subjects have used specific contrasts to test hypotheses about the influence of visual experi- ence on circumscribed regions of VOTC, reporting evidence for both visually dependent and visually independent regions. The goal of the present study was to systematically map out, across the whole VOTC, the influence of visual experience on functional fingerprints (response patterns to 16 object categories) and con- nectional fingerprints (functional connectivity patterns during All subjects were native Mandarin Chinese speakers. None had any history of psychiatric or neurological disorders or suffered from head injuries. All subjects provided informed consent and received monetary compensation for their participation. The study was approved by the institutional review board of the Beijing Normal University Imaging Center for Brain Research. Wang et al. • Function and Connection of Blind Visual Cortex Wang et al. • Function and Connection of Blind Visual Cortex J. Neurosci., September 9, 2015 • 35(36):12545–12559 • 12547 Table 2. Stimulus characteristics Category Exemplar stimulus Word fre- quency (log) Familiarity (1-7,7: most familiar) Sighted Blind Mean SD Mean SD Mean SD Bird Pigeon 0.4 0.4 4.9 1.0 4.1 1.0 Body parts Palm 0.8 0.5 6.7 0.4 6.9 0.1 Bug Ladybug 0.4 0.5 5.2 0.9 4.6 1 Clothing Jacket 0.6 0.5 6.5 0.5 6.3 0.6 Face parts Nose 1.0 0.6 6.6 0.3 6.8 0.3 Fish Goldfish 0.2 0.3 4.3 0.8 3.9 0.9 Flower Rose 0.4 0.4 4.6 0.9 4.1 1.0 Food Dumpling 0.4 0.4 6.4 0.5 6.5 0.5 Furniture Bed 0.6 0.5 6.2 0.4 6.1 0.7 Fruits and vegetables Apple 0.7 0.5 6.6 0.5 6.6 0.5 Mammal Cat 0.6 0.6 5.1 1.2 4.3 0.8 Musical instruments Piano 0.4 0.5 4.3 1.1 4.8 1.0 Reptile Turtle 0.2 0.5 4.1 1.0 3.4 0.8 Scene Kitchen 1.0 0.6 6 0.5 5.7 0.7 Tool Hammer 0.6 0.5 6.4 0.6 6.4 0.7 Vehicle Bus 0.8 0.7 5.9 0.8 5.1 0.9 Table 2. Stimuli For the 34 sighted subjects, a T1-weighted structural MP-RAGE image in sagittal plane (128 slices; TR 2530 ms, TE 3.39 ms, flip angle 7°, matrix size 64 64, voxel size 1.3 1 1.3 mm) and 240 volumes of resting-state functional images using an EPI sequence (33 axial slices, TR 2000 ms, TE 30 ms, flip angle 90°, matrix size 64 64, voxel size 3.1 3.1 3.5 mm with gap of 0.6 mm) were acquired. In the visual object category experiment, stimuli were grayscale pho- tographs (400 400 pixels, subtended 10.55° 10.55° of visual angle); in auditory experiments, they were object names that were digitally re- corded (22,050 Hz, 16 bit), spoken by a female native Mandarin speaker. Stimulus presentation was controlled by E-prime (Schneider et al., 2002). There were no significant differences in word length across the 16 cate- gories (number of one, two, or three syllable words: 2 1.807, df 15, p 0.05). Names of face parts, body parts, scenes, and vehicles tended to be of higher frequency, and those of reptile and fish were of low frequency (Table 2). Familiarity ratings collected from both sighted and blind sub- jects showed that face parts, body parts, clothing, food, fruits and vege- tables, and tools were the most familiar, and fish, reptile, and bird items were the least familiar (Table 2). The rating scores of the 16 object cate- gories were highly correlated between-subject groups (Spearman’s 0.95, p 0.001). Data preprocessing b Object category experiment data were preprocessed using Statistical Para- metric Mapping software (SPM8; http://www.fil.ion.ucl.ac.uk/spm/) and the advanced edition of DPARSF version 2.1 (Chao-Gan and Yu-Feng, 2010). The first six volumes (12 s) of each functional run were discarded for signal equilibrium. Functional data were motion-corrected, low-frequency drifts removed with a temporal high-pass filter (cutoff: 0.008 Hz), and nor- malized to the MNI space using unified segmentation. The functional im- ages were then resampled to 3 mm isotropic voxels and were spatially smoothed using a 6 mm FWHM Gaussian kernel. Image acquisition All fMRI d t t Stimuli Participants Thi Stimulus characteristics size in half of the blocks, whereas for the remaining blocks, the items were presented in random order. The order of the 12 runs and the 18 blocks within each run was pseudo-randomized across subjects with no more than 4 successive blocks in each run being of the same judgment (ascend- ing order or not, see below). Auditory object category experiment. All subjects were asked to keep their eyes closed throughout the experiment and to listen carefully to groups of six spoken words presented binaurally through a headphone. Subjects were instructed to think about each item and press a button with the right index finger if the items were presented in an ascending order in terms of real-world object size and press the other button with the right middle finger if otherwise. Responses were made after the onset of a response cue (300 ms auditory tone) immediately following the last item of the block. Visual object category experiment. Sighted subjects viewed the stimuli through a mirror attached to the head coil. The stimuli were grayscale photographs presented at the center of the screen. Subjects were in- structed to think about the real-world size of each item and press one button with the right index finger if the items were presented in an order of increasing real-world object size and press the other button with the right middle finger if otherwise. Responses were made when the fixation dot at the center of the screen turned from red to green right after the offset of the last item of the block. Data analysis Object category experiment data were analyzed using the GLM in SPM8. For both the auditory and visual object category experiments, the GLM included 18 predictors, convolved with a canonical HRF, corresponding to the 18 category blocks (duration 9 s) along with one regressor of no interest for the button press (duration 0) for each block, and six regressors of no interest corresponding to the six head motion parame- ters in each run. For each subject, runs in which the subject’s head mo- tion was 3 mm or 3° were deleted, and the remaining runs were entered into the analysis. For the group effects, a random-effects analysis was conducted for each experiment separately. Comparing functional fingerprints between sighted and congenitally blind groups. To examine the between-group similarity/dissimilarity of functional fingerprints for each voxel in the VOTC mask, we compared the functional response profile to the 16 object categories between sighted and congenitally blind groups. This analysis was identical to the connectional fingerprint analysis described above, except that the voxel- wise vectors consisted of the responses ( values) to the 16 object cate- gories in the auditory experiment instead of the 180 RSFC values. All the voxelwise analyses were conducted in a VOTC mask. The mask was defined according to three criteria (Mahon et al., 2009): (1) the voxels were activated (random effects, FDR corrected, p 0.05) for the task versus fixation contrast in the sighted visual experiment; (2) the voxels were within the occipitotemporal cortex (37–56 and 79–90) in the Automated Anatomical Labeling template (Tzourio- Mazoyer et al., 2002); and (3) the voxels were at or below a z coordi- nates in the MNI space of 10. Comparing functional fingerprints between modalities, within the sighted group. In addition to comparisons between blind and sighted groups, our data also allowed for comparing response profiles between modalities (auditory, visual) within the sighted group. This analysis examined the degree of between-modality similarity of functional fingerprints within the sighted group. The analysis was otherwise identical to that described for the between-group comparison of functional response profile. Comparing connectional fingerprints between sighted and congenitally blind groups. To examine the between-group similarity/dissimilarity of RSFC patterns for each voxel in the VOTC mask, we compared the in- trinsic functional connectivity patterns between sighted and congenitally blind groups. Design and task d g A size judgment task was adopted (Mahon et al., 2009; He et al., 2013; Peelen et al., 2013) to encourage subjects to retrieve object information. The auditory and the visual version of the experiments had the same following structure. Each object category experiment consisted of 12 runs, with 348 s per run. Each run started and ended with a 12 s fixation (visual experiment) or silence (auditory experiment) block. Between these blocks, eighteen 9 s task blocks, one per category, were presented with an interblock interval of 9 s. For each task block, six 1.5 s items from the same category were presented sequentially. Thus, for each object category, there were a total of 12 blocks across the 12 runs of each exper- iment, yielding a total of 72 presentations per category, with each of the 18 category exemplars repeated 4 times in the experiment. For each cat- egory, items were presented in an order of increasing real-world object Resting-statedatawerepreprocessedandanalyzedusingSPM8,DPARSF version 2.1, and Resting-State fMRI Data Analysis Toolkit version 1.5 (Song etal.,2011).Thefirst6min40softhesightedgroup’sresting-statedatawere analyzedsothatthetwosubjectgroupshadmatchinglengthsofresting-state time-series. Functional data were resampled to 3 mm isotropic voxels. The first 10 volumes of the functional images were discarded. Preprocessing of thefunctionaldataincludedslicetimingcorrection,headmotioncorrection, spatial normalization to the MNI space using unified segmentation, spatial smoothing with 6 mm FWHM Gaussian kernel, linear trend removal, and Wang et al. • Function and Connection of Blind Visual Cortex 12548 • J. Neurosci., September 9, 2015 • 35(36):12545–12559 bandpass filtering (0.01–0.1 Hz). Six head motion parameters, global mean signals, white matter, and CSF signals were regressed out as nuisance cova- riates. Global mean, white matter, and CSF signals were calculated as the mean signals in SPM’s whole-brain mask (brainmask.nii) thresholded at 50%, white matter mask (white.nii) thresholded at 90%, and CSF mask (csf.nii)thresholdedat70%,respectively(Chao-GanandYu-Feng,2010).As described below, validation analyses were performed to examine the effects of global signal removal, gray matter density difference, and head motion. bandpass filtering (0.01–0.1 Hz). Six head motion parameters, global mean signals, white matter, and CSF signals were regressed out as nuisance cova- riates. Global mean, white matter, and CSF signals were calculated as the mean signals in SPM’s whole-brain mask (brainmask.nii) thresholded at 50%, white matter mask (white.nii) thresholded at 90%, and CSF mask (csf.nii)thresholdedat70%,respectively(Chao-GanandYu-Feng,2010).As described below, validation analyses were performed to examine the effects of global signal removal, gray matter density difference, and head motion. the congenitally blind group and within the sighted group, respectively. Data analysis Two of the 14 congenitally blind subjects were excluded because of excessive head motion (2 mm maximum translation or 2° rotation), resulting in 12 blind subjects in the RSFC analysis. There were no significant head motion differences between the remaining blind and sighted subjects: mean/SD of translational motion: blind, 0.36 mm/0.19 mm, sighted, 0.31 mm/0.16 mm; mean/SD of rotational motion: blind, 0.42°/0.23°, sighted, 0.39°/0.26°; two-tailed two-sample t test: p 0.43 for translational, p 0.75 for rotational, p 0.32 for mean framewise displacement computed using the method in Power et al. (2012) and p 0.22 using the method in Jenkinson et al. (2002). Comparing results from connectional and functional fingerprint analyses. Motivated by the view that the functional profile in a brain region is driven by the functional/structural connectivity pattern of this region (Passingham et al., 2002; Mahon and Caramazza, 2009, 2011; Saygin et al., 2012), we directly compared the between-group similarity maps of category response patterns and RSFC patterns. The RSFC pattern maps and category response maps were plotted for regions showing similar between-group similarity of RSFC patterns and category response pat- terns (both highly similar or both dissimilar between-subject groups). We further computed the Pearson correlation coefficient between the between-group category response pattern similarity and the between- group RSFC pattern similarity across the VOTC to measure the corre- spondence between connectional and functional fingerprints. For each voxel in the VOTC mask, its intrinsic functional connectivity pattern was defined as a vector of the strengths of RSFC between the voxel and each of 180 cerebral regions covering the whole cerebrum. The 180 cerebral regions were defined from an atlas that was generated by parcel- lating voxels in the whole brain into 200 regions according to their ho- mogeneity of RSFC patterns (Craddock et al., 2012). Twenty regions in the cerebellum and brainstem were excluded from our analysis. Connec- tional fingerprints were constructed at both individual level and group level. First, the vector of RSFC strengths was obtained for each voxel and each subject by calculating the Pearson correlation of the time series of the voxel and the mean time series of each of the 180 cerebral regions (including the region the seed voxel resided in). For each voxel, the RSFC strengths were then Fisher-transformed and averaged across the subjects of each group (sighted, congenitally blind). Design and task d These values were compared with between-group r values, obtained by correlating the individual-level connectional fingerprint between all pairs of blind and sighted subjects. The bootstrap resampling method (Efron and Tibshirani, 1993; Kriegeskorte et al., 2008) was used to exam- ine whether there was a significant difference between within-group (col- lapsing within-blind and within-sighted) and between-group similarities of connectional fingerprints, separately for each voxel. This method was chosen because it did not require the data being tested to meet distribu- tional assumptions (Kriegeskorte et al., 2008). Specifically, we bootstrap- resampled the within-group and between-group Fisher-transformed r values, recomputing the mean of within-group similarities and the mean of between-group similarities, and the difference between them. The bootstrap resampling process was repeated 1000 times, resulting in a distribution of the difference between the mean of within-group and between-group similarities. For each voxel, the within-group similarity was considered to be significantly higher than the between-group simi- larity at a significance level of if the lower bound of the 1- confidence interval of the difference was positive. A map indicating the significance level ( 0.05, 0.01, 0.001) each voxel could reach was produced. Data analysis For each subject, an individual-level connectional fingerprint was constructed as the vector of 180 RSFC strength values computed based on the subject’s own time-series of resting-state brain activity. For each VOTC voxel, 627 within-group r values (66 within-blind R values, n 12; and 561 within-sighted r values, n 34) and 408 between-group r values were obtained by correlating the individual- level connectional fingerprints of pairs of subjects from the same group or from different groups (see Materials and Methods). Voxels showing higher within-group similarity relative to between-group similarity of connectional fingerprints were identified using the bootstrap resampling method (Efron and Tibshirani, 1993; Krieges- korte et al., 2008) on the within-group and between-group r values. A brain map (Fig. 1C) was obtained to show which VOTC voxels’ connectional fingerprints were significantly different between sighted and blind groups. As shown in Figure 1C (red patches), the differencebetweenthewithin-groupandbetween-groupsimilarities were significant (within between, uncorrected p 0.001) in large portionoftheVOTC,includingthebilateralcalcarine,lingualgyrus, inferior and middle occipital gyrus, posterior fusiform gyrus, and portions of the posterior inferior/middle temporal gyrus. These ar- eas covered almost completely the regions showing the lowest between-group correlations (bottom 15%), indicating that the RSFCpatternsoftheseclustersweresystematicallydifferentbetween thesightedandcongenitallyblindgroups.Bycontrast,thedifference between the within-group similarities and the between-group simi- larities was not significant (uncorrected single voxel, p 0.05) in parts of the bilateral anterior parahippocampal gyrus, anterior me- dial fusiform gyrus, and middle/posterior parts of the inferior and middle temporal gyrus (Fig. 1C, blue patches). These voxels over- lappedwiththeanteriormedialtemporalregionsandmiddle/posterior lateral temporal regions that showed the highest rank (top 15%) of between-groupsimilarities(Fig.1B).Thisanalysiswasalsodoneforthe blind and sighted groups separately (i.e., within-blind correlations vs between-group correlations; within-sighted correlations vs between- group correlations), yielding similar results (data not shown). 4. Between-group age difference. There was a significant age difference between the sighted and the congenitally blind groups in the RSFC anal- ysis. To rule out age effects on the RSFC patterns in the VOTC (Biswal et al., 2010; Zuo et al., 2010; Yan et al., 2011), we performed a control analysis using resting-state data of a new group of 7 sighted subjects who were age-matched with our congenitally blind subjects. The scanning time and parameters were identical for the two groups. 5. SVM classification analyses on identified clusters of interest. Data analysis p g p Finally, we examined the consistency of the between-group and between-modality similarity maps of category response patterns by plot- ting overlapping regions and calculating their Pearson correlation coef- ficients across all voxels in the VOTC mask. Control and validation analyses. We additionally performed four control analyses to address the potentially confounding effects of global signal removal, gray matter density, head motion and subject age, and one validation analysis to consolidate the major identified clusters of interest using an alternative approach (support vector ma- chine classification). In a first analysis, these group-level data were directly compared using correlation analysis: a correlation map was generated by calculating, for each voxel, the Pearson correlation coefficient between the group-level connectional fingerprints (i.e., the 180 values reflecting RSFC strengths) of the sighted and congenitally blind groups. The resulting map repre- sents the degree of between-group RSFC pattern similarity, with voxels showing a relatively high correlation having relatively similar connec- tional fingerprints in the blind and sighted groups. 1. Global signal removal. Whether global signal should be removed during resting-state fMRI preprocessing is currently controversial. Although previous studies have suggested that global signal should be removed because it is associated with non-neuronal activity such as respiration (e.g., Fox et al., 2009), this procedure introduces wide- spread negative functional connectivities and may alter the intrinsic brain network structure (e.g., Murphy et al., 2009). To explore the effects of global signal removal on our results, we reanalyzed the correlation between the between-group similarity of category re- sponse patterns and the between-group similarity of RSFC patterns without global signal removal. In a second analysis, putative group differences and similarities were examined in more detail by statistically comparing within-group and between-group similarities using individual subject data. Specifically, for each voxel, within-blind and within-sighted r values were calculated by correlating each pair of individual-level connectional fingerprints within J. Neurosci., September 9, 2015 • 35(36):12545–12559 • 12549 Wang et al. • Function and Connection of Blind Visual Cortex (180 pairs of RSFC strength values) for each voxel. For better visualization, the resulting r map (Fig. 1A) was transformed to a percentile rank map in which the value of each voxel denoted its rank of between-group RSFC pattern similarity among all voxels in the VOTC mask (Fig. 1B). 2. Group difference of gray matter density. Data analysis For the potential influence of gray matter density on RSFC patterns (He et al., 2007; Wang et al., 2011), we calculated the partial correlation between the between-group similar- ity of RSFC patterns and the between-group similarity of category re- sponse patterns by including the between-group differences of gray matter density as a covariate. The between-group difference of gray mat- ter density for each voxel was the absolute value of the effect size (esti- mated) (Hedges, 1982) in the comparison between the gray matter density of blind subjects and sighted subjects. As shown in Figure 1A, voxels showing the highest between- group similarity of RSFC patterns (top 15%, Pearson r 0.88; Fig. 1B, dark red clusters) were observed in the bilateral anterior medial parts and posterior lateral parts of the VOTC, whereas voxels showing the lowest between-group RSFC pattern similar- ity (bottom 15%, Pearson r 0.67; Fig. 1B, dark blue regions) were mainly found in the posterior occipital cortex, including the bilateral inferior occipital gyrus, lingual gyrus, and the left poste- rior lateral fusiform gyrus. 3. Head motion confounds. Head motion can exert a confounding effect on functional connectivity patterns (Power et al., 2012, 2014; Van Dijk et al., 2012; Satterthwaite et al., 2013). As reported above, we found no significant difference in head motion between our sighted and congeni- tally blind groups. To further exclude any potential effects of head mo- tion, we repreprocessed the resting-state functional images using a scrubbing method (Power et al., 2012; Yan et al., 2013). Specifically, for each subject, resting-state functional volumes were removed based on a threshold of framewise displacement (Power et al., 2012) 0.2 mm as well as the 1 back and 2 forward neighbors (Power et al., 2013). Scrubbing resulted in different lengths of time-series across subjects. Nine sighted and 3 congenitally blind subjects were removed due to too few remaining time points (150 volumes, i.e., 5 min) (Power et al., 2012). For the remaining 25 sighted and 9 congenitally blind subjects, the first 150 time points were analyzed to match the lengths of time-series between groups. gy To statistically examine where the two subject groups differed significantly, we compared the between-group correlations and the within-group correlations of RSFC patterns across all VOTC voxels. Data analysis To antic- ipate, our results revealed visual regions in bilateral posterior occipital regions and left posterior fusiform region, and polymodal regions, in- cluding the bilateral anterior medial temporal regions and bilateral mid- dle/posterior lateral temporal regions. To verify these results, we performed group classification in each cluster based on the RSFC pat- terns or category response patterns of the sighted and blind groups using a linear SVM (LIBSVM: http://www.csie.ntu.edu.tw/~cjlin/libsvm) with standard parameters. We used a leave-one-subject-out cross-validation scheme. In each iteration of the cross-validation, each dimension of fea- ture vectors of both training and test examples was z-normalized within each subject. The classifier was then trained on the training set and tested on the remaining one subject by classifying each subject into either the blind or the sighted groups. At the end of the procedure, we obtained the decoding accuracy in terms of the percentage of corrected predictions of group label in all predictions. The statistical significance of decoding accuracy was assessed using permutation tests (1000 iterations), in which group labels for all subjects were shuffled. The p value for decoding accuracy was calculated as the fraction of accuracies from all permuta- tions that were equal to or greater than the actual accuracy when using correct group labels. All results in this paper are shown in the MNI space and projected onto the MNI brain surface using the BrainNet viewer (http://www.nitrc.org/ projects/bnv/) (Xia et al., 2013). Comparing functional fingerprints between sighted and congenitally blind groups B, Percentile rank map of Pearson correlation coefficients of RSFC patterns betweenblindandsightedgroups.Colorbarrepresentsthepercentilerankvalue.Warmercolorsrepresentgreaterbetween-groupsimilarity.Blackarrowsinthecolorbarindicatethebottomand the top 15% similarity. C, Brain map indicating the significance level each voxel reached for the difference between within-group similarity and between-group similarity of RSFC patterns. The contours of bilateral fusiform gyrus (solid black lines), bilateral parahippocampal gyrus (dashed black lines), and bilateral inferior temporal gyrus (dash-dot black lines) are imposed on the brain surface for a clear reference of significant anatomical locations. (top 15%, Pearson r 0.71; Fig. 2B, dark red clusters) were the anterior medial parts of the bilateral VOTC, including the para- hippocampal gyrus and anterior medial fusiform gyrus and mid- dle/posterior lateral parts of the bilateral VOTC, including the posterior inferior/middle temporal regions, whereas voxels showing the lowest between-group category response similarity (bottom 15%, Pearson r 0.02; Fig. 2B, dark blue regions) mainly located in the bilateral posterior lingual gyrus and inferior occipital gyrus, with the clusters in the left hemisphere extending anteriorly to the left posterior lateral fusiform. lapped largely with regions showing the lowest between-group correlations (bottom 15%), indicating that the category response rankings of these clusters were systematically different between the sighted and congenitally blind groups. The VOTC regions showing the highest between-group similarities (top 15%) did not exhibit significant within- versus between-group differences (uncorrected single voxel, p 0.05), confirming similar cate- gory response patterns between the two groups in these bilat- eral anterior medial and middle/posterior lateral VOTC regions (Fig. 2C, blue patches). This analysis was also done for the blind and sighted groups separately (i.e., within-blind cor- relations vs between-group correlations; within-sighted cor- relations vs between-group correlations), yielding similar results (data not shown). Similar to the connectional fingerprint comparison, we statis- tically compared the within-group and between-group similari- ties calculated from individual-level functional fingerprints. Bootstrap resampling was repeated 1000 times on 198 within- group r values: 78 within-blind r values for the blind group (n 13) and 120 within-sighted r values for the sighted group (n 16) and 208 between-group r values for each VOTC voxel. As shown in Figure 2C, a small number of voxels (red patches) in bilateral posterior inferior occipital and lingual gyri showed significant within- versus between-group differences (within between; uncorrected p 0.001). Comparing functional fingerprints between sighted and congenitally blind groups By lowering the thresholds of signifi- cance to uncorrected p 0.01 (yellow patches) and p 0.05 (cyan patches), voxels exhibiting difference of category response patterns between-subject groups were still mainly observed in these bilateral posterior occipital regions. These regions over- Comparing functional fingerprints between sighted and congenitally blind groups For each voxel in the VOTC mask, strengths of RSFCs (Fisher- transformed Pearson correlation coefficients) between the voxel and 180 cerebral regions (Craddock et al., 2012, their parcellation atlas) covering the whole brain were computed for each subject and averaged across subjects in the sighted and congenitally blind groups separately, rendering a vector of 180 mean RSFC strength values as the RSFC pattern for each voxel in each group; then, the similarity of RSFC patterns between the blind and sighted groups was calculated as the Pearson correlation between the 2 vectors To explore the degree of between-group similarity of functional response patterns in VOTC, we calculated, for each voxel, the correlation of the response profiles to 16 object categories (rep- resented by the spoken names of category exemplars) between the sighted and blind groups. The r map (Fig. 2A) was transformed to a percentile rank map for an overview of the pattern of between- group similarity across the VOTC (Fig. 2B). Regions showing the highest between-group similarity of category response patterns 12550 • J. Neurosci., September 9, 2015 • 35(36):12545–12559 Wang et al. • Function and Connection of Blind Visual Cortex Figure1. ComparisonoftheconnectionalfingerprintsoftheVOTCvoxelsintheblindandsightedgroups.A,OriginalmapofPearsoncorrelationcoefficientsofRSFCpatternsbetweenb sighted groups. Color bar represents the Pearson r value. Warmer colors represent greater between-group similarity. B, Percentile rank map of Pearson correlation coefficients of RSFC p betweenblindandsightedgroups.Colorbarrepresentsthepercentilerankvalue.Warmercolorsrepresentgreaterbetween-groupsimilarity.Blackarrowsinthecolorbarindicatethebott the top 15% similarity. C, Brain map indicating the significance level each voxel reached for the difference between within-group similarity and between-group similarity of RSFC patte contours of bilateral fusiform gyrus (solid black lines), bilateral parahippocampal gyrus (dashed black lines), and bilateral inferior temporal gyrus (dash-dot black lines) are imposed on th surface for a clear reference of significant anatomical locations. Figure1. ComparisonoftheconnectionalfingerprintsoftheVOTCvoxelsintheblindandsightedgroups.A,OriginalmapofPearsoncorrelationcoefficientsofRSFCpatternsbetweenblindand sighted groups. Color bar represents the Pearson r value. Warmer colors represent greater between-group similarity. B, Percentile rank map of Pearson correlation coefficients of RSFC patterns betweenblindandsightedgroups.Colorbarrepresentsthepercentilerankvalue.Warmercolorsrepresentgreaterbetween-groupsimilarity.Blackarrowsinthecolorbarindicatethebottomand the top 15% similarity. C, Brain map indicating the significance level each voxel reached for the difference between within-group similarity and between-group similarity of RSFC patterns. The contours of bilateral fusiform gyrus (solid black lines), bilateral parahippocampal gyrus (dashed black lines), and bilateral inferior temporal gyrus (dash-dot black lines) are imposed on the brain surface for a clear reference of significant anatomical locations. Figure1. ComparisonoftheconnectionalfingerprintsoftheVOTCvoxelsintheblindandsightedgroups.A,OriginalmapofPearsoncorrelationcoefficientsofRSFCpatternsbetweenblindand sighted groups. Color bar represents the Pearson r value. Warmer colors represent greater between-group similarity. Comparing functional fingerprints between modalities, within the sighted group Re- gions showing the lowest correlations (bottom 15%, r values, Pearson r 0.27; Fig. 3B, dark blue clusters) between modality of inputs in sighted group were mainly located in the bilateral posterior lingual gyrus, posterior lateral fusiform gyrus, and in- ferior occipital gyrus. Spearman’s rank order correlations gener- ated highly similar results. (top 15%, Pearson r 0.63; Fig. 3B, dark red clusters) were the anterior medial parts of the bilateral VOTC, including the poste- rior parahippocampal gyrus, anterior medial fusiform gyrus, and middle/posterior lateral parts of the bilateral VOTC, including the posterior parts of the middle/inferior temporal regions. Re- gions showing the lowest correlations (bottom 15%, r values, Pearson r 0.27; Fig. 3B, dark blue clusters) between modality of inputs in sighted group were mainly located in the bilateral posterior lingual gyrus, posterior lateral fusiform gyrus, and in- ferior occipital gyrus. Spearman’s rank order correlations gener- ated highly similar results. rankings of these clusters were indeed systematically different be- tween auditory and visual modalities. By contrast, in bilateral para- hippocampalgyrus,medialanteriorfusiformgyrus,andthemiddle/ posterior parts of the lateral inferior and middle temporal cortex (Fig. 3C, blue regions), the within-modality correlations and the between-modality correlations did not differ significantly (uncor- rected single voxel, p 0.05). These regions correspond well to the bilateral anterior medial temporal and middle/posterior lateral tem- poral regions showing the highest rank (top 15%) of between- modality similarities. This analysis was also done for the sighted visual and sighted auditory experiments separately (i.e., within- visual correlations vs between-modality correlations; within- auditory correlations vs between-modality correlations), yielding similar results (data not shown). Similar to the other measures, we statistically compared the within-modality and between-modality similarities (240 within- vs 256between-modality)usingthebootstrapresamplingmethod.The resulting binary map (Fig. 3C) showed that the differences between the within-modality correlations and between-modality correla- tions were significant (within between) in vast regions of the VOTC (Fig. 3C, red patches), mainly including the bilateral calcar- ine, lingual gyrus, inferior and middle occipital gyrus, middle and posterior fusiform gyrus, and the posterior tip of the inferior and middle temporal gyri (uncorrected, p 0.001). These areas largely overlapped with bilateral posterior lingual, lateral fusiform, and in- ferior occipital regions showing the lowest between-modality corre- lations (bottom 15%), confirming that the category response Comparing functional fingerprints between modalities, within the sighted group To explore the degree of between-modality similarity of func- tional fingerprints in VOTC, for each voxel we calculated the Pearson correlation of the category response levels for the 16 categories between the sighted-visual and sighted-auditory ex- periments. The r map (Fig. 3A) was transformed to a percentile rank map for an overview of the pattern of between-modality similarity across the VOTC (Fig. 3B). Regions showing the high- est between-modality similarity of category response patterns J. Neurosci., September 9, 2015 • 35(36):12545–12559 • 12551 Wang et al. • Function and Connection of Blind Visual Cortex Figure 2. Comparison of the functional fingerprints of the VOTC voxels in the blind and sighted auditory experiments. A, Original map of Pearson correlation coefficients of category response patterns between blind and sighted groups. Color bar represents the Pearson r value. Warmer colors represent greater between-group similarity. B, Percentile rank map of Pearson correlation coefficientsofcategoryresponsepatternsbetweenblindandsightedgroups.Colorbarrepresentsthepercentilerankvalue.Warmercolorsrepresentgreaterbetween-groupsimilarity.Blackarrows in the color bar indicate the bottom and the top 15% similarity. C, Brain map indicating the significance level each voxel reached for the difference between within-group similarity and between-group similarity of category response patterns. The contours of bilateral fusiform gyrus (solid black lines), bilateral parahippocampal gyrus (dashed black lines), and bilateral inferior temporal gyrus (dash-dot black lines) are imposed on the brain surface for a clear reference of significant anatomical locations. Figure 2. Comparison of the functional fingerprints of the VOTC voxels in the blind and sighted auditory experiments. A, Original map of Pearson correlation coefficients of category response patterns between blind and sighted groups. Color bar represents the Pearson r value. Warmer colors represent greater between-group similarity. B, Percentile rank map of Pearson correlation coefficientsofcategoryresponsepatternsbetweenblindandsightedgroups.Colorbarrepresentsthepercentilerankvalue.Warmercolorsrepresentgreaterbetween-groupsimilarity.Blackarrows in the color bar indicate the bottom and the top 15% similarity. C, Brain map indicating the significance level each voxel reached for the difference between within-group similarity and between-group similarity of category response patterns. The contours of bilateral fusiform gyrus (solid black lines), bilateral parahippocampal gyrus (dashed black lines), and bilateral inferior temporal gyrus (dash-dot black lines) are imposed on the brain surface for a clear reference of significant anatomical locations. (top 15%, Pearson r 0.63; Fig. 3B, dark red clusters) were the anterior medial parts of the bilateral VOTC, including the poste- rior parahippocampal gyrus, anterior medial fusiform gyrus, and middle/posterior lateral parts of the bilateral VOTC, including the posterior parts of the middle/inferior temporal regions. Comparing results from connectional and functional fingerprint analyses The maps of voxels showing various degrees of similarity be- tween the blind and the sighted subject were highly similar in terms of RSFC and category response patterns (Fig. 4A). The bilateral medial parts of anterior temporal cortex, including the parahippocampal and anterior medial fusiform gyri, and the bilateral posterior parts of lateral temporal cortex, includ- ing the posterior middle and inferior temporal gyri, showed 12552 • J. Neurosci., September 9, 2015 • 35(36):12545–12559 Wang et al. • Function and Connection of Blind Visual Cortex Figure 3. Comparison of the functional fingerprints of the VOTC voxels in the sighted visual and auditory experiments. A, Original map of Pearson correlation coefficients of category response patterns between visual and auditory modalities. Color bar represents the Pearson r value. Warmer colors represent greater between-modality similarity. B, Percentile rank map of Pearson correlation coefficients of category response patterns between visual and auditory modalities. Color bar represents the percentile rank value. Warmer colors represent greater between-modality similarity.Blackarrowsinthecolorbarindicatethebottomandthetop15%similarity.C,Brainmapindicatingthesignificanceleveleachvoxelreachedforthedifferencebetweenwithin-modality similarity and between-modality similarity of category response patterns. The contours of bilateral fusiform gyrus (solid black lines), bilateral parahippocampal gyrus (dashed black lines), and bilateral inferior temporal gyrus (dash-dot black lines) are imposed on the brain surface for a clear reference of significant anatomical locations. Figure 3. Comparison of the functional fingerprints of the VOTC voxels in the sighted visual and auditory experiments. A, Original map of Pearson correlation coefficients of category response patterns between visual and auditory modalities. Color bar represents the Pearson r value. Warmer colors represent greater between-modality similarity. B, Percentile rank map of Pearson correlation coefficients of category response patterns between visual and auditory modalities. Color bar represents the percentile rank value. Warmer colors represent greater between-modality similarity.Blackarrowsinthecolorbarindicatethebottomandthetop15%similarity.C,Brainmapindicatingthesignificanceleveleachvoxelreachedforthedifferencebetweenwithin-modality similarity and between-modality similarity of category response patterns. The contours of bilateral fusiform gyrus (solid black lines), bilateral parahippocampal gyrus (dashed black lines), and bilateral inferior temporal gyrus (dash-dot black lines) are imposed on the brain surface for a clear reference of significant anatomical locations. terns. The distribution of voxels showing various degrees of similarity of category response patterns between blind and sighted auditory experiments (i.e., across groups with differ- ent visual experiences) were highly similar with those between visual and auditory experiments within the sighted group (i.e., across different task input modality in the same subject group) (Fig. 4B). Comparing results from connectional and functional fingerprint analyses The bilateral medial parts of anterior temporal cor- tex, including the parahippocampal gyrus and anterior medial fusiform gyrus, and the bilateral posterior parts of lateral temporal cortex, including the posterior middle and inferi- or temporal gyrus, showed the greatest similarity (top 15%) of task-induced object category response patterns between the blind and sighted auditory experiments, as well as between visual and auditory experiments within the sighted group (Fig. 4B, top row of the left panel, red patches). Bilateral posterior occipital regions, with the left-side cluster extending anteri- orly to the left fusiform gyrus, showed the least similarity (bottom 15%) of task-induced object category response pat- terns both between the blind and sighted auditory experi- ments and between the visual and auditory experiments within the sighted group (Fig. 4B, bottom row of the left panel, purple patches). There was a high correlation (Pearson r 0.71) between the between-group similarity and the between- modality similarity of category response patterns across the the greatest similarity (top 15%) between the blind and sighted groups for both category response patterns and RSFC patterns (Fig. 4A, top row of the left panel, red patches). Be- low, these regions will be considered to be “polymodal.” Bilat- eral posterior occipital regions and a small region in the left posterior lateral fusiform gyrus showed the least similarity (bottom 15%) in both category response patterns and RSFC patterns (Fig. 4A, bottom row of the left panel, purple patches) and will be considered to be “visual” regions. There was a positive correlation (Pearson r 0.56) between the between-group RSFC pattern similarity and the between- group category response profile similarity across the VOTC mask. As can be seen from the scatter plot (Fig. 4A, right panel), the coupling between the functional response pattern and the intrinsic functional connectivity pattern seemed to be a general pattern across the VOTC rather than driven by a few functionally specific clusters. We also examined the relationship between the category response patterns in VOTC in two types of visual input anal- yses: the between-group similarity (blind and sighted auditory experiments), which reflects how visual input across life af- fects response patterns, and the between-modality similarity (sighted visual and auditory experiments), which measures how visual input during the task setting affects response pat- J. Neurosci., September 9, 2015 • 35(36):12545–12559 • 12553 Wang et al. • Function and Connection of Blind Visual Cortex Figure 4. Comparing results from connectional and functional fingerprint analyses Top row of left panel, Voxels with the highest percentile rank (85) of between-group category response pattern similarity (yellow) overlap with voxels showing highest percentile rank (85) of between- modalitycategoryresponsepatternsimilarity(cyan),overlappingvoxelsinred.Bottomrowofleftpanel,Voxelswiththelowestpercentilerank(15)ofbetween-groupcategoryresponsepattern similarity (blue) overlap with voxels with the lowest percentile rank (15) of between-modality similarity of category response (green) patterns, with overlapping voxels in purple. Right panel, Scatterplotofthecorrelationbetweenbetween-modalitycategoryresponsepatternsimilarity( y-axis,Fisher-transformedrvalues)andbetween-groupcategoryresponsepatternsimilarity(x-axis, Fisher-transformedrvalues).Thecontoursofbilateralfusiformgyrus(solidblacklines),bilateralparahippocampalgyrus(dashedblacklines),andbilateralinferiortemporalgyrus(dash-dotblack lines) are imposed on the brain surface for a clear reference of significant anatomical locations. VOTC mask. These results show that the effect of visual input through life and the effect of visual input in task settings mod- ulate VOTC voxels responses similarly. global signal removal. Polymodal voxels, those exhibiting high between-group similarities in terms of both RSFC patterns and cat- egory response patterns, were in bilateral medial parts of anterior temporalcortexandlateralpartsofposteriortemporalcortex.Visual voxels,thoseexhibitingthelowestbetween-groupsimilarityinterms of both RSFC patterns and category response patterns, were in the posterior occipital cortex. There was a positive correlation (Pearson r 0.44) between the between-group RSFC pattern similarity and Comparing results from connectional and functional fingerprint analyses Relationship between connectional fingerprints similarity and functional fingerprints similarity, and between two types of visual input effects on functional fingerprints similarity, in VOTC. A, Relationship between the between-group similarity of RSFC patterns and between-group similarity of category response patterns in VOTC. Top row of left panel, Voxels with the highest percentilerank(85)ofbetween-groupRSFCpatternsimilarity(cyan)overlapwithvoxelsshowinghighestpercentilerank(85)ofbetween-groupcategoryresponsepatternsimilarity(yellow), with overlapping voxels in red. Bottom row of left panel, Voxels with the lowest percentile rank (15) of between-group RSFC pattern similarity (green) overlap with voxels with the lowest percentilerank(15)ofbetween-groupsimilarityofcategoryresponsepatterns(blue),withoverlappingvoxelsinpurple.Rightpanel,Scatterplotofthecorrelationbetweenbetween-groupRSFC patternsimilarity( y-axis,Fisher-transformedRvalues)andbetween-groupcategoryresponsepatternsimilarity(x-axis,Fisher-transformedRvalues).B,Relationshipbetweencategoryresponse pattern similarities between groups (blind and sighted auditory experiments) and between task input modalities (visual and auditory experiments within sighted group) in VOTC. Top row of left Figure 4. Relationship between connectional fingerprints similarity and functional fingerprints similarity, and between two types of visual input effects on functional fingerprints similarity, in VOTC. A, Relationship between the between-group similarity of RSFC patterns and between-group similarity of category response patterns in VOTC. Top row of left panel, Voxels with the highest percentilerank(85)ofbetween-groupRSFCpatternsimilarity(cyan)overlapwithvoxelsshowinghighestpercentilerank(85)ofbetween-groupcategoryresponsepatternsimilarity(yellow), with overlapping voxels in red. Bottom row of left panel, Voxels with the lowest percentile rank (15) of between-group RSFC pattern similarity (green) overlap with voxels with the lowest percentilerank(15)ofbetween-groupsimilarityofcategoryresponsepatterns(blue),withoverlappingvoxelsinpurple.Rightpanel,Scatterplotofthecorrelationbetweenbetween-groupRSFC patternsimilarity( y-axis,Fisher-transformedRvalues)andbetween-groupcategoryresponsepatternsimilarity(x-axis,Fisher-transformedRvalues).B,Relationshipbetweencategoryresponse pattern similarities between groups (blind and sighted auditory experiments) and between task input modalities (visual and auditory experiments within sighted group) in VOTC. Top row of left panel, Voxels with the highest percentile rank (85) of between-group category response pattern similarity (yellow) overlap with voxels showing highest percentile rank (85) of between- modalitycategoryresponsepatternsimilarity(cyan),overlappingvoxelsinred.Bottomrowofleftpanel,Voxelswiththelowestpercentilerank(15)ofbetween-groupcategoryresponsepattern similarity (blue) overlap with voxels with the lowest percentile rank (15) of between-modality similarity of category response (green) patterns, with overlapping voxels in purple. Right panel, Scatterplotofthecorrelationbetweenbetween-modalitycategoryresponsepatternsimilarity( y-axis,Fisher-transformedrvalues)andbetween-groupcategoryresponsepatternsimilarity(x-axis, Fisher-transformedrvalues).Thecontoursofbilateralfusiformgyrus(solidblacklines),bilateralparahippocampalgyrus(dashedblacklines),andbilateralinferiortemporalgyrus(dash-dotblack lines) are imposed on the brain surface for a clear reference of significant anatomical locations. Figure 4. Relationship between connectional fingerprints similarity and functional fingerprints similarity, and between two types of visual input effects on functional fingerprints similarity, in VOTC. A, Relationship between the between-group similarity of RSFC patterns and between-group similarity of category response patterns in VOTC. Top row of left panel, Voxels with the highest percentilerank(85)ofbetween-groupRSFCpatternsimilarity(cyan)overlapwithvoxelsshowinghighestpercentilerank(85)ofbetween-groupcategoryresponsepatternsimilarity(yellow), with overlapping voxels in red. Bottom row of left panel, Voxels with the lowest percentile rank (15) of between-group RSFC pattern similarity (green) overlap with voxels with the lowest percentilerank(15)ofbetween-groupsimilarityofcategoryresponsepatterns(blue),withoverlappingvoxelsinpurple.Rightpanel,Scatterplotofthecorrelationbetweenbetween-groupRSFC patternsimilarity( y-axis,Fisher-transformedRvalues)andbetween-groupcategoryresponsepatternsimilarity(x-axis,Fisher-transformedRvalues).B,Relationshipbetweencategoryresponse pattern similarities between groups (blind and sighted auditory experiments) and between task input modalities (visual and auditory experiments within sighted group) in VOTC. We validated these results with five additional analyses. Two-way mixed-design ANOVAs were per- formed on the functional responses in these two clusters, with object category as within-group factor and subject group as between-group factor. A significant main effect of category was observed in both clusters (left: F(7,189) 18.74, p 10 18; right: F(8,222) 13.55, p 10 15; Greenhouse-Geisser corrected). Post hoc comparisons revealed that both clusters exhibited signifi- cantly stronger responses to daily scenes and furniture compared with the average of the responses to the remaining 15 object categories (Fs(1,27) 30.12, Bonferroni corrected, ps 0.05). The main effect of group and the interaction between category and group were not significant in either cluster (ps 0.07). For the bilateral posterior lateral temporal cortex, the left (Fig. 6A) had significant positive connections (FDR corrected, p 0.05) with abutting middle and inferior temporal gyrus, bilateral inferior and middle frontal cortex, right superior frontal cortex, left pre- central gyrus, bilateral superior and inferior parietal lobule, and adjacent postcentral gyrus in both the blind and the sighted groups. Two-way mixed-design ANOVA identified signifi- cant main effects of category (F(8,223) 11.53, p 10 13, Greenhouse-Geisser corrected) as well as group (F(1,27) 6.43, p 0.05) in this region, although the interaction was not signif- icant (F(8,223) 0.56, p 0.83, Greenhouse-Geisser corrected). Post hoc comparisons revealed that this cluster showed signifi- cantly stronger responses to body parts, face parts, and tools com- pared with the average of the responses to the remaining 15 object 4. To control for age differences, we performed an additional analysis on resting-state data of 7 sighted subjects who were age- matched with the congenitally blind subjects. Again, we found a very similar pattern of results. Voxels exhibiting high between- group similarity of RSFC patterns located in bilateral medial parts of anterior temporal cortex and lateral parts of posterior tempo- ral cortex, whereas voxels exhibiting low between-group similar- ity of RSFC patterns were in the posterior occipital cortex. A positive correlation (Pearson r 0.45) was again observed be- tween the between-group RSFC pattern similarity and the between-group category response profile similarity across the VOTC mask. 5. We performed group classification based on the functional or connectional fingerprints using a linear SVM (LIBSVM: http://www.csie.ntu.edu.tw/~cjlin/libsvm) with standard pa- rameters to confirm the characteristics of the visual (Fig. 4A, purple patches in the bottom row) and polymodal (Fig. We validated these results with five additional analyses. When the between-group differences (indexed by the absolute value of effect size) in terms of gray matter probability of each voxel were included as a covariate, the partial correlation between the between-group RSFC pattern similarity and the between- group category response profile similarity across the VOTC mask was still high (Pearson r 0.53), indicating that the results were not driven by cross-voxel variations of gray matter density differ- ences between blind and sighted subjects. To plot the RSFC maps, for each cluster, one-sample t tests were conducted separately for the blind and sighted groups on the RSFC strengths between the cluster and each of the 180 re- gions covering the whole brain. The t values were then mapped onto each region of the brain for a direct visual inspection of the RSFC patterns in the blind and sighted groups. To depict the functional fingerprints (category response profiles) for each clus- ter, values of activation strength to the 16 object categories in the auditory experiments were extracted and plotted. 3. We ruled out possible head motion confounds by perform- ing the analysis using head-motion scrubbed resting-state data (see Materials and Methods). The overall results remained stable. Bilateral medial parts of anterior temporal cortex and lateral parts of posterior temporal cortex exhibited high between-group sim- ilarity in terms of both RSFC patterns and category response patterns, whereas voxels in the posterior occipital cortex exhib- ited low between-group similarity in terms of both RSFC patterns and category response patterns. There was again a positive corre- lation (Pearson r 0.42) between the between-group RSFC pat- tern similarity and the between-group category response profile similarity across the VOTC mask. y As shown in Figures 5 and 6, the whole-brain RSFC patterns as well as the category response patterns (in the auditory experi- ments) for the bilateral anterior medial temporal clusters and the bilateral posterior lateral temporal clusters were highly similar between the congenitally blind and sighted groups. Bilateral an- terior medial temporal clusters (Fig. 5) were significantly con- nected (FDR corrected, p 0.05) with bilateral precuneus and posterior cingulate cortex and adjacent calcarine and lingual re- gions, the ventral medial and anterior temporal cortex, middle occipital gyrus, and medial prefrontal cortex in both the blind and sighted groups. We validated these results with five additional analyses. A significant main effect of category was observed in both clusters (left: F(7,189) 18.74, p 10 18; right: F(8,222) 13.55, p 10 15; Greenhouse-Geisser corrected). Post hoc comparisons revealed that both clusters exhibited signifi- cantly stronger responses to daily scenes and furniture compared with the average of the responses to the remaining 15 object categories (Fs(1,27) 30.12, Bonferroni corrected, ps 0.05). The main effect of group and the interaction between category and group were not significant in either cluster (ps 0.07). For the bilateral posterior lateral temporal cortex, the left (Fig. 6A) had significant positive connections (FDR corrected, p 0.05) with abutting middle and inferior temporal gyrus, bilateral inferior and middle frontal cortex, right superior frontal cortex, left pre- central gyrus, bilateral superior and inferior parietal lobule, and able 3. ROI-based SVM results Left posterior occipital Right posterior occipital Left posterior fusiform Left anterior medial temporal Right anterior medial temporal Left posterior lateral temporal Right posterior lateral temporal lassification accuracy (p value) RSFC 93.48% (0.001) 93.48% (0.001) 78.26% (0.017) 69.57% (0.198) 60.87% (0.601) 69.57% (0.188) 76.09% (0.053) Category response 75.86% (0.027) 79.31% (0.015) 55.17% (0.356) 48.28% (0.548) 48.28% (0.566) 34.48% (0.889) 65.52% (0.120) the between-group category response profile similarity across the VOTC mask. between-group similarity in both RSFC patterns and category re- sponse patterns; Fig. 4A, red patches in the top row) and visual re- Table 3. ROI-based SVM results Left posterior occipital Right posterior occipital Left posterior fusiform Left anterior medial temporal Right anterior medial temporal Left posterior lateral temporal Right posterior lateral temporal Classification accuracy (p value) RSFC 93.48% (0.001) 93.48% (0.001) 78.26% (0.017) 69.57% (0.198) 60.87% (0.601) 69.57% (0.188) 76.09% (0.053) Category response 75.86% (0.027) 79.31% (0.015) 55.17% (0.356) 48.28% (0.548) 48.28% (0.566) 34.48% (0.889) 65.52% (0.120) between-group similarity in both RSFC patterns and category re- sponse patterns; Fig. 4A, red patches in the top row) and visual re- gions (showing the lowest percentile rankings, 15%, of between- group similarity in both RSFC patterns and category response patterns; Fig. 4A, purple patches in the bottom row) identified above.Inthecurrentanalyses,thetimeseries(inRSFCanalyses)and values (in category response analyses) were averaged across all voxels within each region for exploration and visualization pur- poses.Itshouldbenotedthattheseanalysesdonotconsiderpossibly heterogeneous subregions within the clusters. 2. We considered the possible influence of gray matter density. We validated these results with five additional analyses. 4A, red patches in the top row) clusters we identified above. Results (Ta- ble 3) showed that the bilateral posterior occipital “visual” clus- ters could successfully discriminate between blind and sighted groups based on both the connectional and functional finger- prints, the left posterior fusiform cluster distinguished success- fully the blind from sighted group based on the connectional fingerprints but not the functional fingerprints. In contrast, the temporal “polymodal” clusters could not successfully discrimi- nate between groups based on connectional fingerprints or based on functional fingerprints. Thus, the ROI validation analyses confirmed the “visual” and “polymodal” characteristics of these VOTC clusters we identified. We validated these results with five additional analyses. We validated these results with five additiona 1.WecalculatedtheRSFCstrengthswithoutremovingtheglobal signal. The overall pattern of results was largely similar to those with Wang et al. • Function and Connection of Blind Visual Cortex Wang et al. • Function and Connection of Blind Visual Cortex 12554 • J. Neurosci., September 9, 2015 • 35(36):12545–12559 Wang et al. • Function and Connection of Blind Visual Cortex he between-group category response profile similarity across the VOTC mask. 2. We considered the possible influence of gray matter density. When the between-group differences (indexed by the absolute value of effect size) in terms of gray matter probability of each voxel were included as a covariate, the partial correlation between he between-group RSFC pattern similarity and the between- group category response profile similarity across the VOTC mask was still high (Pearson r 0.53), indicating that the results were not driven by cross-voxel variations of gray matter density differ- ences between blind and sighted subjects. 3. We ruled out possible head motion confounds by perform- ng the analysis using head-motion scrubbed resting-state data see Materials and Methods). The overall results remained stable. Bilateral medial parts of anterior temporal cortex and lateral parts of posterior temporal cortex exhibited high between-group sim- larity in terms of both RSFC patterns and category response patterns, whereas voxels in the posterior occipital cortex exhib- ted low between-group similarity in terms of both RSFC patterns and category response patterns. There was again a positive corre- ation (Pearson r 0.42) between the between-group RSFC pat- ern similarity and the between-group category response profile imilarity across the VOTC mask. 4. To control for age differences, we performed an additional analysis on resting-state data of 7 sighted subjects who were age- matched with the congenitally blind subjects. Again, we found a very similar pattern of results. Voxels exhibiting high between- group similarity of RSFC patterns located in bilateral medial parts of anterior temporal cortex and lateral parts of posterior tempo- al cortex, whereas voxels exhibiting low between-group similar- ty of RSFC patterns were in the posterior occipital cortex. A positive correlation (Pearson r 0.45) was again observed be- ween the between-group RSFC pattern similarity and the between-group category response profile similarity across the VOTC mask. 5. We performed group classification based on the functional or connectional fingerprints using a linear SVM (LIBSVM: http://www.csie.ntu.edu.tw/~cjlin/libsvm) with standard pa- ameters to confirm the characteristics of the visual (Fig. We validated these results with five additional analyses. 4A, purple patches in the bottom row) and polymodal (Fig. 4A, red patches in the top row) clusters we identified above. Results (Ta- ble 3) showed that the bilateral posterior occipital “visual” clus- ers could successfully discriminate between blind and sighted groups based on both the connectional and functional finger- prints, the left posterior fusiform cluster distinguished success- ully the blind from sighted group based on the connectional ingerprints but not the functional fingerprints. In contrast, the between-group similarity in both RSFC patterns and category re- sponse patterns; Fig. 4A, red patches in the top row) and visual re- gions (showing the lowest percentile rankings, 15%, of between- group similarity in both RSFC patterns and category response patterns; Fig. 4A, purple patches in the bottom row) identified above.Inthecurrentanalyses,thetimeseries(inRSFCanalyses)and values (in category response analyses) were averaged across all voxels within each region for exploration and visualization pur- poses.Itshouldbenotedthattheseanalysesdonotconsiderpossibly heterogeneous subregions within the clusters. To plot the RSFC maps, for each cluster, one-sample t tests were conducted separately for the blind and sighted groups on the RSFC strengths between the cluster and each of the 180 re- gions covering the whole brain. The t values were then mapped onto each region of the brain for a direct visual inspection of the RSFC patterns in the blind and sighted groups. To depict the functional fingerprints (category response profiles) for each clus- ter, values of activation strength to the 16 object categories in the auditory experiments were extracted and plotted. As shown in Figures 5 and 6, the whole-brain RSFC patterns as well as the category response patterns (in the auditory experi- ments) for the bilateral anterior medial temporal clusters and the bilateral posterior lateral temporal clusters were highly similar between the congenitally blind and sighted groups. Bilateral an- terior medial temporal clusters (Fig. 5) were significantly con- nected (FDR corrected, p 0.05) with bilateral precuneus and posterior cingulate cortex and adjacent calcarine and lingual re- gions, the ventral medial and anterior temporal cortex, middle occipital gyrus, and medial prefrontal cortex in both the blind and sighted groups. Two-way mixed-design ANOVAs were per- formed on the functional responses in these two clusters, with object category as within-group factor and subject group as between-group factor. Figure6. Connectionalfingerprintsandfunctionalfingerprints(categoryresponseintheauditoryexperiments)ofthe(A)leftand(B)rightposteriorlateraltemporalclusters.Colorscalesinthe RSFC maps represent the strengths of RSFC between regions (t values). The radial distance in the functional fingerprints reflects the activation strength ( values). Figure6. Connectionalfingerprintsandfunctionalfingerprints(categoryresponseintheauditoryexperiments)ofthe(A)leftand(B)rightposterio RSFC maps represent the strengths of RSFC between regions (t values). The radial distance in the functional fingerprints reflects the activation strengt blind and sighted groups were rather different (Fig. 7). One- sample t tests showed that the posterior occipital clusters were significantly connected with the precentral and postcentral cortex and superior temporal regions in the sighted group; whereas in the blind group, they exhibited significant func- tional connectivity with the left inferior/middle frontal cortex and adjacent precentral gyrus (both FDR corrected, ps 0.05). The left posterior lateral fusiform cluster was signifi- cantly connected with vast regions in the bilateral occipital and ventral temporal cortex and regions in the bilateral pre- central and postcentral cortex and supplementary motor areas in the sighted group (FDR corrected, p 0.05). For the blind group, the left posterior lateral fusiform cluster was signifi- cantly connected with bilateral Heschl’s gyrus and superior temporal regions corresponding to the primary and secondary auditory cortices, bilateral superior parietal regions, postcen- tral and precentral cortices and supplementary motor areas, categories (Fs(1,27) 16.95, Bonferroni corrected, ps 0.05). The right posterior lateral temporal cluster (Fig. 6B) was significantly positively connected (FDR corrected, p 0.05) with the bilateral inferior and middle temporal gyrus, inferior and middle frontal cortex, supplementary motor area, precentral and postcentral cortex, and inferior/superior parietal cortex in both the sighted and the blind groups. A significant main effect of category was identified by the two-way mixed-design ANOVA (F(15,405) 8.08, p 10 15). Post hoc comparisons revealed that the right posterior lateral temporal cluster showed significantly stronger responses to body parts, face parts, and clothing compared with the average of the responses to the remaining 15 object categories (Fs(1,27) 13.04, Bonferroni corrected, ps 0.05). The main effect of group and the interaction between category and group were not significant (ps 0.22). categories (Fs(1,27) 16.95, Bonferroni corrected, ps 0.05). The right posterior lateral temporal cluster (Fig. 6B) was significantly positively connected (FDR corrected, p 0.05) with the bilateral inferior and middle temporal gyrus, inferior and middle frontal cortex, supplementary motor area, precentral and postcentral cortex, and inferior/superior parietal cortex in both the sighted and the blind groups. Characterization of visual and polymodal regions In this section, we map the connectional and functional fingerprints of the polymodal (showing the highest percentile rankings, 85, of J. Neurosci., September 9, 2015 • 35(36):12545–12559 • 12555 Wang et al. • Function and Connection of Blind Visual Cortex Figure5. Connectionalfingerprintsandfunctionalfingerprints(categoryresponseintheauditoryexperiments)ofthe(A)leftand(B)rightanteriormedialtemporalclusters.Colorscalesinthe RSFC maps represent the strengths of RSFC between regions (t values). The radial distance in the functional fingerprints reflects the activation strength ( values). Figure5. Connectionalfingerprintsandfunctionalfingerprints(categoryresponseintheauditoryexperiments)ofthe(A)leftand(B)rightanteriormedialtemporalclusters.Colorscalesinthe RSFC maps represent the strengths of RSFC between regions (t values). The radial distance in the functional fingerprints reflects the activation strength ( values). Figure6. Connectionalfingerprintsandfunctionalfingerprints(categoryresponseintheauditoryexperiments)ofthe(A)leftand(B)rightposteriorlateraltemporalclusters.Colorscalesinthe RSFC maps represent the strengths of RSFC between regions (t values). The radial distance in the functional fingerprints reflects the activation strength ( values). Discussion We mapped out the extent to which visual experience modulates connectional and functional fingerprints of VOTC by comparing RSFC patterns and category response profiles between congeni- tally blind and sighted subjects. Large-scale continuous maps of functional and connectional similarity revealed which parts of VOTC are shaped by visual input/experience and which parts are not. We identified highly “visual” areas in bilateral posterior oc- cipital and left posterior lateral fusiform clusters, where RSFC patterns and object category responses were significantly differ- ent between congenitally blind and sighted individuals, and dif- ferent between sighted subjects performing visual and auditory tasks. We also revealed “polymodal” regions in bilateral anterior medial temporal clusters and posterior lateral temporal clusters, where highly similar functional and connectivity patterns be- tween sighted and blind individuals were obtained. Regions with medium levels of visual dependency were intermixed. The effects of visual input in shaping occipital and part of the posterior lateral fusiform regions are in line with previous studies showing plastic changes in this territory. Studies on visually de- prived animals have identified functional and structural reorga- nization in occipital visual cortices (Hubel et al., 1977; Hyva¨rinen et al., 1981; Price et al., 1994). In humans, cross-modal activation has been robustly reported in occipital visual cortices in blind individuals when performing nonvisual tasks (for reviews, see Noppeney, 2007; Ricciardi et al., 2014). We here not only ob- served stronger activation in blind subjects in the auditory exper- iments relative to the sighted group but also showed that the categorical response patterns are different between blind and sighted, further confirming the visual characteristic of these pos- terior regions and indicating that the cross-modal reorganization does not follow a simple additive pattern (compare Lewis et al., 2010). Studies on RSFC have found that visual deprivation is associated with reduced connectivity within the occipital cortices and between the occipital cortex and other primary sensory/mo- tor regions, and with increased connectivity between occipital cortices and inferior frontal cortices (Liu et al., 2007; Butt et al., 2013; Burton et al., 2014; for review, see Bock and Fine, 2014). We here showed that in sighted, but not in congenitally blind, poste- rior occipital regions are significantly synchronized with post- central/precentral cortices and superior temporal regions; whereas in congenitally blind, but not in sighted, posterior occip- ital regions are functionally connected with left inferior frontal cortex. A significant main effect of category was identified by the two-way mixed-design ANOVA (F(15,405) 8.08, p 10 15). Post hoc comparisons revealed that the right posterior lateral temporal cluster showed significantly stronger responses to body parts, face parts, and clothing compared with the average of the responses to the remaining 15 object categories (Fs(1,27) 13.04, Bonferroni corrected, ps 0.05). The main effect of group and the interaction between category and group were not significant (ps 0.22). blind and sighted groups were rather different (Fig. 7). One- sample t tests showed that the posterior occipital clusters were significantly connected with the precentral and postcentral cortex and superior temporal regions in the sighted group; whereas in the blind group, they exhibited significant func- tional connectivity with the left inferior/middle frontal cortex and adjacent precentral gyrus (both FDR corrected, ps 0.05). The left posterior lateral fusiform cluster was signifi- cantly connected with vast regions in the bilateral occipital and ventral temporal cortex and regions in the bilateral pre- central and postcentral cortex and supplementary motor areas in the sighted group (FDR corrected, p 0.05). For the blind group, the left posterior lateral fusiform cluster was signifi- cantly connected with bilateral Heschl’s gyrus and superior temporal regions corresponding to the primary and secondary auditory cortices, bilateral superior parietal regions, postcen- tral and precentral cortices and supplementary motor areas, For the bilateral posterior occipital and left posterior lateral fusiform clusters, the whole-brain RSFC patterns between Wang et al. • Function and Connection of Blind Visual Cortex 12556 • J. Neurosci., September 9, 2015 • 35(36):12545–12559 Figure7. RSFCpatternmapsandfunctionalfingerprints(categoryresponseintheauditoryexperiments)ofthe(A)left,(B)rightposterioroccipital,and(C)posteriorlateralfusiformvisualregions (lowbetween-groupsimilarityofbothRSFCpatternsandcategoryresponsepatterns).ColorscalesintheRSFCmapsrepresentthestrengthsofRSFCbetweenregions(tvalues).Theradialdistance in the functional fingerprints reflects the activation strength ( values). Figure7. RSFCpatternmapsandfunctionalfingerprints(categoryresponseintheauditoryexperiments)ofthe(A)left,(B)rightposterioroccipital,and(C)posteriorlateralfusiformvisualregions (lowbetween-groupsimilarityofbothRSFCpatternsandcategoryresponsepatterns).ColorscalesintheRSFCmapsrepresentthestrengthsofRSFCbetweenregions(tvalues).Theradialdistance in the functional fingerprints reflects the activation strength ( values). and small parts of the right inferior frontal cortex (FDR cor- rected, p 0.05). Two-way mixed-design ANOVAs showed a significant main effect of group in all three clusters (left pos- terior lateral FG: F(1,27) 8.84, p 0.006; left posterior occip- ital: F(1,27) 9.07, p 0.006; right posterior occipital: F(1,27) 11.34, p 0.002), with blind subjects exhibiting stronger overall activity than sighted subjects. The main effect of cate- gory was not significant (ps 0.24), but there was a significant interaction between group and category in the left posterior fusiform cluster (F(15,405) 1.70, p 0.048). Further analyses revealed that only the responses to fish, flower, musical instru- ment, and reptile categories were significantly stronger in con- genitally blind relative to sighted groups (ts(27) 3.26, Bonferroni corrected, ps 0.05). variable plasticity reflecting the degree to which they are typically programmed to process visual or polymodal information (or computation in the metamodal hypothesis; e.g., Pascual-Leone and Hamilton, 2001). The coupling between a region’s functional profile and its intrin- sic connectional fingerprint observed in this study corroborates a previous finding that selectivity to faces in VOTC is accurately pre- dicted by voxelwise patterns of whole-brain structural connectivity patterns(Sayginetal.,2012).Ourstudyextendsthisfindingtofunc- tional connectivity and to categories other than faces, and most im- portantly, showed that drastic changes in visual experience affect local activity and functional connectivity patterns together. These results are in accord with the general hypothesis that local activity is determined by large-scale connectivity patterns (Passingham et al., 2002; Mahon and Caramazza, 2009, 2011; Behrens and Sporns, 2012). Discussion In other words, typically the type of representation computed in early visual cortex by dedicated neural circuits may be intrinsic and exclusive to the visual modality, and this representation is therefore not directly recoverable from other sensory inputs. Thus, the plastic changes associated with congenital blindness become more drastic here, responding to language and auditory stimuli without similarity to the visual characteristics. An unex- pected hemispheric asymmetry was present in the occipital cor- tex: the between-group similarities of both the connectional and functional fingerprints were more prominent in the right hemi- sphere than the left (Figs. 1, 2), perhaps due to more extensive reorganization in the left occipital cortex (Raz et al., 2005; Bedny et al., 2011; Watkins et al., 2012; Butt et al., 2013). The transition between visual and polymodal cortical regions appears to be continuous rather than discrete. The cortical re- gions with medium levels of visual dependence may contain neu- rons that are more strongly tuned to visual input, but they are also modulated by nonvisual input. Alternatively, they may contain a mixture of visual neurons and polymodal neurons that is not distinguishable at the current spatial scale. In conclusion, by comparing congenitally blind and sighted individuals, we provide a large-scale mapping of the degree to which connectional and functional fingerprints of the “visual” cortex depend on visual experience/input. In addition to reveal- ing regions that are strongly dependent on visual experience (early visual cortex and posterior fusiform gyrus), our results point to regions in which connectional and functional patterns are surprisingly similar in blind and sighted individuals (anterior medial and posterior lateral parts of the VOTC). Although there is general consensus that representations become more “high- level” and more “abstract” as one moves anteriorly from the primary visual system, our results show where exactly such tran- sitions happen. Finally, our results help define the limits of plas- ticity: plasticity is maximal in those regions that are most strongly modality-specific. In the absence of relevant input, these regions may become “available” for alternative functions. The other major result concerns the bilateral anterior medial temporal regions and posterior lateral temporal regions, where highly similar patterns of intrinsic functional connectivity and response preferences for various types of objects were observed between congenitally blind and sighted subjects, as well as similar categorical response patterns between visual and auditory verbal input within sighted subjects. Discussion These findings are consistent with previous studies and may underlie the recruitment of this occipital region for verbal The first result to emphasize is the convergence between the connectional fingerprints and the two types of functional finger- prints, strengthening confidence in the generality of our findings. We also found strong convergence between the effect of visual input during the task and the effect of visual input during life. The map of similarity between input modalities for sighted individu- als (sighted visual experiment and auditory experiment) was sim- ilar to that of between-subject groups with and without visual experience (blind auditory experiment and sighted auditory ex- periment). This correspondence invites the inference that a uni- tary explanation may be appropriate for the modality effect within the sighted group and for the visual experience effect across groups. One possibility is that regions of VOTC exhibit J. Neurosci., September 9, 2015 • 35(36):12545–12559 • 12557 Wang et al. • Function and Connection of Blind Visual Cortex (e.g., Bedny et al., 2011) or other higher cognitive functions in early blind adults. RSFC patterns of early visual cortex with other visual regions have been reported to be similar between groups (Burton et al., 2014; Striem-Amit et al., 2015), and the intriguing differences of RSFC patterns within the visual cortex and bet- ween early visual cortex and nonvisual regions warrant further investigation. cluding tactile and verbal input, such as objects’ shape (Peelen et al., 2014) or size (Konkle and Oliva, 2012; Konkle and Cara- mazza, 2013). Thus, these regions respond in similar ways when sighted subjects receive visual and nonvisual stimuli and when blind subjects receive verbal or tactile stimuli. Taking away inputs from one modality does not change the function of these regions perhaps because they can develop functional specificity through input from other modalities. g These findings are well explained by the assumption that neu- rons in early visual cortex are genetically programmed to process stimuli from the visual modality and represent characteristic fea- tures within this modality, such as orientation and spatial rela- tions. Stimuli presented in other modalities (e.g., auditory input) are not expressible in terms of those basic units because they depend on other stimulus properties, such as the temporal di- mension in this case. Even in the case of tactile input, where spatial relations are important, the construction of object repre- sentation requires integration of tactile features over time. References Amedi A, Raz N, Pianka P, Malach R, Zohary E (2003) Early “visual” cortex activation correlates with superior verbal memory performance in the blind. 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Medline We expect that the polymodal nature of the regions revealed here is closely related to the type of information and/or compu- tation these regions represent: information that is abstracted away from vision-specific properties is suitable for interaction with representations computed through other modalities, in- Burton H, Snyder AZ, Raichle ME (2014) Resting state functional connec- tivity in early blind humans. Front Syst Neurosci 8:51. Discussion For the bilateral anterior medial temporal regions, we observed that, for both blind and sighted subjects, the activity was strongest for scenes and furniture, converging with previous findings of selectivity for tactile exploration of scenes and verbal comprehension of large nonma- nipulable objects in the parahippocampal cortex of early blind adults (Wolbers et al., 2011; He et al., 2013). Furthermore, in both subject groups, these regions were functionally connected with bilateral precuneus and posterior cingulate cortex, ventral medial temporal cortex, and middle occipital gyrus. This connec- tivity pattern is in line with the proposal that this section of VOTC is part of a navigation network that processes scenes and large objects that may serve as landmarks (Epstein, 2008; He et al., 2013). The categorical response profile in bilateral posterior lat- eral temporal regions is less transparent. Here, relatively stronger activation was observed for diverse categories, including human face and body parts, clothing, and tools. One possibility is that these regions represent one or more properties that are common to these categories. Alternatively, these clusters may consist of voxels with diverse functional properties; our tests of visual de- pendence did not require voxels of a cluster to be functionally homogeneous. 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https://openalex.org/W2609992360	https://ejurnal.its.ac.id/index.php/teknik/article/download/22775/3633	Indonesian	null	Perancangan Aplikasi Berbasis Android untuk Pemeriksaan Pengecatan Kapal Bangunan Baru	Jurnal Teknik/Jurnal Teknik ITS	2,017	cc-by-sa	4,227	Perancangan Aplikasi Berbasis Android untuk Pemeriksaan Pengecatan Kapal Bangunan Baru Dandy Adrianto dan Triwilaswandio Wuruk Pribadi Departemen Teknik Perkapalan, Fakultas Teknologi Kelautan, Institut Teknologi Sepuluh Nopember (ITS) Jl. Arief Rahman Hakim, Surabaya 60111 Indonesia e-mail: triwilas.its@gmail.com Dandy Adrianto dan Triwilaswandio Wuruk Pribadi Perkapalan, Fakultas Teknologi Kelautan, Institut Teknologi Sepuluh Nopember (ITS) Jl. Arief Rahman Hakim, Surabaya 60111 Indonesia e-mail: triwilas.its@gmail.com pengetahuan literatur mengenai standart-standart yang harus dilakukan. Aspek pengalaman juga berperan penting dalam hal ini, oleh karena itu seorang inspector harus memiliki pengetahuan literatur yang baik diimbangi dengan pengalaman yang banyak pula. Hal tersebut yang menjadi permasalahan junior coating inspector. Abstrak— Proses pemeriksaan pengecatan kapal yang ada saat ini dilakukan secara manual dimana coating inspector dan pihak terkait lainnya yang melakukan pemeriksaan mengacu pada form pemeriksaan yang telah disetujui. Penginputan dan pelaporan hasil pemeriksaan dilakukan dengan menggunakan kertas yang ditulis secara manual sehingga tidak efektif dan terdapat resiko besar jika terjadi kehilangan dokumen karena tidak memiliki backup. Selain itu, proses pemeriksaan cat yang dilakukan kurang efektif dikarenakan antar pihak terkait yang melakukan pemeriksaan tidak memiliki pengetahuan dan pengalaman yang sama. Tujuan dari pengerjaan Studi ini adalah untuk merancang aplikasi berbasis android yang berfungsi sebagai sarana untuk memudahkan praktisi di lapangan dalam proses pemeriksaan dan dokumentasi pemeriksaan pengecatan kapal. Dalam pengerjaan Studi ini, pertama dilakukan observasi pada kegiatan pemeriksaan pengecatan kapal bangunan baru yang dilakukan oleh coating inspector di lapangan. Kemudian yang kedua adalah studi literatur tentang lingkup pekerjaan dari coating inspector, standart pemeriksaan dari tahap perisapan hingga final, dan standart untuk melaporkan hasil pemeriksaan cat. Setelah itu dilakukan perancangan aplikasi. Perancangan aplikasi dilakukan dengan pembuatan kerangka dasar aplikasi, diagram alir data dan entitas, mock up aplikasi, perancangan database, dan pengkodingan aplikasi yang dirancang. Pada aplikasi yang dirancang dapat dilakukan penginputan hasil pemeriksaan pada form pemeriksaan yang didalamnya terdapat batasan pemeriksaan yang telah ditentukan, review hasil pemeriksaan, penyimpanan hasil pemeriksaan dalam bentuk file, dan fitur search dokumen hasil pemeriksaan dan dokumen lain yang terkait. Uji coba aplikasi dilakukan kepada responden yang merupakan praktisi di lapangan yang melakukan pemeriksaan pengecatan kapal bangunan baru. Dari hasil pengujian kuisoner yang diperoleh dapat ditarik kesimpulan bahwa aplikasi yang dirancang perlu diaplikasikan sebagai sarana pendukung proses pemeriksaan pengecatan kapal bangunan baru. Tugas dari coating inspector belum selesai hingga dokumentasi proyek dan laporan (report) dikirimkan ke pihak owner dan direview oleh klas [1]. Dalam prosesnya, pembuatan laporan membutuhkan waktu yang relatif lama untuk penyelesaiannya karena berkas yang dibutuhkan tidak sedikit selain itu proses dokumentasi dan pelaporan dilakukan secara manual sehingga ada resiko terjadinya kehilangan laporan yang akan menghambat produktifitas dari proses pembangunan kapal tersebut. Perancangan Aplikasi Berbasis Android untuk Pemeriksaan Pengecatan Kapal Bangunan Baru Dandy Adrianto dan Triwilaswandio Wuruk Pribadi Departemen Teknik Perkapalan, Fakultas Teknologi Kelautan, Institut Teknologi Sepuluh Nopember (ITS) Jl. Arief Rahman Hakim, Surabaya 60111 Indonesia e-mail: triwilas.its@gmail.com Oleh karena itu dibutuhkan aplikasi berbasis android yang dapat digunakan untuk memberikan panduan pekerjaan inspeksi yang harus dilakukan meliputi surface preparation, mixing dan thinning, dan proses inspeksi lainnya sehingga diharapkan seorang coating inspector dapat bekerja sesuai standard dan menghindari adanya repair work akibat proses aplikasi yang salah. Dengan aplikasi ini seorang coating inspector dapat lebih mudah dalam melakukan penyusunan laporan karena di dalam aplikasi ini terdapat form standard yang dapat disisipi dengan keterangan atau gambar disertai standardnya sesuai inspeksi yang telah dilakukan dan dapat dikirimkan langsung melalui koneksi internet pada pihak owner, shipyard, klas dan pihak terkait lainnya. G-15 G-15 JURNAL TEKNIK ITS Vol. 6, No. 1, (2017) ISSN: 2337-3539 (2301-9271 Print) A. Pengecatan Kapal Bangunan Baru Kata Kunci— Aplikasi Android, Pengecatan Kapal, Pemeriksaan Pengecatan Kapal, Coating inspector. Kata Kunci— Aplikasi Android, Pengecatan Kapal, Pemeriksaan Pengecatan Kapal, Coating inspector. Pengecatan kapal bangunan baru dimulai dari material datang hingga kapal selesai dilakukan erection. Pengecatan kapal ini berguna untuk melindungi kulit kapal dari proses pengkaratan dan juga binatang laut, karena hampir semua material penyusun kapal adalah logam (pelat baja) dan daerah kerja kapal adalah di laut maka sifat logam (pelat baja) reaktif terhadap korosi. Sebelum melakukan pengerjaan pengecatan terlebih dahulu material yang akan dicat harus bersih dari kotoran-kotoran minyak maupun sisa-sisa cat dan debu. Gambar 1 menunjukkan urutan proses pengecatan kapal bangunan baru. B. Surface Preparation Surface preparation ini merupakan proses untuk menghilangkan semua kotoran dari permukaan material yang dapat mengganggu daya rekat coating atau dapat membuat coating mudah terkelupas. Selain menghilangkan kotoran, surface preparation juga berfungsi untuk menghilangkan karat, mill scale, surface defect seperti sisa pengelasan dan permukaan yang tajam yang dapat menyebabkan coating rusak [2]. A. Studi Literature Studi literatur yang dilakukan untuk mengerjakan studi ini terdiri dari: 1) Mempelajari item-item pekerjaan inspeksi yang dilakukan oleh coating inspector pada pembangunan kapal bangunan baru. 2) Alur inspeksi coating inspector. 2) Alur inspeksi coating inspector. 3) Mempelajari standard yang mengatur kegiatan inspeksi coating inspector. 4) Mempelajari proses dokumentasi dan report hasil inspeksi yang sesuai standard. III. METODOLOGI PENELITIAN Tabel 2. Item pemeriksaan surface preparation Nama Proses Item Pemeriksaan Surface Preparation (Blasting dan Power Tool) Kondisi Lingkungan:  Cek relative humidity  Cek dew point  Cek temperatur material Kontaminan Cleanliness grade Surface Profile Cek Dust level Cek Soluble Salt Tabel 2. D. Coating Inspector Gambar 1. Urutan proses pengecatan kapal Gambar 1. Urutan proses pengecatan kapal Coating Inspector memiliki tanggung jawab yang bervariasi dalam setiap pekerjaan. Peranan inspector adalah sebagai quality control technician yang memiliki tanggung jawab untuk melakukan observasi dan report tentang aspek teknis dari proyek coating dan untuk mengecek kesesuaian atau tidaknya project dari project specification [1]. Coating Inspector harus dapat menjamin bahwa project specification telah dilakukan dengan benar sesuai dengan yang dibuat. Selain itu, Coating Inspector harus mendokumentasikan hasil pemeriksaan. JURNAL TEKNIK ITS Vol. 6, No. 1, (2017) ISSN: 2337-3539 (2301-9271 Print) JURNAL TEKNIK ITS Vol. 6, No. 1, (2017) ISSN: 2337-3539 (2301-9271 Print) G-16 Raw Material Shop Primer Fabrikasi dan Assembly Rust Preventive Painting Subsequent Painting Final Inspection Primary Surface Preparation Secondary Surface Preparation Stripe Coat Tabel 3. Item pemeriksaan pengecatan kapal Nama Proses Item Pemeriksaan Painting Inspeksi Visual Dry Film thickness (DFT) Measurement Cleanliness dan Curing Time Tiap Coating Holiday detection D. Coating Inspector Raw Material Shop Primer Fabrikasi dan Assembly Rust Preventive Painting Subsequent Painting Final Inspection Primary Surface Preparation Secondary Surface Preparation Stripe Coat D Raw Material Shop Primer Fabrikasi dan Assembly Rust Preventive Painting Subsequent Painting Final Inspection Primary Surface Preparation Secondary Surface Preparation Stripe Coat D Tabel 3. Item pemeriksaan pengecatan kapal Nama Proses Item Pemeriksaan Painting Inspeksi Visual Dry Film thickness (DFT) Measurement Cleanliness dan Curing Time Tiap Coating Holiday detection Coating Inspector Fabrikasi dan Assembly E. Aplikasi Android Tabel 1. Item pemeriksaan pre-surface preparation Nama Proses Item Pemeriksaan Steel Surface Preparation (Pre- Surface Preparation) Inspeksi Visual:  Shrap edges  Miss weld  Weld defect  Imperfect steel surface Tabel 1. Item pemeriksaan pre-surface preparation Nama Proses Item Pemeriksaan Steel Surface Preparation (Pre- Surface Preparation) Inspeksi Visual:  Shrap edges  Miss weld  Weld defect  Imperfect steel surface Android merupakan system operasi berbasis Linux yang bersifat terbuka (open source) dan dirancang untuk perangkat seluler layar sentu seperti smartphone dan computer tablet [5]. Aplikasi Android dikembangkan dalam bahasa pemrograman Java dengan menggunakan kit pengembangan perangkat lunak Android (SDK). SDK ini terdiri dari seperangkat perkakas pengembangan, termasuk debugger, perpustakaan perangkat lunak, emulator handset yang berbasis QEMU, dokumentasi, kode sampel, dan tutorial. Didukung secara resmi oleh lingkungan pengembangan terpadu (IDE) Eclipse, yang menggunakan plugin Android Development Tools (ADT) Pada Tabel 1 dijelaskan item-item yang dilakukan pemeriksaan pada tahap pre-surface preparation [3]. Metode- metode surface preparation yang umum digunakan adalah solvent cleaning, abrasive blasting, waterjetting, hand tool cleaning, power tool cleaning, dan lain-lain. Pada Tabel 2 dijelaskan item-item pemeriksaan pada proses surface preparation [3]. C. Pengecatan Kapal 5) Mempelajari pengantar sistem informasi mengenai sistem database dan MySQL. Pengecatan kapal pada block stage umumnya dilakukan indoor dan kondisi lingkungan yang terkontrol [4]. Metode yang digunakan sesuai dengan spesifikasi kontrak yang disetujui. Metode pengecatan kapal dapat dilakukan dengan airless spray, roller, brush, dan conventional air spray. Pada Tabel 3 dijelaskan item-item pemeriksaan pengecatan kapal 6) Mempelajari sistem aplikasi android. 6) Mempelajari sistem aplikasi android. I. PENDAHULUAN A r D ALAM proses pembangunan kapal baru tidak terkecuali reparasi kapal terdapat pekerjaan coating di dalamnya. Coating merupakan tahapan yang harus dilalui pada proses pembangunan kapal. Tahapan ini harus dilakukan dengan benar karena penerapan coating yang salah berpengaruh pada umur cat dan biaya operasional kapal tersebut. Pekerjaan reparasi (repair work) sering terjadi pada setiap instalasi coating baru. Untuk menghindari coating failure dibutuhkan D E. Perancangan Aplikasi Android Tahap perancangan aplikasi ini antara lain: Tahap perancangan aplikasi ini antara lain: 1) Perancangan permodelan aplikasi Pembuatan entitiy relationship diagram, data flow diagram, dan system interface diagram. 2) Perancangan aplikasi Berikut merupakan proses perancangan aplikasi yang dilakukan: Tabel 4. Item pemeriksaan pengecatan kapal bangunan baru Tahapan Tahapan Item Pemeriksaan Tabel 4. Item pemeriksaan pengecatan kapal bangunan baru Tahapan Konstruksi Tahapan Pengecatan Item Pemeriksaan Fabrikasi (Raw material) Primary Surface Preparation  Cek relative humidity  Cek dew point  Cek temperatur permukaan material Kontaminan Cleanliness grade Surface Profile (Roughness) Cek Dust level Cek Soluble Salt Shop primer Application  Cek relative humidity  Cek dew point  Cek temperatur permukaan material Inspeksi Visual Dry Film thickness (DFT) Measurement Cleanliness dan Curing Time Tiap Coating Assembly (Block Stage) Steelwork Preparation Inspeksi Visual:  Shrap edges  Weld defect  Imperfect steel surface Secondary Surface Preparation  Cek relative humidity  Cek dew point  Cek temperatur permukaan material Kontaminan Cleanliness grade Cek Dust level Surface Profile (Roughness) Cek Soluble Salt Coating  Cek relative humidity a. Pembuatan mock up aplikasi. b. Perancangan interface atau tampilan antarmuka aplikasi. c. Perancangan database pemeriksaan pengecatan kapal bangunan baru. d. Pengcodingan aplikasi. 3) Pengujian Aplikasi a. Uji coba aplikasi pada praktisi di lapangan b. Uji kepuasan dalam penggunaan program dengan kuisoner c. Uji perbandingan kinerja aplikasi dengan sistem eksisting. C. Pengumpulan Data dilaksanakan. Coating Inspector Pemilik Kapal Badan Klasifikasi Kapal QC Galangan Owner Representative (Owner Surveyor) Inspeksi Pengecatan Kapal Report inspeksi area IMO PSPC Inspection Report, Coating Technical File Gambar 2. Alur proses pemeriksaan dan pelaporan hasil pemeriksaan Owner Representative (Owner Surveyor) QC Galangan Tahapan ini adalah pengumpulan data yang menunjang dalam penulisan studi ini yang terdiri dari: Tahapan ini adalah pengumpulan data yang menunjang dalam penulisan studi ini yang terdiri dari: 1) List pekerjaan pemeriksaan coating inspector pada pembangunan kapal baru. Inspeksi Pengecatan Kapa 2) Standard yang mengatur kegiatan coating inspector untuk kapal bangunan baru. 3) Form laporan dan dokumentasi hasil inspeksi coating inspector. bangunan baru. bangunan baru. persetujuan apa saja yang harus dilakukan inspeksi (inspection agreement) yang terdapat pada persetujuan tiga pihak (tripartite agreement). Setelah persetujuan dilakukan barulah kemudian inspeksi pengecatan kapal dapat dilaksanakan. 2) Mengidentifikasi alur proses inspeksi pengecatan kapal bangunan baru. 3) Mempelajari form dan dokumen laporan inspeksi. D. Pengolahan Data Gambar 2. Alur proses pemeriksaan dan pelaporan hasil pemeriksaan Berikut merupakan tahap pengolahan data yang dilakukan : Pada Gambar 2 dijelaskan inspeksi pengecatan kapal dilakukan oleh tiga pihak yaitu Coating inspector, Owner, dan QC Galangan. Pekerjaan inspeksi yang dilakukan sesuai dengan inspection agreement pada tripartie agreement. Coating inspector bertanggung jawab untuk melakukan pelaporan hasil inspeksi pada area yang diatur IMO PSPC kepada badan klasifikasi kapal. Sedangkan QC Galangan memiliki tugas untuk melakukan pelaporan hasil inspeksi harian (daily inspection report) dan pembuatan Coating Technical File kepada pemilik kapal. Owner surveyor juga ditugaskan untuk melakukan pemeriksaan jika diperlukan dan merupakan salah satu pihak yang melakukan approval atau submit laporan inspeksi. Proses pemeriksaan pengecatan kapal bangunan baru dapat dilihat pada Tabel 4 dibawah ini. 1) Melakukan pengelompokkan data pekerjaan pemeriksaan. 2) Melakukan pembuatan list pekerjaan yang harus dilakukan. 3) Pembuatan list panduan untuk pemeriksaan. 4) Pembuatan struktur laporan. B. Studi Lapangan Studi lapangan dilakukan di galangan PT. Lamongan Marine Industry. Berikut merupakan studi lapangan yang dilakukan: 1) Mengidentifikasi item pemeriksaan pengecatan kapal 1) Mengidentifikasi item pemeriksaan pengecatan kapal JURNAL TEKNIK ITS Vol. 6, No. 1, (2017) ISSN: 2337-3539 (2301-9271 Print) G-17 A. Penjelasan Umum Aplikasi Android Aplikasi yang dirancang memiliki 2 fungsi login yaitu administrator dan user. Fungsi login administrator adalah melakukan input item komponen/blok/area yang hendak dilakukan inspeksi, memberikan standard parameter inspeksi, memberikan file panduan inspeksi dan mengakses laporan hasil inspeksi. Sedangkan login user yang pada hal ini dioperasikan oleh coating inspector dan pihak terkait lainnya yang hendak melakukan pemeriksaan dan pengawasan pengecatan kapal bangunan baru memiliki fungsi untuk melakukan pengisian form pemeriksaan yang telah dirancang dan sesuai dengan proses inspeksi yang telah dipilih oleh administrator. Setelah melakukan pengisian form pemeriksaan, user dapat melakukan persetujuan form inspeksi dan melihat hasil report yang telah jadi berdasarkan pengisian form pemeriksaan yang telah dilakukan. Untuk satu proyek kapal yang didaftarkan oleh administrator, dapat memiliki maksimal 4 orang user yang dapat melakukan pengisian form pemeriksaan. User tersebut sebelumnya didaftarkan oleh administrator dengan menyantumkan Nomer Induk Karyawan (NIK) sebagai verifikasinya. C. Kendala Pemeriksaan Pengecatan Kapal Bangunan Baru Saat Ini Setelah dilakukan studi lapangan dan interview kepada praktisi di lapangan, kendala yang sering dihadapi adalah kurang praktisnya proses dokumentasi inspeksi dan pelaporan hasil inspeksi. Pembuatan form inspeksi dan input data masih dilakukan secara manual sehingga jika terjadi kehilangan coating inspector tidak memiliki backup data inspeksi dan harus melakukan input ulang data inspeksi. Selain itu, proses pelaporan kepada pihak terkait seperti pemilik kapal dan klas tidak dapat dilakukan secara langsung sehingga kurang praktis. B. Form Pemeriksaan Form pemeriksaan merupakan form yang telah disetujui oleh tiga pihak sebagai acuan inspektor untuk melakukan pekerjaannya. p j y Gambar 3. Contoh form pemeriksaan pada proses surface preparation A. Pemeriksaan Pengecatan Kapal Bangunan Baru Kendala Pemeriksaan Pengecatan Kapal Bangunan Baru Saat Ini C. Kendala Pemeriksaan Pengecatan Kapal Bangunan Baru Saat Ini A. Pemeriksaan Pengecatan Kapal Bangunan Baru A. Pemeriksaan Pengecatan Kapal Bangunan Baru Proses pengecatan kapal perlu adanya pengawasan dari coating inspector yang bertujuan untuk menjamin proyek kapal yang dibangun dapat sesuai dengan kualitas dan standard yang diinginkan. Untuk pembagian aktifitas pekerjaan pemeriksaan pengecatan, sebelumnya tiga pihak (three parties) yang terdiri dari owner, galangan dan manufaktur coating telah menentukan perjanjian atau JURNAL TEKNIK ITS Vol. 6, No. 1, (2017) ISSN: 2337-3539 (2301-9271 Print) G-18  Cek dew point  Cek temperatur permukaan material Tahapan Konstruksi Tahapan Pengecatan Item Pemeriksaan Inspeksi Visual Dry Film thickness (DFT) Measurement Cleanliness dan Curing Time After Erection Steelwork Preparation (Erection Joint) Inspeksi Visual:  Shrap edges  Weld defect  Imperfect steel surface Secondary Surface Preparation  Cek relative humidity  Cek dew point  Cek temperatur permukaan material Kontaminan Cleanliness grade Cek Dust level Surface Profile (Roughness) Cek Soluble Salt Stripe Coat  Cek relative humidity  Cek dew point  Cek temperatur permukaan material Inspeksi Visual Coating Selanjutnya  Cek relative humidity  Cek dew point  Cek temperatur permukaan material Inspeksi Visual Dry Film thickness (DFT) Measurement Cleanliness dan Curing Time Tiap Coating Inspeksi Final Inspeksi Visual Dry Film thickness (DFT) Measurement Cleanliness dan Curing Time Tiap Coating Holiday detection  Cek dew point  Cek temperatur permukaan material Tahapan Konstruksi Tahapan Pengecatan Item Pemeriksaan Inspeksi Visual Dry Film thickness (DFT) Measurement Cleanliness dan Curing Time After Erection Steelwork Preparation (Erection Joint) Inspeksi Visual:  Shrap edges  Weld defect  Imperfect steel surface Secondary Surface Preparation  Cek relative humidity  Cek dew point  Cek temperatur permukaan material Kontaminan Cleanliness grade Cek Dust level Surface Profile (Roughness) Cek Soluble Salt Stripe Coat  Cek relative humidity  Cek dew point  Cek temperatur permukaan material Inspeksi Visual Coating Selanjutnya  Cek relative humidity  Cek dew point  Cek temperatur permukaan material Inspeksi Visual Dry Film thickness (DFT) Measurement Cleanliness dan Curing Time Tiap Coating Inspeksi Final Inspeksi Visual Dry Film thickness (DFT) Measurement Cleanliness dan Curing Time Tiap Coating Holiday detection Gambar 3 diatas merupakan contoh format form pemeriksaan yang telah disetujui oleh owner, shipyard, dan coating inspector. Setiap proyek memiliki form pemeriksaan yang berbeda-beda sehingga perlu dilakukan persetujuan sebelum proyek pengecatan kapal dimulai. Form pemeriksaan yang telah diisi harus dilaporkan ke pihak-pihak terkait untuk dilakukan persetujuan/approval dan disimpan untuk keperluan Coating Technical File (CTF) kapal yang dibangun. C. C. Simulasi Aplikasi Android C. Simulasi Aplikasi Android 1) Administrator Administrator merupakan salah satu entitas dari aplikasi ini yang memiliki kewenangan untuk mendaftarkan proyek, memanajemen pengguna aplikasi, memasukkan file bantuan, menginput blok/area yang akan diperiksa, dan pemilihan proses pengecatan disertai dengan pengisian parameter pada setiap form pemeriksaan proses pengecatan yang dipilih, (a) (b) Gambar 4. Menu awal aplikasi (a) dan halaman input data proyek kapal (b) (a) (b) Gambar 5. Menu input file panduan (a) dan halaman input data blok Gambar 5 (a) merupakan menu penginputan file bantuan untuk user yang nantinya dimaksudkan untuk dapat membantu user dalam proses pemeriksaan. Sedangkan pada Gambar 5 (b) merupakan form pengisian data blok yang akan dilakukan pemeriksaan dan juga pemilihan proses pemeriksaan yang akan dilakukan pada blok. Setelah blok terdaftar, selanjutnya administrator memberikan batasan atau parameter pada setiap form pemeriksaan yang telah dipilih. User pada aplikasi ini adalah coating inspector, qc shipyard, owner surveyor, dan klas. Kegiatan yang dilakukan oleh user adalah memiliki kapal yang akan dilakukan pemeriksaan, mengisi form pemeriksaan pada blok /area yang telah diinput sebelumnya oleh administrator, dan mendokumentasikan proses pemeriksaan yang telah dilakukan. ( ) ( ) Gambar 4. Menu awal aplikasi (a) dan halaman input data proyek kapal (b) Gambar 4 (a) merupakan tampilan dari menu awal setelah administrator melakukan log in. Pada halaman menu ini, administrator dapat mendaftarkan proyek kapal yang akan dilakukan pemeriksaan cat. Setelah melakukan pemilihan menu tambah proyek akan muncul tampilan pengisian data kapal seperti Gambar 4 (b) diatas. Selain data kapal, administrator juga mendaftarkan user yang memiliki dengan cara memasukkan NIK user ke data proyek tersebut. Gambar 4 (a) merupakan tampilan dari menu awal setelah administrator melakukan log in. Pada halaman menu ini, administrator dapat mendaftarkan proyek kapal yang akan dilakukan pemeriksaan cat. Setelah melakukan pemilihan menu tambah proyek akan muncul tampilan pengisian data kapal seperti Gambar 4 (b) diatas. Selain data kapal, administrator juga mendaftarkan user yang memiliki dengan cara memasukkan NIK user ke data proyek tersebut. Gambar 4 (a) merupakan tampilan dari menu awal setelah administrator melakukan log in. Pada halaman menu ini, administrator dapat mendaftarkan proyek kapal yang akan dilakukan pemeriksaan cat. Setelah melakukan pemilihan menu tambah proyek akan muncul tampilan pengisian data kapal seperti Gambar 4 (b) diatas. Selain data kapal, administrator juga mendaftarkan user yang memiliki dengan cara memasukkan NIK user ke data proyek tersebut. (a) (b) Gambar 6. Menu pilih proyek (a) dan halaman list blok (b) (a) (a) (b) Gambar 6. B. Perancangan Aplikasi Android Perancangan aplikasi dilakukan dengan tiga program utama yaitu Android Studio, MySQL, dan phpMyAdmin. Android Studio digunakan untuk merancang aplikasi sesuai dengan permintaan desainer. Sedangkan MySQL digunakan untuk merancang database aplikasi yang dirancang. Sebelum program tersebut digunakan, sebelumnya desainer memberikan diagram alir dan mockup aplikasi. Diagram- diagram yang dibutuhkan antara lain Entitiy Relationship Diagram, Data flow Diagram, dan System Interface Diagram. Gambar 3. Contoh form pemeriksaan pada proses surface preparation JURNAL TEKNIK ITS Vol. 6, No. 1, (2017) ISSN: 2337-3539 (2301-9271 Print) G-19 (a) (b) Gambar 5. Menu input file panduan (a) dan halaman input data blok Setelah diagram selesai dibuat, implementasikan DFD dan SID pada program Android Studio. Kemudian implementasikan ERD pada database MySQL dengan tools phpMyAdmin. Setelah itu, aplikasi android dan database dihubungkan secara online dengan file php. VII. KESIMPULAN/RINGKASAN Kesimpulan yang didapatkan dari pengerjaan studi ini antara lain: 1) Pemeriksaan pengecatan kapal bangunan baru yang ada saat ini memiliki beberapa kelemahan diantaranya memerlukan coating inspector yang memiliki pengalaman dan pengetahuan yang memadai untuk melakukan pemeriksaan dengan benar, form pemeriksaan dibuat secara manual sehingga setiap proyek memiliki form yang berbeda-beda, proses penyusunan laporan dan pelaporan hasil pemeriksaan dilakukan secara manual sehingga kurang efektif. Gambar 7. Halaman pengisian form pemeriksaan p g p Gambar 7 merupakan contoh tampilan form pemeriksaan yang telah dipilih. Pada form ini terdapat parameter pemeriksaan yang sebelumnya diinput oleh administrator sehingga dapat menjadi panduan inspektor dalam pengambilan keputusan. 1) Administrator Menu pilih proyek (a) dan halaman list blok (b) Gambar 6 (a) merupakan menu awal aplikasi yang pada menu ini user dapat melakukan pemilihan proyek kapal yang akan dilakukan pemeriksaan. Setelah dilakukan pemilihan Gambar 6 (a) merupakan menu awal aplikasi yang pada menu ini user dapat melakukan pemilihan proyek kapal yang akan dilakukan pemeriksaan. Setelah dilakukan pemilihan G-20 JURNAL TEKNIK ITS Vol. 6, No. 1, (2017) ISSN: 2337-3539 (2301-9271 Print) proyek kapal, selanjutnya dipilih komponen yang akan di inspeksi dan akan muncul halaman seperti pada Gambar 6 (b). Kemudian user memilih proses pemeriksaan yang akan dilakukan. Dari Tabel 5 diatas dapat dilihat perbandingan antara sistem eksisting dengan sistem aplikasi yang dirancang. Pada tabel tersebut terdapat kelebihan maupun kekurangan dari sistem eksisting dan sistem aplikasi. Gambar 7. Halaman pengisian form pemeriksaan B. Uji Coba Aplikasi Untuk mengetahui kelayakan aplikasi, dilakukan uji coba aplikasi terlebih dahulu kepada praktisi di lapangan yang masih memiliki keterkaitan dalam pemeriksaan pengecatan kapal bangunan baru. Setelah dilakukan uji coba, selanjutnya pengujian dilakukan dengan memberikan kuisoner untuk mengetahui respon praktisi mengenai aplikasi yang dirancang dan penilaian dari kuisoner ini akan digunakan sebagai acuan kelayakan aplikasi. A. Analisis Sistem Analisis perbandingan kelebihan dan kekurangan sistem Aspek Sistem Eksisting Aplikasi Koneksi Tanpa Koneksi Menggunakan koneksi internet (selalu online) Penulisan form inspeksi Penulisan form inspeksi dilakukan secara manual dan format form pemeriksaan setiap proyek berbeda-beda Melakukan checklist dan penulisan secara cepat karena form pemeriksaan telah didesain sesuai standard Penyusunan laporan dan dokumentasi hasil inspeksi Jika ditemukan ketidaksesuaian harus di foto terlebih dahulu mengirimkannya ke laptop untuk diinput pada laporan Pada aplikasi terdapat fitur upload file gambar yang dapat dimasukkan secara langsung ke dalam laporan sehingga lebih efektif Proses pelaporan Proses pelaporan lama (memindahkan data tertulis ke komputer terlebih dahulu baru dilaporkan) Proses pelaporan lebih ringkas dan cepat (setelah laporan inspeksi di submit, dapat dibuka oleh user lain yang terintegrasi) SDM Diperlukan seseorang yang memiliki pengetahuan dan pengalaman yang memadai seperti senior coating inspector Terdapat fitur bantuan yang berisikan panduan dan standard pemeriksaan yang dapat membantu proses pengawasan Keamanan Penyimpanan laporan terdapat pada lemari penyimpanan dan laporan pengawasan ada kemungkinan hilang/rusak Penyimpanan laporan terdapat pada database server dan dapat dibackup di HP dengan fitur save report pada 3) Uji coba aplikasi ini dilakukan kepada praktisi di lapangan seperti coating inspector, quality control painting galangan, dan owner surveyor. Dalam pengujian kelayakan aplikasi ini digunakan data kuisoner. Dari hasil kuisoner yang telah dilakukan pengolahan data, didapati nilai kelayakan sebesar 29,4 dari nilai maksimum 35 yang mengartikan bahwa aplikasi yang dirancang layak untuk diaplikasikan di lapangan sebagai sarana pendukung proses pemeriksaan pengecatan kapal bangunan baru. 3) Uji coba aplikasi ini dilakukan kepada praktisi di lapangan seperti coating inspector, quality control painting galangan, dan owner surveyor. Dalam pengujian kelayakan aplikasi ini digunakan data kuisoner. Dari hasil kuisoner yang telah dilakukan pengolahan data, didapati nilai kelayakan sebesar 29,4 dari nilai maksimum 35 yang mengartikan bahwa aplikasi yang dirancang layak untuk diaplikasikan di lapangan sebagai sarana pendukung proses pemeriksaan pengecatan kapal bangunan baru. 3) Uji coba aplikasi ini dilakukan kepada praktisi di lapangan seperti coating inspector, quality control painting galangan, dan owner surveyor. Dalam pengujian kelayakan aplikasi ini digunakan data kuisoner. Dari hasil kuisoner yang telah dilakukan pengolahan data, didapati nilai kelayakan sebesar 29,4 dari nilai maksimum 35 yang mengartikan bahwa aplikasi yang dirancang layak untuk diaplikasikan di lapangan sebagai sarana pendukung proses pemeriksaan pengecatan kapal bangunan baru. [1] NACE. (2011). Coating inspector Program Level 1. Houston: NACE International. [2] Berendsen, A. M. (1989). Marine Painting Manual. Netherlands: Kluwer Academic Publisher. [3] ASTM D3276. (2000). A. Analisis Sistem 2) Aplikasi android yang dirancang memiliki kelebihan yang diantaranya terdapat file panduan pemeriksaan yang berisikan prosedur pemeriksaan sesuai standard ISO, ASTM, dan IMO PSPC, form pemeriksaan yang disusun sesuai IMO PSPC dan observasi di PT. Lamongan Marine Industry, fitur penyampaian laporan yang terintegrasi antara pihak-pihak terkait, dan data history pemeriksaan sebelumnya yang telah dilakukan yang dapat digunakan sebagai acuan pemeriksaan selanjutnya. Analisis sistem dilakukan dari hasil interview dan komentar praktisi di lapangan setelah dilakukan pengujian aplikasi. Berikut hasil perbandingan sistem ditunjukkan pada Tabel 5 di bawah ini. Tabel 5. Analisis perbandingan kelebihan dan kekurangan sistem Aspek Sistem Eksisting Aplikasi Koneksi Tanpa Koneksi Menggunakan koneksi internet (selalu online) Penulisan form inspeksi Penulisan form inspeksi dilakukan secara manual dan format form pemeriksaan setiap proyek berbeda-beda Melakukan checklist dan penulisan secara cepat karena form pemeriksaan telah didesain sesuai standard Penyusunan laporan dan dokumentasi hasil inspeksi Jika ditemukan ketidaksesuaian harus di foto terlebih dahulu mengirimkannya ke laptop untuk diinput pada laporan Pada aplikasi terdapat fitur upload file gambar yang dapat dimasukkan secara langsung ke dalam laporan sehingga lebih efektif Proses pelaporan Proses pelaporan lama (memindahkan data tertulis ke komputer terlebih dahulu baru dilaporkan) Proses pelaporan lebih ringkas dan cepat (setelah laporan inspeksi di submit, dapat dibuka oleh user lain yang terintegrasi) SDM Diperlukan seseorang yang memiliki pengetahuan dan pengalaman yang memadai seperti senior coating inspector Terdapat fitur bantuan yang berisikan panduan dan standard pemeriksaan yang dapat membantu proses pengawasan Keamanan Penyimpanan laporan terdapat pada lemari penyimpanan dan laporan pengawasan ada kemungkinan hilang/rusak Penyimpanan laporan terdapat pada database server dan dapat dibackup di HP dengan fitur save report pada Tabel 5. [4] American Bureau of Shipping (ABS). (2007). Guidance Notes on The Inspection, Maintenance and Application of Marine Coating Systems, Third Edition. United States: ABS. [5] Salbino, S. (2013). Buku Pintar Gadget Android untuk Pemula. Lembar Langit Indonesia. JURNAL TEKNIK ITS Vol. 6, No. 1, (2017) ISSN: 2337-3539 (2301-9271 Print) A. Analisis Sistem Standard Guide for Painting Inspectors (Metal Substrates). New York: American Society for Testing and Materials. G-21 JURNAL TEKNIK ITS Vol. 6, No. 1, (2017) ISSN: 2337-3539 (2301-9271 Print)
https://openalex.org/W2138966318	https://revistas.unal.edu.co/index.php/ingeinv/article/download/39521/41407	Latin	null	An academic value-added mathematical model for higher education in Colombia	Ingeniería e Investigación/Ingeniería e Investigación	2,013	cc-by	5,602	1 José Daniel Bogoya. Ingeniero Químico and Magister en Ingeniería de Sistemas, Universidad Nacional de Colombia, Colombia. Affiliation: Universidad Jorge Tadeo Lozano, Colombia. E-mail: daniel.bogoya@utadeo.edu.co 2 Johan Manuel Bogoya Ramírez. Ingeniería Mecánica and Matemáticas Puras, Universidad de los Andes, Colombia. M.Sc. y Ph.D. en Matemáticas puras, CINVES- TAV, México. Affiliation: Pontificia Universidad Javeriana, Colombia. E-mail: jbogoya@javeriana.edu.co How to cite: Bogoya, J. D., Bogoya, J. M., An academic value-added mathematical model for higher education in Colombia., Ingeniería e Investigación. Vol. 33, No. 2. August 2013, pp. 76 – 81. RESUMEN El concepto de valor académico agregado puede ser asociado con la variación en el desarrollo cognitivo de estudiantes que completaron un ciclo educativo en una institución perteneciente a un universo de referencia, con relación a la te ndencia mostra- da por todos los individuos del mencionado universo. Para estimar tal variación es necesario evaluar cierto conocimiento al co- mienzo y final del ciclo. La literatura existente dice que el conocimiento es el resultado de las capacidades intele ctuales del estu- diante, profesores y compañeros de clase, principalmente en el contexto familiar e institucional. Este trabajo propone un mod elo matemático lineal que representa el valor académico agregado en Colombia para el nivel de un estudiante o de un grupo de estudiantes en una institución, en términos de dos vectores: los resultados de exámenes estandarizados a nivel nacional aplicados al comienzo y final de un programa de educación superior, y el contexto estimado como el estrato socioeconómico. Palabras clave: M odelo M atemático, Valor académico agregado, educación superior, evaluación. Received: October 22th 2012 Accepted: July 16th 2013 the Colombian standardised exam for senior high school stu- dents. The methods and types of classroom practice based on a variety of pedagogical models whose objective is an academic exercise going beyond the classroom. Higher education timeta- bles seek to reduce face-to-face classes and increase autono- mous work supported by tutors or academic advisors. Curricu- lum plans tend to optimise the scope of conceptual and cognitive domain development, including content enabling the foundations of higher education programmes. Evaluation systems enable students’ level of learning to be determined at different times regarding educational effectiveness, make necessary adjustments, verify the degree of accomplishment regarding established stand- ards and map students’ achievements. ABSTRACT The concept of academic added-value can be associated with a variation in the cognitive development of students w ho com- plete an educational cycle at a given institution belonging to a reference universe, related to the tendency shown by all students in the aforementioned universe. Certain knowledge at the beginning and end of the cycle must be evaluated to estimate such varia- tion. The pertinent literature states that learning is the result of students, teachers, and classmates’ intellectual capacity, mainly within the family and an institution. This article proposes an academic value-added linear mathematical model representing a single stu- dent and sets of students in Colombia, grouped by institution, in terms of tw o vectors: learning outcome, regarding standardised national exams at the beginning and end of higher education programmes, and context, estimated as socioeconomic strata. Keywords: M athematical model, academic value added effectiveness, higher education, evaluat Un modelo matemático para el valor académico agregado: Caso de la educación superior en Colombia J. D. Bogoya1 and J. M. Bogoya2 INGENIERÍA E INVESTIGACIÓN VOL. 33 No. 2, AUGUST - 2013 (76-81) INGENIERÍA E INVESTIGACIÓN VOL. 33 No. 2, AUGUST - 2013 (76-81) Towards a conceptual model Evaluation entails structuring arguments for maintaining the initial design or preparing guidelines for making adjustments during such development, whenever corrections of unexpected aspects are necessary, to guarantee that proposed goals are achieved (Stufflebeam & Shinkfield, 1995, p. 199). The specified relationship (statistical formulation) allowed esti- mating explained variance for a response by means of the coeffi- cient value in equation (2), which was found by mathematical regression: Product evaluation consists of interpreting results achieved throughout a project, during different stages of development and the final stage, showing the degree of proposed goal fulfilment and the quality of obtained results. Such evaluation entails com- parison with other projects’ products in similar contexts and observing project significance. Activity effectiveness can be seen in changes in initial conditions regarding context (Stufflebeam & Shinkfield, 1995, pp. 201-202). (2) (2) where was output showing the performance of student I, variables estimations of arguments in equation (1) for student I, parameters of educational process, and the portion of variance for performance which could not be explained by (Hanushek & Kain, 1972, p. 124). Figure 1 shows the meaning of academic added-value for univer- sities A and B within a set of 116 institutions. It presents the relationship between the students’ average scores, grouped by university, on assessment on two different occasions, i.e. at the beginning and end of a higher education programme. The average result of the first assessment is represented on the horizontal axis and that of the second assessment on the vertical axis. With minor nomenclature modifications, equation (1) was pro- posed for estimating variation in student performance during a given time, starting at moment t* and ending at t: ( ( ) ( ) ( ) ) (3) ( ) (3) ) represented change in family influence for student ( ) where ( ) represented change in family influence for student i during period ( ), ( ) reflected the change in classmate influence for student i during period ( ), ( ) indicated the change in school input relevant for student i during period ( ) and showed natural skills for student i (Hanushek, 1979, pp. 363-364). Towards a conceptual model Figure 1. The academic value added effectiveness for university A and B, denoted by VA and VB, respectively, within a set of results for 116 institu- tions regarding assessment on two different occasions Assessment 1 Assessment 2 Assessment 1 Assessment 2 A model regarding the study and evaluation of educational effec- tiveness considers information from four entities: context, input, process and product. Evaluating context implies identifying and assessing project framework characteristics regarding either an individual or a group concerning a particular programme or institution, anticipating observed strengths and aiming to rectify any detected difficulties. It must be ascertained whether the foreseen goals and priorities are relevant when evaluating con- text and that they are in accordance with specified needs which must be satisfied. Such evaluation should provide reliable and sufficient information for designing the required adjustments and guaranteeing improvement (Stufflebeam & Shinkfield, 1995, p. 196). Figure 1. The academic value added effectiveness for university A and B, denoted by VA and VB, respectively, within a set of results for 116 institu- tions regarding assessment on two different occasions Evaluating input aims at recognising initial conditions, the proce- dures being used and those which could be used. When evaluat- ing input, every fresh possibility is checked so that it meets es- tablished requirements and the most likely paths which would be taken according to a hypothetical benchmarking exercise are defined. The most sensitive differences between the current state of each path and that of a typical one are also outlined (Stufflebeam & Shinkfield, 1995, p. 197). The following relationship for possible aspects intervening in an educational project was proposed for analysing primary schools subsequent to a critical study of the Coleman report (1996): ( ) (1) (1) where was output, i.e. educational performance of student i during time t, individual and family characteristics for student i accumulated during time t, characteristics for the student population (classmate effects) of the institution to which student i belonged, accumulated during time t, initial endowments for student i and school input relevant to student i, accumulated during time t (Hanushek & Kain, 1972, p. 123). Verifying the degree of a given plan’s fulfilment represents evalua- tion aimed at providing the main actors and decision-makers with ongoing information in terms of required time, execution oppor- tunities, displayed method quality, the suitability of the people responsible for executing planned activities and the resources used. Introduction1 The learning outcome of higher education programmes being offered by Colombian universities, involving varied starting condi- tions and different processes taking place, raises the need for proposing and exploring relationships amongst some variables enabling educational project effectiveness and significance to be identified. These variables represent the influence of the socio- economic and cultural environment, the students’ level of learn- ing at the beginning of their studies and a wide variety of such projects’ academic value added elements (Bogoya, 2006, p. N18). Some such conditions are worth highlighting. The performance of students entering universities is observable in their results in This article proposes a mathematical model representing higher education overall effectiveness in Colombia, viewed as academic added-value. Variables such as input, context, academic added- value and output are defined. Input variable has been estimated using the score obtained by students on the Colombian Higher Education Admission Exam (Saber 11). Students’ socioeconomic strata at the end of their university studies was assigned to con- text, their scores on the Colombian Higher Education Exit Exam (Saber Pro) was assigned to output and the difference between 76 BOGOYA AND BOGOYA scores on the Saber Pro exam and the value of the national tendency function defined as academic value added effectivess (Bogoya, 2011, p. 32). value VA. University B was below the line of tendency because its average result on the second assessment was lower than the value indicated by the reference line, indicating negative academic added-value -VB (Goldstein, 2001, p. 4). AN ACADEMIC VALUE-ADDED MATHEMATICAL MODEL FOR HIGHER EDUCATION IN COLOMBIA Vectors and were defined as follows: ( ) ( ( ) ( ) ( )) and ( ). The model in equation (10) was thus redefined as: The model in equation (10) was thus redefined as: (6) (11) ( ) ( ) ̂( ) (11) (6) In an ideal situation ( ̂ ), the objective would be to calculate vector , solving equations in (11). In geometric terms, the problem consisted of calculating the normal for a plane in , containing points ( ( ) ( )), where was output, representing the performance of student i in school j, school characteristics for the school to which student i belonged, family background for student i and stochastic error (Krueger, 1997, p. 12). The real situation would be different: points ( ( ) ( )), were not coplanar. Thus, the objective would be written as an optimisation problem: Another example using initial condition, contributing to discus- sion of academic added-value models in England aimed at stu- dents aged 4 to 16, involved a two-level model (see equation 7): would be the Euclidean norm for , defined as: (7) (7) ( ) \| ( ) ( )\| (12) (12) where was as described in equation (6), average score at the beginning of a period as an explanatory variable, the component of unexplained variance corresponding to the school, and the component of unexplained variance attributed to the student (Ray, 2006, p. 73). In this case, academic value added effectiveness could be estimated by subtraction( ). where was as described in equation (6), average score at the beginning of a period as an explanatory variable, the component of unexplained variance corresponding to the school, and the component of unexplained variance attributed to the student (Ray, 2006, p. 73). In this case, academic value added effectiveness could be estimated by subtraction( ). It should be noted that ( ) was the distance from point ( ( ) ( )) to the plane passing through the origin determined by normal vector ( ). The function was minimised by following the base of the least square method: Mathematical model ( ) ∑ ( ) ∑( ( ) ( )) (13) Colombia has two mandatory national state examinations; one, called Saber 11, is used on students finishing the high school cycle as a university admission requirement, whereas the second, known as Saber Pro, is used for students finishing the higher education cycle. The results obtained by a student on these two exams enables verifying the validity of an academic value added model. (13) The four partial derivatives of were cancelled to find vector . The previous approach involved an algorithm known as multi- lineal regression which can be found as a standard function in most software (Wolfram Mathematica was used here). Students taking a given Saber Pro exam in Colombia were num- bered from 1 to n, and the programmes or universities attended by these individuals were numbered from 1 to m; ( ), ( ) and ( ) were the results for student i on Saber Pro and Saber 11 exams and his/her socioeconomic stratum, respectively. 1 The public database at ftp://ftp.icfes.gov.co was used. Towards a conceptual model i during period ( ), ( ) reflected the change ( Taking the line of tendency describing average scores for Co- lombian universities on exams taking place at the beginning and end of a higher education programme as reference, it can be observed that institutions having a low average on the first as- sessment (Figure 1, left) tended to also have a low average on the second assessment. By contrast, institutions shown on the right maintained high averages on both assessments. Figure 1 also shows that university A was located above the line of tendency, having a higher average score on the second assessment than that traced by the line of tendency and positive academic added- Modelling equation (1) by level, academic added-value could be estimated as an indication of teaching quality: (4) (4) INGENIERÍA E INVESTIGACIÓN VOL. 33 No. 2, AUGUST - 2013 (76-81) 77 INGENIERÍA E INVESTIGACIÓN VOL. 33 No. 2, AUGUST - 2013 (76-81) 77 INGENIERÍA E INVESTIGACIÓN VOL. 33 No. 2, AUGUST - 2013 (76-81) 77 AN ACADEMIC VALUE-ADDED MATHEMATICAL MODEL FOR HIGHER EDUCATION IN COLOMBIA AN ACADEMIC VALUE-ADDED MATHEMATICAL MODEL FOR HIGHER EDUCATION IN COLOMBIA ( ) ( ( )) ( ( )) ( ) ( ) (8) where output value indicated educational performance for student i in grade level u, school factors, family and con- text input, , and unknown parameters, a stochastic term expressing unconsidered influences and value added for teach- er j (Hanushek & Rivkin, 2010, p. 267). (8) l where real constants, ( ) academic value added effectiveness for programme s, continuous functions determining the model’s complexity and ( ) estimated error for student i. For example, the linear model was obtained when ( ) i.e.: After incorporating fixed and stochastic effects, a general linear mixed model with one association level was proposed, (5) (5) ( ) ( ) ( ) (9) (9) where output variable was observation, a known matrix × in size, an unknown fixed-effect vector, a known fixed- effect matrix and and unobservable random variable vectors (Henderson, 1973, p. 16; Sanders & Horn, 1994, p. 305; Pinheiro & Bates, 2000, p. 58). where output variable was observation, a known matrix × in size, an unknown fixed-effect vector, a known fixed- effect matrix and and unobservable random variable vectors (Henderson, 1973, p. 16; Sanders & Horn, 1994, p. 305; Pinheiro & Bates, 2000, p. 58). Equation (9) was proposed in three steps. The following equation determined the values of : ( ) ( ) ( ) ̂( ) (10) (10) The second step involved defining ( ) as the average of ̂ for the students in each program , . In the third step, ( ) was defined as ̂( ) ( ): The second step involved defining ( ) as the average of ̂ for the students in each program , . In the third step, ( ) was defined as ̂( ) ( ): An example regarding fixed and stochastic effects on two associ- ation levels can be found in an analysis of results in the STAR project (Tennessee Student/Teacher Achievement Ratio experi- ment) by means of a model summarising kindergarten students’ learning in terms of school and family characteristics (see equa- tion 6). If the model were used on two consecutive occasions and a subtraction made, change in output can be found and asso- ciated with academic value added effectiveness: ( ) . Vectors and were defined as follows: ( ) . Case study Students’ performance on the 2009 Business Administration Saber Pro exam in Colombia was used for evaluating a set of corresponding programmes. Only institutions having 20 or more students were taken into account because of reliability issues. The database used3 reported 120 programmes having these The academic value added for a programme was defined as the positive or negative influence of the programme on students’ learning when results at two different moments were compared, represented by variables and , assumed to be related as follows: INGENIERÍA E INVESTIGACIÓN VOL. 33 No. 2, AUGUST - 2013 (76 - 81) 78 BOGOYA AND BOGOYA characteristics for a universe of 10,782 students. Equation (9) was used for estimating . characteristics for a universe of 10,782 students. Equation (9) was used for estimating . Figure 4. Error [see equation (9)] after omitting 2% of the outliers Figure 5. The distribution of the number of students per decile on the error scale Students ε Number of students Decile Figure 4. Error [see equation (9)] after omitting 2% of the outliers Students ε ( ) was a sample having mean and standard deviation . ( ) was a sample having mean and standard deviation . ̂( ) (14) ( ) (14) Sample ̂( ) was thus located on a scale having mean 0 and standard deviation 1; transformation ̂ is known as normalisation. Figure 4. Error [see equation (9)] after omitting 2% of the outliers Figure 2. Error [see equation (9)] for each of the 10,782 Business Ad- ministration students whose results were analysed Students ε Figure 5. The distribution of the number of students per decile on the error scale Number of students Decile Figure 2. Error [see equation (9)] for each of the 10,782 Business Ad- ministration students whose results were analysed Figure 5. The distribution of the number of students per decile on the error scale Variables , and were normalised to compare the values of constants . Absolute error value (see Figure 2) had a maximum of 4.95 and a mean of 0.55 points on a normal- ised scale. Based on the information in Figure 3, 2% of the data farthest from the line of tendency (equivalent to 216 records) were removed (i.e. the outliers). After data depuration, the absolute error value had a maximum of 1.67 and a mean of 0.52 points (see Figures 4 and 5). Results Equation ( ) ( ) ( ) was defined [see equation (10)], where represented the estimated value for variable (Saber Pro score) according to the model in equation (9). Figure 6 shows the depurated score for the 10,566 students who took the 2009 Business Administration Saber Pro exam (vertical axis) and estimated value (horizontal axis). The data line of tendency divided the universe of students considered into two sets: individuals whose results were located above the line (having positive academic value added effectiveness because their scores on the Saber Pro were higher than the value estimated by means ) and individuals whose results were below the line (having negative academic value added effectiveness because estimated a result higher than their actual score). According to the normalised approach, the results for each student were relative to the universe they belonged to. Quadratic, cubic and exponential models were studied during the course of the investigation, but it was found that these models did not substantially reduce (about a hundredth) lineal model error. Other variables were also included, such as the number of semesters studied, the type of high-school education and the students’ gender; however, the results showed these variables to be irrelevant. Figure 6. 2009 Business Administration Saber Pro results compared to estimated value per student, after omitting 2% of the outliers Table 1 shows position and dispersion calculated for , and . Pearson’s correlation coefficient between the values for Saber Pro ( ) and the corresponding estimated values ( ) was 0.71; the line of tendency had a 1.00 slope and 0.00 intercept. Estimated value w Saber Pro y Figure 6. 2009 Business Administration Saber Pro results compared to estimated value per student, after omitting 2% of the outliers Estimated value w Saber Pro y Figure 3. Maximum absolute value pattern for ( ), [see equation (9)] for every set taken into account, compared to the percentage of omitted outliers % outliers omitted max {\|ɛ(i)\|} % outliers omitted max {\|ɛ(i)\|} Figure 6. 2009 Business Administration Saber Pro results compared to estimated value per student, after omitting 2% of the outliers % outliers omitted Table 1 shows position and dispersion calculated for , and . Pearson’s correlation coefficient between the values for Saber Pro ( ) and the corresponding estimated values ( ) was 0.71; the line of tendency had a 1.00 slope and 0.00 intercept. Figure 3. Case study The normalised score for 10,566 students on the Saber Pro exam had a minimum of -4.24 and a maximum of 3.68 points; the model in equation (9) predicted the score on this exam having 0.52 mean error, equal to 6.57% of range y. The solution found the following values for the con- stants: Results 2009 Business Administration Saber Pro results compared to estimated value per programme or university Figure 8 shows the distribution of academic value added effec- tiveness for the 120 programmes considered in the analysis, arranged in ascending order from left to right, whereas Tables 2, 3 and 4 show the set of values for and for three groups of five programmes, respectively. The first group had the highest observed value, the second a value close to zero and the third the lowest academic value added effectiveness. Figure 9. The distribution of the number of programmes per decile on the academic value added scale. Results 2009 Business Administration Saber Pro results compared to estimated value per programme or university Figure 8 shows the distribution of academic value added effec- tiveness for the 120 programmes considered in the analysis, arranged in ascending order from left to right, whereas Tables 2, 3 and 4 show the set of values for and for three groups of five programmes, respectively. The first group had the highest observed value, the second a value close to zero and the third the lowest academic value added effectiveness. Estimated av erage value Saber Pro av erage effectiveness ranged from 0.66 to 0.82 and that having the lowest ranged from -0.83 to -0.66 points. Table 1. Mean and standard deviation for and after omitting 2% of the outliers Parameter y w V Mean 1.80x10-3 1.80x10-3 -2.25x10-2 Standard deviation 9.80x10-1 6.94x10-1 2.84x10-1 Table 1. Mean and standard deviation for and after omitting 2% of the outliers Table 1. Mean and standard deviation for and after omitting 2% of the outliers Parameter y w V Mean 1.80x10-3 1.80x10-3 -2.25x10-2 Standard deviation 9.80x10-1 6.94x10-1 2.84x10-1 Table 2. and for five programmes having the highest academic value added effectiveness Figure 7 presents the relationship between the average score obtained on the 2009 Business Administration Saber Pro exam by students grouped in each of the 120 considered programmes (vertical axis) and the corresponding average of the values esti- mated by means of function w (horizontal axis). The line of ten- dency again divided the universe of programmes into two sets: those above the line were individuals having positive academic value added effectiveness because their actual average was higher than the estimated value and those below the line having negative academic value added effectiveness because their estimated average foresaw a higher value than the average which they actually achieved. Table 3. and for five programmes having academic value added effectiveness close to zero Pearson’s correlation coefficient between the average values on the Saber Pro ( ) and the corresponding average for the esti- mated value ( ) was 0.90; the line of tendency had a 1.35 slope and 3.24 × 10-3 intercept. Table 4. and for five programmes having the lowest aca- demic value added effectiveness Figure 7. 2009 Business Administration Saber Pro results compared to estimated value per programme or university Estimated av erage value Saber Pro av erage Figure 7. Results Maximum absolute value pattern for ( ), [see equation (9)] for every set taken into account, compared to the percentage of omitted outliers INGENIERÍA E INVESTIGACIÓN VOL. 33 No. 2, AUGUST - 2013 (76-81) 79 AN ACADEMIC VALUE-ADDED MATHEMATICAL MODEL FOR HIGHER EDUCATION IN COLOMBIA effectiveness ranged from 0.66 to 0.82 and that having the lowest ranged from -0.83 to -0.66 points. Table 2. and for five programmes having the highest academic value added effectiveness University 1 1.20 0.54 -0.08 0.37 0.83 2 1.21 0.70 -0.12 0.48 0.73 3 1.46 1.09 -0.05 0.75 0.72 4 0.84 0.40 0.24 0.29 0.55 5 1.34 1.20 0.32 0.84 0.50 Table 3. and for five programmes having academic value added effectiveness close to zero University 53 1.51 2.05 1.60 1.49 0.02 54 -0.10 -0.20 0.20 -0.12 0.01 55 -0.37 -0.48 -1.09 -0.38 0.01 56 -0.37 -0.54 -0.30 -0.38 0.01 57 -0.16 -0.25 0.25 -0.15 -0.01 Table 4. and for five programmes having the lowest aca- demic value added effectiveness University 116 -0.54 -0.16 -0.09 -0.11 -0.43 117 -0.81 -0.49 0.58 -0.30 -0.51 118 -0.02 0.86 0.26 0.61 -0.63 119 -1.23 -0.51 -0.80 -0.39 -0.85 120 -1.39 -0.72 -0.84 0.53 -0.86 Figure 9. The distribution of the number of programmes per decile on the academic value added scale. C l i Decile Number of programmes Table 1. Mean and standard deviation for and after omitting 2% of the outliers Parameter y w V Mean 1.80x10-3 1.80x10-3 -2.25x10-2 Standard deviation 9.80x10-1 6.94x10-1 2.84x10-1 Figure 7 presents the relationship between the average score obtained on the 2009 Business Administration Saber Pro exam by students grouped in each of the 120 considered programmes (vertical axis) and the corresponding average of the values esti- mated by means of function w (horizontal axis). The line of ten- dency again divided the universe of programmes into two sets: those above the line were individuals having positive academic value added effectiveness because their actual average was higher than the estimated value and those below the line having negative academic value added effectiveness because their estimated average foresaw a higher value than the average which they actually achieved. Pearson’s correlation coefficient between the average values on the Saber Pro ( ) and the corresponding average for the esti- mated value ( ) was 0.90; the line of tendency had a 1.35 slope and 3.24 × 10-3 intercept. Figure 7. Conclusions Academic value added effectiveness represents a valuable tool for recognising an educational institution’s effectiveness as the students achieve higher average learning results on external standardised exams in relation to the value estimated by the line of tendency describing the universe of institutions considered as reference. Figure 8. Academic value added effectiveness for the 120 programmes analysed Programme Academic v alue added Programme Academic v alue added A linear mathematical model involving four variables was devel- oped in the current research for estimating academic value add- ed effectiveness for higher education programmes in Colombia. Two variables established the learning level of students enrolled in a given programme (the first at the beginning and the second at the end of their studies), a third variable incorporated the the students’ socioeconomic strata and a fourth covered academic value added effectiveness. e e ect ve ess o g e e ucat o p og a es Co o b a. Two variables established the learning level of students enrolled in a given programme (the first at the beginning and the second at the end of their studies), a third variable incorporated the the students’ socioeconomic strata and a fourth covered academic value added effectiveness. Figure 8. Academic value added effectiveness for the 120 programmes analysed To solve the model and show its application, academic value added effectiveness ( ) was calculated for a sample of 10,782 Business Administration students who finished their higher edu- cation in Colombia. The data was depurated to 10,566 records; the students were grouped into 120 programmes according to the university they were attending. Student data included score obtained on Saber 11 required for university admission ( ), From a different perspective, Figure 9 shows the distribution of the number of programmes per decile on the scale of academic value added effectiveness. Of the 120 Business Administration programmes considered, 99 were located in the middle area, having academic value added effectiveness ranging from -0.33 to 0.32; the programme having the highest academic value added INGENIERÍA E INVESTIGACIÓN VOL. 33 No. 2, AUGUST - 2013 (76 - 81) 80 BOGOYA AND BOGOYA socioeconomic strata ( ) and score obtained on the 2009 Saber Pro exam ( ). tion Functions. Industrial Relations Section W orking Paper No. 379, Princeton: Princeton University. Pinheiro, J. C. & Bates, D. M. (2000). Mix ed Effects Models in S and S-Plus. Secaucus: Springer. References Hanushek, E. A. & Kain, J. F. (1972). On the Value of Equality of Educational Opportunity as a Guide to Public Policy. In: On Equality of Educational Opportunity, Eds Mosteller, F. & Moyni- han, D. P. New York: Random House. Bogoya, D. (2006). Evaluación Educativa en Colombia. Seminario Internacional de Evaluación, N1-N27. Cartagena: ICFES. Bogoya, D. (2011). Impacto académico de la Facultad de Inge- niería de la Universidad Nacional de Colombia – Sede Bogotá. Ingeniería e Investigación, 31, 29-39. Hanushek, E. A. (1979). Conceptual and Empirical Issues in the Estimation of Educational Production Functions. The Journal of Human Resources, 14, 351-388. Coleman, J. S., Campbell, E. Q., Hobson, C. F., McPartland, J., Mood, A. M., W einfeld, F. D. & York, R. L. (1966). Equality of Edu- cational Opportunity. W ashington DC: US Government Printing Office. Hanushek, E. A. & Rivkin, S. G. (2010). Generalizations about Using Value-Added Measures of Teacher Quality. American Econom- ics Review : Papers & Proceedings, 100, 267-271. Henderson, C. R. (1973). Sire Evaluation and Genetic Trends. Journal of Animal Science, 1973, 10-41. Goldstein, H. (2001). League Tables and Schooling. Science in Parliament, 58-2, 4-5. Krueger, A. B. (1997). Ex perimental Estimates of Education Produc- tion Functions. Industrial Relations Section W orking Paper No. 379, Princeton: Princeton University. Hanushek, E. A. & Kain, J. F. (1972). On the Value of Equality of Educational Opportunity as a Guide to Public Policy. En: On Equality of Educational Opportunity, eds Mosteller, F. & Moyni- han, D. P. New York: Random House. Pinheiro, J. C. & Bates, D. M. (2000). Mix ed Effects Models in S and S-Plus. Secaucus: Springer. Hanushek, E. A. (1979). Conceptual and Empirical Issues in the Estimation of Educational Production Functions. The Journal of Human Resources, 14, 351-388. Ray, A. (2006). School Value Added Measures in England. A paper for the OECD Project on the Development of Value- Added Models in Education Systems. Hanushek, E. A. & Rivkin, S. G. (2010). Generalizations about Using Value-Added Measures of Teacher Quality. American Econom- ics Review : Papers & Proceedings, 100, 267-271. Sanders, W . L. & Horn, S. P. (1994). The Tennessee Value-Added Assessment System (TVAAS): Mix ed-Model Methodology in Edu- cational Assessment. Journal of Personnel Evaluation in Educa- tion, 8, 299-311. Henderson, C. R. (1973). Sire Evaluation and Genetic Trends. Journal of Animal Science, 1973, 10-41. Stufflebeam, D. & Shinkfield, A. (1995). Conclusions An event’s location on a scale was relative to the universe being considered due to the normalised approach adopted in this research. For higher education benchmarking exercises, one programme will be more effective and more significant for stu- dent learning than another when the first programme has higher academic value added effectiveness than the second one, provid- ed that identified crucial context variables are controlled. Ray, A. (2006). School Value Added Measures in England. A paper for the OECD Project on the Development of Value- Added Models in Education Systems. Sanders, W . L. & Horn, S. P. (1994). The Tennessee Value-Added Assessment System (TVAAS): Mix ed-Model Methodology in Edu- cational Assessment. Journal of Personnel Evaluation in Educa- tion, 8, 299-311. Sanders, W . L. & Horn, S. P. (1994). The Tennessee Value-Added Assessment System (TVAAS): Mix ed-Model Methodology in Edu- Assessment System (TVAAS): Mix ed-Model Methodology in Edu- cational Assessment. Journal of Personnel Evaluation in Educa- tion, 8, 299-311. cational Assessment. Journal of Personnel Evaluation in Educa- tion, 8, 299-311. The values found for constants and were 0.6837 and 0.0526 in the mathematical model. A statistical interpretation based on data from the sample of 10,782 students showed that variable (Saber 11) was more powerful (13 times more) in explaining variance for variable (Saber Pro) than variable (socioeconomic strata). Stufflebeam, D. & Shinkfield, A. (1995). Evaluación sistemática – Guía teórica y práctica. Barcelona: Ediciones Paidós.Coleman, J. S., Campbell, E. Q., Hobson, C. F., McPartland, J., Mood, A. M., W einfeld, F. D. & York, R. L. (1966). Equality of Educational Opportunity. W ashington DC: US Government Printing Office. Stufflebeam, D. & Shinkfield, A. (1995). Evaluación sistemática – Guía teórica y práctica. Barcelona: Ediciones Paidós.Coleman, J. S., Campbell, E. Q., Hobson, C. F., McPartland, J., Mood, A. M W einfeld F D & York R L (1966) Equality of Educational M., W einfeld, F. D. & York, R. L. (1966). Equality of Educational Opportunity. W ashington DC: US Government Printing Office. Goldstein, H. (2001). League Tables and Schooling. Science in Parliament, 58-2, 4-5. References Evaluación sistemática – Guía teórica y práctica. Barcelona: Ediciones Paidós. Krueger, A. B. (1997). Ex perimental Estimates of Education Produc- INGENIERÍA E INVESTIGACIÓN VOL. 33 No. 2, AUGUST - 2013 (76-81) 81
https://openalex.org/W3167505271	https://www.researchsquare.com/article/rs-551083/latest.pdf	English	null	Paracrine activity of adipose derived stem cells on limbal epithelial stem cells	Scientific reports	2,021	cc-by	8,884	Research Article Posted Date: June 15th, 2021 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read F ll License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/20 Page 1/20 Abstract Limbal stem cells deficiency (LSCD) is an eye disease caused by the loss of stem cells in the corneal limbus as a succession of an injury due physical, biological, or chemical agents. Current therapies of LSCD are focused on the transplantation of donor corneas or tissue equivalents produced from autologous limbal stem cells. Every year there are waiting millions of people for the cornea transplantation all over the world and the list of waiting patients is growing due to the relatively low number of cornea donors. On the other hand, the transplantation of tissue or cells into the recipient’s body is associated with the higher risk of possible side effects. The possibility of the application of an indirect treatment using the properties of the paracrine activity of stem cells, would be beneficial for the patients with transplant failures. This study was to evaluate the paracrine effect of mesenchymal stem cells derived from adipose tissue (ADSC) on the viability of limbal epithelial stem cells (LESC). The paracrine effect was assessed by treating LESC with conditioned medium collected from ADSC culture. Cell viability, cytotoxicity, apoptosis and proliferation were evaluated using in vitro assays in standard conditions and induced inflammation. After the exposure to the examined conditions, the expression of genes related to pro- and anti- inflammatory factors was evaluated and compared to the secretion of selected cytokines by ELISA test. Moreover, the changes in LESC phenotype were assessed using of phenotype microarrays. Our findings suggest that paracrine activity of ADSC on LESC promotes its proliferation and mitigates the adverse impact of induced inflammation. Introduction Cornea is front, transparent part of eye responsible for transmission and refraction of light. It possesses high self-regenerative ability due to the presence of limbal epithelial stem cells (LESC) what makes the cornea easy to be transplanted. LESC are localized in corneal limbus which is a thin, transparent cell zone in the junction between cornea, sclera and conjunctiva [1]. Limbal Stem Cell Deficiency (LSCD) may occur due to chemical or thermal burns and physical destruction. LSCD is also a congenital disease like aniridia-associated keratopathy (AAK), corneal leukoma or Fuchs’ dystrophy [2–4]. Cornea is one of the most frequently transplanted organs. Unfortunately, despite the possibility of transplantation, insufficient number of donors in comparison to number of recipients is still a huge problem of transplant medicine. Statistical report of Eye Bank Association of America (EBAA) showed that 85441 corneal transplantations were done in year 2018 [5]. Reported cases of corneal transplantations in 2019 reached 85601. In 2020 due the worldwide pandemic of COVID-19 reported procedures dramatically decreased to the number of 66278 surgeries. The pandemic has influenced on performing many surgical procedures and the implemented sanitary restrictions in most countries all over the world had slower healthcare system what appeared in lower number of all surgical procedures. Instead of the successfully ended cornea transplantation reported by EBAA, every year there is a huge number of waiting patients for the corneal transplantation all over the world. In 2016 it was reported about 12.7 million of waiting patients [6]. Page 2/20 Treatment of the corneal disorders may embrace partial or whole cornea transplantation. Unfortunately, the number of patients waiting for the graft indicates that there are not enough cornea donors, thus newer and newer medicinal products for corneal reconstruction are being introduced. When other treatment fails, standard surgical procedure of damaged cornea replacement with a donor tissue is being displaced by artificial cornea transplantation [7–10]. As well the use of xenogeneic corneas is being considered in the absence of cornea donors [11–13]. However, each medicinal product of such matter is produced with active stem cells usually harvested from small biopsy taken from the healthy limbal region of patients’ eye. Such a biopsy could potentially broaden stem cells deficiency and cause extensive damage to the corneal epithelium. On the other hand, not all the corneal injures require immediate replacement. Introduction New strategies may focus on improving residual limbal stem cells viability and supporting them with factors or drugs aimed to activate their proliferation and thus tissue reconstruction. Therefore, new strategies on corneal treatment, based on supporting the residual stem cells should be developed. Regarding these concerns we assume that new strategy of corneal treatment which will be based on supporting residual healthy stem cells niche in patients’ eye will be the future therapy of limbal stem cells deficiency. Mesenchymal stem cells (MSCs) are somatic cells with wide regenerative properties. Research focus on using them e.g. in bone and cartilage failures regeneration or cardiovascular disease therapies. One of the common sources of MSCs is adipose tissue. Adipose tissue is a rich source of mesenchymal stem cells. Its collection is an easy procedure and the probable amount of stem cells which can be gathered during the isolation procedure is high. Considering other sources of mesenchymal stem cells, liposuction is burdened with low risk of complications in opposite to the bone marrow biopsy. Mesenchymal stem cells, besides its plasticity, exhibit immunomodulatory abilities by secreting numerous cytokines [14, 15]. ADSC affects neighboring cells also via secretion of growth factors [16, 17] and extracellular vesicles [18–20]. Secretome of ADSC promotes proliferation and reduces inflammation [21, 22]. MSCs secrete anti-scarring factors as KGF, SDF-1, MIP-1a, MIP-1b, anti-apoptotic agents as STC-1, SFRP2, TGF-β1, VEGF and HGF, molecules associated to angiogenesis like VEGF, TGF-β1 and mitogenic factors as TGF-α, TGF-β, HGF, IGF-1, FGF-2 or EGF [15, 23]. Research indicated that MSCs secretome act positively on corneal epithelium [24]. It suggests that ADSC can be used clinically in the treatment of LSCD. Our experiment evaluates the paracrine activity of ADSC on LESC’S dysfunction model in vitro. The effect of ADSC’S secreted factors on the proliferation, metabolic activity, and transcriptome of limbal stem cells was analyzed under conditions of induced inflammation using bacterial lipopolysaccharide (LPS). MTT assay 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, USA) was used to evaluate the viability of the cells after exposure to LPS to determine the optimal concentration for cells stimulation [27]. Absorbance referring to the concentration of formazan was measured using Wallac 1420 VICTOR plate reader (Perkin Elmer, USA) after 24 h of exposure to 0,1 µg/ml; 0,2 µg/ml; 0,5 µg/ml; 0,7 µg/ml; 1 µg/ml and 2 µg/ml of LPS and compared to control. All groups were analyzed in six replicates. Experiment Paracrine activity of ADSC was assessed in an in vitro model of LESC dysfunction. Experiment was conducted in two aspects. First, the direct influence of conditioned medium (CM) from ADSC on LESC activity was assessed. The examined cells were compared to the control cells cultured in standard medium (SM). Then, LESC were pretreated with LPS to induce inflammation and again cultured in CM to Page 3/20 assess its influence under the condition of induced inflammation (CM_LPS group). The control cells were cultured in SM after analogous pretreatment with LPS (SM_LPS group). Cell culture conditions ADSC (PT-5006) were obtained from Lonza company (Switzerland) and LESC were provided by courtesy of Department of Microbial Biotechnology and Cell Biology, Faculty of Science, University of Debrecen (Hungary) where they were isolated and identified [25]. ADSC and LESC were cultured in a Dulbecco's Modified Eagle Medium (DMEM, Lonza, Switzerland) with 10% of fetal bovine serum (FBS, EuroClone, Italy) and 1% of antibiotics: amphotericin B with penicillin-streptomycin (Lonza, Switzerland) at 37˚C in a 5% CO2 incubator (Direct Heat CO2; Thermo Fisher Scientific, USA). The medium was changed every 48 h. Identification of ADSC was made with real time RTqPCR analysis of genes: CD73, CD90 and CD105 and flow cytometry with Human Mesenchymal Stem Cell Marker Verification Multi-Color Flow Cytometry Kit for the analysis of surface proteins: CD73, CD90 and CD105 (R&D Systems, USA). Cell fluorescence was measured with FACS Aria 2 (Becton Dickinson, USA) and the data was analyzed using software (Becton Dickinson, USA) (not shown). Conditioned medium preparation CM was collected from 24 h culture of ADSC at 60–70% confluency. It was centrifuged at 3500 rpm for 5 min to pellet down dead cells and cell debris. Then supernatants were filtered through 0.22 µm filters. The filtered supernatants were diluted with a SM (DMEM, 10% FBS, 1% antibiotics) in a ratio 1:1. [26]. The SM for the experiments was prepared simultaneously in analogous manner. First portion was incubated for 24 h in clean culture dishes and then it was centrifuged, filtered, and diluted in a 1:1 ratio with fresh portion of medium. Inflammation inducement To mimic the inflammation, LESC were pretreated with LPS (lipopolysaccharide from E. coli, Sigma- Aldrich, USA) at concentration of 2 µg/ml for 24 hours. ApoTox-Glo Triplex Assay To measure cell viability, cytotoxicity and apoptosis in the samples, the ApoTox-Glo Triplex Assay (Promega, USA) was used according to the manufacturer’s instructions. Viability, cytotoxicity, and apoptosis were evaluated after 24 h of cells exposure to examined conditions. LESC were cultured in SM, Page 4/20 CM after pretreatment with LPS (groups: SM_LPS, CM_LPS) and compared to standard conditions (groups: SM, CM). Viability and cytotoxicity were measured by the fluorescent signal, which was emitted due to the cleavage of the added substrates glycyl-phenylalanyl-aminofluorocoumarin (GF-AFC) for viability and bis-alanyl-alanyl-phenylalanyl-rhodamine 110 (bis-AAF-R110) for cytotoxicity by specific proteases. GF-AFC enters the cells where it is cleaved via the live-cell protease activity to generate a fluorescent signal that is proportional to the number of living cells. AAF-R110 was used to measure the dead-cell protease activity, which was released from the cells that have lost membrane integrity. Both substrates have different excitation (400 nm, 485 nm) and emission (505 nm, 520 nm) spectra. CM after pretreatment with LPS (groups: SM_LPS, CM_LPS) and compared to standard conditions (groups: SM, CM). Viability and cytotoxicity were measured by the fluorescent signal, which was emitted due to the cleavage of the added substrates glycyl-phenylalanyl-aminofluorocoumarin (GF-AFC) for viability and bis-alanyl-alanyl-phenylalanyl-rhodamine 110 (bis-AAF-R110) for cytotoxicity by specific proteases. GF-AFC enters the cells where it is cleaved via the live-cell protease activity to generate a fluorescent signal that is proportional to the number of living cells. AAF-R110 was used to measure the dead-cell protease activity, which was released from the cells that have lost membrane integrity. Both substrates have different excitation (400 nm, 485 nm) and emission (505 nm, 520 nm) spectra. Apoptosis was measured by the addition of a Caspase-Glo 3/7 Reagent. It is a luminogenic substrate that contains the tetrapeptide sequence DEVD in the reagent to assess caspase activity, luciferase activity and cell lysis. Fluorescence and luminescence were measured using a plate reader Triad LTMultimode Detector (Dynex Technologies, USA) [28]. All groups were analyzed in six replicates. Apoptosis was measured by the addition of a Caspase-Glo 3/7 Reagent. It is a luminogenic substrate that contains the tetrapeptide sequence DEVD in the reagent to assess caspase activity, luciferase activity and cell lysis. Fluorescence and luminescence were measured using a plate reader Triad LTMultimode Detector (Dynex Technologies, USA) [28]. All groups were analyzed in six replicates. Scratch assay LESC’S proliferation rate was analyzed in all study groups (SM, SM_LPS, CM, CM_LPS). For this purpose, a scratch assay (wound healing test) was done. The LESC were grown in standard condition to 80% confluence and then cells were exposed to LPS for 24 hours to induce the inflammation. Control cells were cultured without LPS. After reaching the full confluence, the scratch was made using 200 µl pipette tip and cells were washed with PBS to remove non-adherent cells. After that medium was replaced for SM or CM [29]. Then the scratch area was photographed with an Olympus IX81 microscope (Japan) with a DP70 Olympus camera (Japan) and the wound area was measured using Image J software in time points: 0 h, 6 h, 12 h, 24 h, 36 h. All groups were analyzed in six replicates. Enzyme-linked immunosorbent assay (ELISA) The concentration of interleukins (IL-2, IL-4, IL-10, IL-6, IL-1α) secreted by LESC to culture medium after exposure to the examined conditions, was analyzed with the use of immunoenzymatic tests (R&D Systems Quantikine ELISA Kits, USA) according to manufacturer protocol, as was described previously [31]. Optical density (OD) was read at 450 nm using Wallac 1420 VICTOR2™ (PerkinElmer Inc., USA). For each biological replicate three technical replicates were performed. Phenotype microarrays (PMM) The phenotype of LESC was assessed under the influence of SM, CM and SM with the addition of LPS (SM + LPS) with phenotype microarrays (PMM, Biolog, USA). Phenotype microarrays are 96-well microplates coated with different substrates in appropriate solvents e.g. ions, hormones and metabolic effectors. PMM show how cells metabolism, growth and productivity is affected by these agents. In order to the composition of substrates located on microarray, the PMM7 and PMM8 plates were used. These arrays are microplates coated with growth factors (e.g. IGF, FGF, PDGF), hormones (e.g. leptin, somatotropin, calcitonin, TSH) and cytokines (e.g. IL-1β, IL-6, IL-8, TNFα, IFNγ). This method enables the demonstration of cell preferences in the uptake of substrate added to the well of microarray. It also gives an information about possible cytotoxic effect of single compound. For this method cells previously were cultured in examined media for 24 h. Then, cells were collected by trypsinization and seeded on microarrays at the density of 10000 cells per well. For the each medium (SM, CM, SM + LPS) one microarray of each type was used. LESC were cultured on microarrays for 24 h and after this period a dedicated tetrazolium salt (provided by the producer) was added to the wells for 3 h of further incubation. Added salt was reduced to soluble formazan what appeared in color-change reaction. The measured absorbance value corresponded to cell viability. The absorbance was measured with a Wallac 1420 VICTOR plate reader (Perkin Elmer, USA) at 590 nm [30]. The phenotype of LESC was assessed under the influence of SM, CM and SM with the addition of LPS (SM + LPS) with phenotype microarrays (PMM, Biolog, USA). Phenotype microarrays are 96-well microplates coated with different substrates in appropriate solvents e.g. ions, hormones and metabolic effectors. PMM show how cells metabolism, growth and productivity is affected by these agents. In order to the composition of substrates located on microarray, the PMM7 and PMM8 plates were used. These arrays are microplates coated with growth factors (e.g. IGF, FGF, PDGF), hormones (e.g. leptin, Page 5/20 Page 5/20 Statistical analysis Statistical analysis was performed using Statistica 13.0 software (StatSoft, USA). A one-way ANOVA test with Tukey’s post hoc test and two-way ANOVA test or T-test were applied to evaluate any significant differences in the examined groups for normally distributed data. For non-normally distributed data Kruskal-Wallis test was applied. The level of significance was set at p < 0.05 for all statistical tests. Quantitative real-time polymerase chain reaction assay (real time RTqPCR) Total RNA was extracted from the cells using a TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions. The nucleic acid concentration was determined using a MaestroNano Spectrophotometer (Maestrogen, Taiwan). The expression of mRNAs related to inflammation (IL6, IL-10, IL-2, IL-1α, IL-1β, IFNγ) was detected using the real time RT-qPCR technique with SYBR Green chemistry (SensiFAST™ SYBR® No-ROX Kit, Bioline, USA) and an Opticon™ DNA Engine Continuous Fluorescence detector (MJ Research Inc., USA) as was previously described [27]. For each biological replicate three technical replicates were performed. Enzyme-linked immunosorbent assay (ELISA) Inflammation inducement Viability was evaluated after 24 h of LPS treatment at following concentrations: 0.1 µg/ml; 0.2 µg/ml; 0.5 µg/ml; 0.7 µg/ml; 1 µg/ml and 2 µg/ml in comparison to control (0 µg/ml of LPS). Control cells were taken as 100% (Fig. 1.). We have not observed statistically significant difference of mitochondrial activity in examined cells. However, meaningfully decreased viability was observed in 0.2 µg/ml (p = 0.0337) and 2 µg/ml (p = 0.0009) groups compared to cells treated by 0.5 µg/ml of LPS. In addition, viability of 2 µg/ml group was also lower in comparison to 0.7 µg/ml group (p = 0.0351). Based on the most meaningful biological effect, which was observed, the LPS concentration of 2 µg/ml was selected as optimal and nontoxic for the inflammation inducement in further analyses. Effect of ADSC secretome on viability, cytotoxicity, and apoptosis of LESC Impact of ADSC secretome on the transcriptome of LESC The expression of pro-inflammatory cytokines IL-1α (p = 0,0075) and IL-1β (p = 0,0098) was significantly lower in cells cultured in CM_LPS in comparison to CM. The IL-6 mRNA level was lower in CM_LPS cells (p = 0,0177) versus SM. Level of IFNγ mRNA in SM_LPS was higher (p = 0,0374) than in SM group. IL-2 and IL-10 mRNA did not differ in analyzed groups (Fig. 5.). Impact of ADSC secretome on inflammatory cytokines secretion by LESC Impact of ADSC secretome on phenotype Impact of ADSC secretome on phenotype of LESC Obtained results are presented in heatmaps (Fig. 4). The pattern of heatmap for each group significantly differs what shows that the preferences of cells has changed due to the exposure of examined media. A detailed analysis showed a diverse reaction to the presence of hormones and metabolic activators (growth factors and cytokines) present on microarrays, such as: insulin, resistin, glucagon, ghrelin, leptin, gastrin, exendin-3, hGH, IGF-I, FGF-1, PDGF-AB, IL-1β, IL-2, Il-6, Il- 8, PTH, calcitonin, LH, HCG, TSH, IFN-γ, adenosine, vasopressin, prolactin, calcitriol, LH-RH, ACTH, TRH, TNF-α, and Gly-His-Lys tripeptide. LESC showed decreased activity in the presence of insulin, IL-8, and prolactin in all examined groups. Cells cultured in CM showed less activity in response to IL-6 and the Gly-His-Lys tripeptide. Whereas cells after the culture in SM showed decreased activity in response to IL-2. Finally, cells cultured in SM + LPS showed less activity in the presence of glucagon. The above results varied in lower concentrations. A detailed description of our findings is included in discussion. Impact of ADSC secretome on the transcriptome of LESC Impact of ADSC secretome on proliferation and migration f LESC Impact of ADSC secretome on proliferation and migration of LESC Effect of ADSC secretome on viability, cytotoxicity, and apoptosis of LESC Page 6/20 Cells treated with CM showed a significant increase in viability (p = 0.023) and decrease in cytotoxicity (p = 0.033) comparing to the control. We have not observed significant changes in apoptosis of cells cultured in SM and CM. We did not find statistically significant differences in viability, cytotoxicity and apoptosis of cells pretreated with LPS (Fig. 2). Impact of ADSC secretome on proliferation and migration of LESC Scratch assay was made to assess an influence of ADSC secretome on LESC which were pretreated with LPS for 24 hours. The results of microscopic observations are presented in the Fig. 3A. Wound areas were calculated as the fold change of measured surface area (total number of pixels, N = 6) in examined groups (CM, SM, CM_LPS, SM_LPS) after 6 h, 12 h, 24 h and 36 h compared to the initial time point (0 h). Significant differences were observed between the examined groups. The two-way analysis of variance (ANOVA) (p < 0.05) showed that cells treated with CM had higher proliferation rate compared to the cells grown in SM, both in standard conditions and in induced inflammation. It was noticed that cells treated with LPS showed lower proliferation rate than non-stimulated cells, however the change was not statistically significant compared to standard conditions (Fig 3B) Discussion The first stage of experiment included the choice of lipopolysaccharide concentration for the inducement of inflammation. The effect of a series of concentrations of LPS on LESC was assessed, however MTT assay showed no statistically significant differences between the examined groups compared to the control. Similarly Kukolj et al. [32] observed that LPS did not affect proliferation and viability of periodontal ligament stem cells and it did not change their immunophenotype and cell cycle. Kukolj et al. showed that LPS acts on differentiation potential of cells and it inhibits osteogenesis and promotes chondrogenesis and adipogenesis [32]. Cell viability may change under the influence of LPS depending on the time of incubation and its concentration [33]. Many studies suggest the application of different concentrations from very low like 0,1 µg/ml [34]; through 0,5 µg/ml [35], 1 µg/ml [33] to 10 µg/ml [33, 34] or even 24 µg/ml [36]. Moreover, the suggested incubation time which is necessary for the induction of inflammation is diverse and takes e.g. from 2 h to 24 h [33]. Some studies indicate that a proper time to induce the inflammation in mesenchymal stem cells is 24 h with 1 µg/ml of LPS concentration [37]. Other suggest increasing the concentration of LPS to 2 µg/ml, but shortening the incubation time to 6 h in morphologically similar cells like normal human skin fibroblasts (NHDF) [38]. In contrast, LPS at concentration of 0,1 µg/ml was used for the induction of inflammation in macrophages for 24 h [39]. Therefore, based on numerous literature data and our findings, the concentration selected for this experiment was 2 µg/ml of LPS. Next, the analysis with ApoTox-Glo Triplex Assay showed that LESC viability increased after the treatment of conditioned medium from ADSC. The test showed also that the CM was not toxic to cells and as well there were no differences in cells apoptosis between groups. These results suggest that factors secreted by ADSC improve LESC’S viability. In the condition of induced inflammation, no statistically significant differences in cell viability, cytotoxicity and apoptosis were noticed under the influence of CM. These findings suggest that LPS inhibits the positive effect of ADSC’S secretome on limbal stem cells. Probably different signaling pathways were activated in the presence of LPS in the cells, not necessarily associated with the promotion of cell viability. It indicates that the addition of LPS does not significantly reduce cell activity. Impact of ADSC secretome on inflammatory cytokines secretion by LESC The secretion of cytokines (IL-2, IL-10, IL-6, IL-1α, IFNγ and IL-4) was quantified by ELISA. Only the presence of IL-6 and IL-1α was detectable, the secretion of IL2, IL10, IL4 and INFy has not been noticed. (Fig. 6). The concentration of IL-6 was significantly lower in cells cultured in CM both in standard conditions (p = 0,0002) and in induced inflammation (CM_LPS) (p = 0,0002) compared to SM and SM_LPS groups. The concentration of IL-1α was significantly lower both in CM (p = 0,0006) and CM_LPS (p = 0,0066) groups compared to SM_LPS. We also noticed lower level of IL-1α in SM (p = 0,0002) group compared to SM_LPS. There were no differences between CM, CM_LPS and SM groups. Discussion The secretion of cytokines (IL-2, IL-10, IL-6, IL-1α, IFNγ and IL-4) was quantified by ELISA. Only the presence of IL-6 and IL-1α was detectable, the secretion of IL2, IL10, IL4 and INFy has not been noticed. (Fig. 6). The concentration of IL-6 was significantly lower in cells cultured in CM both in standard conditions (p = 0,0002) and in induced inflammation (CM_LPS) (p = 0,0002) compared to SM and SM_LPS groups. The concentration of IL-1α was significantly lower both in CM (p = 0,0006) and CM_LPS (p = 0,0066) groups compared to SM_LPS. We also noticed lower level of IL-1α in SM (p = 0,0002) group compared to SM_LPS. There were no differences between CM, CM_LPS and SM groups. Discussion The secretion of cytokines (IL-2, IL-10, IL-6, IL-1α, IFNγ and IL-4) was quantified by ELISA. Only the presence of IL-6 and IL-1α was detectable, the secretion of IL2, IL10, IL4 and INFy has not been noticed. (Fig. 6). The concentration of IL-6 was significantly lower in cells cultured in CM both in standard conditions (p = 0,0002) and in induced inflammation (CM_LPS) (p = 0,0002) compared to SM and SM_LPS groups. The concentration of IL-1α was significantly lower both in CM (p = 0,0006) and CM_LPS (p = 0,0066) groups compared to SM_LPS. We also noticed lower level of IL-1α in SM (p = 0,0002) group compared to SM_LPS. There were no differences between CM, CM_LPS and SM groups. Discussion Discussion Probably a longer cell culture in the above-mentioned conditions could result in similar findings as in the case of cells cultured in standard conditions. However, these results show that the presence of LPS changes the activity of cellular proteases and caspase 3/7 in LECSs. Similar findings were reported by Chen and his team who investigated the effect of conditioned medium from mesenchymal stem cells derived from Wharton jelly on epithelial cells. It was shown that conditioned medium increased cells proliferation and migration by activation of stress response kinase JNK (c-Jun N- Page 8/20 terminal Kinase) and isoform of p38 protein [40]. Farahmand et al. also confirmed these results by observing higher expression of proliferation-related genes after culture in conditioned medium [41]. Some data indicated using a conditioned medium in the therapy of e.g. arthritis. In this case, a reduction of damages in cartilage tissue was shown and an inhibition of the immune response in cells has been noted [42]. Li et al. investigated using conditioned medium as a promoter of wound healing in diabetic patients in whom this process is usually disturbed. Research showed that the use of LPS reduces the rate of migration and proliferation in keratinocytes which grown in the presence of high glucose concentration, as the disease model for analyzing the effect of type 2 diabetes on cells. It was proven that the use of CM eliminates the negative effect of LPS and also negative effect of high glucose levels [43]. Also, the high potential of using CM in the treatment of bronchopulmonary dysplasia in oxygen-induced alveolar damage model showed that MSCs support tissue with antioxidant substances which therefore can lead to new therapy [44]. Scratch wound healing assay was performed to assess the rate of cell proliferation. LESC cultured in CM compared to SM, both under standard conditions and induced inflammation, showed better proliferation rate. It was noticed that cells stimulated with LPS and cultured in CM showed lower proliferative potential compared to unstimulated cells. These results indicate inhibition of cells proliferation by LPS, which is consistent with previous findings and the literature data [43]. Zhang et al. in in vivo studies showed that injection of ADSC promoted wound healing in rabbits [45]. Zeppieri et al. presented that mesenchymal stem cells promote corneal wound healing in rats [46]. Similarly, Galindo et al. demonstrated that ADSC have a therapeutic effect on LSCD in rabbits [47]. Discussion Phenotype microarrays showed that LESC in all study groups (SM, CM, SM + LPS) had various activity. PMM7 and PMM8 arrays were coated with six replicates of the same compound but in variable concentration. Producer provides an information that these concentrations are increasing from left to right, but does not give the information about the number and the unit, so we do not really know how much of each compound is in the well. We have numbered these concentrations from 1 to 6, where 1 is the lowest concentration and 6 is the highest. Due to a lot of data on cells activity, the results are presented on heat maps what ensures clear readability. All readings are presented as the fold change compared to control which was taken as 100%. We noticed that the activity of LPS significantly impact on LESC viability. This group of cells became more sensitive for bigger concentrations of each compound what appeared in the highest spread of fold change values. Interestingly, we noticed that LESC cultured in SM with the addition of LPS were more viable in presence of IL-6, while LESC cultured in SM and CM showed neutral or toxic effect of this cytokine. The lowest viability in presence of IL-6 was noticed in CM. We assume that while ADSC secrete IL-6, the final concentration of this cytokine in one well could be high enough to act negatively on LESC [48–50]. Surprisingly, the highest toxic effect was observed at the concentration “4”. LESC showed decreased activity in response to IL-8 in all examined groups. The toxic effect was more readable in higher concentrations and the highest values were noticed in CM group, but there were no differences between SM. Similarly, IL-2 in higher concentrations showed a toxic effect on LESC viability in all groups, but in opposite, lower concentrations of IL-2 induced LESC activity what was mostly notable in CM group. We observed that INFγ exerted a neutral response of LESC. In SM and CM Page 9/20 Page 9/20 groups we noted slightly increasing viability in direct proportion to the INFγ concentration. LESC cultured in SM + LPS showed decreased viability in the presence of INFγ. groups we noted slightly increasing viability in direct proportion to the INFγ concentration. LESC cultured in SM + LPS showed decreased viability in the presence of INFγ. Discussion Phenotype microarrays brought plenty of information which could help composing supplementation of culture media to improve cells viability. These factors could be potentially cross-linked in biopolymer scaffolds adapted for carrying ADSC to strengthen the response to secreted factors. However, further studies on the influence of presented factors are needed for better understanding of variable effect on LESC activity. The last stage included the assessment of mRNA expression and cytokine secretion of pro and anti- inflammatory cytokines in LESC due to the examined conditions. Increased expression of IL-1α and IL-1β was observed in cells cultured in CM at standard conditions compared to cells with induced inflammation (CM_LPS). IL-1α and IL-1β role is associated with the regulation of an immune response as a result of ongoing infection [51, 52]. IL-1 also acts by activating the secretion of many different cytokines and chemokines, e.g. IL-6, TNFα and IFNγ [51, 52]. IL-1 participate in the regulation of stem cell activity. In hematopoietic stem cells (HSCs), it exhibits radioprotective activity, induces their proliferation and differentiation [52]. Our findings showed no differences in the expression of IL-1α and IL-1β between cells cultured in CM and SM in both standard conditions and induced inflammation. The lower expression of both interleukins in CM_LPS group suggests that CM has a significant impact on IL-1 release during the inflammation. This activity was confirmed by ELISA detection of IL-1α. The concentration of this IL-1α in LESC’S culture medium was lower during induced inflammation under the influence of CM (CM_LPS group). Furthermore, significant difference in the secretion of IL-1α between LECSs with induced inflammation and LESC in standard conditions cultured in SM indicates that LPS has meaningful impact on IL-1α release. Finally, observed no differences in IL-1α secretion between CM, CM_LPS and SM group suggest that CM downregulates the release of IL-1α which means that ADSC secrete factors which mitigates the inflammation. Some studies suggest that an increase in interleukin-1 secretion was observed under the influence of cellular stress e.g. in a state of hypoxia, chemical or physical damage. During the apoptosis which is natural, programmed cell death IL-1α is not released by the cells [51]. Solomon et al. reported that stroma of amniotic membrane inhibits the level of IL-1α and IL-1β, after LPS stimulation, in cell culture of limbal epithelial stem cells [53]. Discussion It suggests that the use of CM simultaneously with amniotic membrane could give better therapeutic results in cornea treatment by achieving a synergistic effect. The analysis of the IL-6 mRNA expression showed decreased copy number in cells with induced inflammation cultured in CM compared to SM. ELISA also indicated a significant decrease of IL-6 secretion by LESC with induced inflammation compared to cells cultured in SM. Similarly, lower concentration of IL-6 in the same group was noticed at LESC in standard conditions. Studies indicate that IL-6 secretion increase in cells during inflammation [54, 55]. However, it was proven that MSCs naturally secrete IL-6 [56, 57]. This interleukin is responsible for the regulation of the immune response, hematopoiesis, apoptosis, proliferation and cell viability [58]. It plays important role in regulation of homeostasis in corneal limbus cells niche. IL-6 may help in wound healing of epithelial cells in vivo [59, 60]. Perhaps a significant increase in the level of this interleukin may be the result of cellular responses to unfavorable environmental conditions e g like LPS induced inflammation Lower levels of Similarly, lower concentration of IL-6 in the same group was noticed at LESC in standard conditions. Studies indicate that IL-6 secretion increase in cells during inflammation [54, 55]. However, it was proven that MSCs naturally secrete IL-6 [56, 57]. This interleukin is responsible for the regulation of the immune response, hematopoiesis, apoptosis, proliferation and cell viability [58]. It plays important role in regulation of homeostasis in corneal limbus cells niche. IL-6 may help in wound healing of epithelial cells in vivo [59, 60]. Perhaps a significant increase in the level of this interleukin may be the result of cellular responses to unfavorable environmental conditions e.g. like LPS induced inflammation. Lower levels of Page 10/20 Page 10/20 IL-6 at LESC cultured in CM suggest a beneficial property of ADSC secreted factors. Probably factors present in the conditioned medium from ADSC promote the LESC regeneration. Perhaps the use of IL-6 as a supplement for cell culture could provide better proliferation. However, this issue requires further study. The analysis also revealed an increase of IFNγ mRNA expression in LESC with induced inflammation cultured in SM compared to the LECSs in standard conditions cultured in the same medium. This indicates that LPS stimulation probably caused upregulation of this gene expression. However, IFNγ was not detected by ELISA. Discussion It should be remembered that the increase in mRNA level is ahead of the protein concentration. Gene expression depends on many transcription factors which activation changes the level of mRNA in cells. Thus, when translation product is at demanded level, the DNA transcription can be suppressed. In opposite, when there is a low level of protein, gene expression can be continuously upregulated. Conclusion Our results showed that the paracrine activity of ADSC on LESC promotes its proliferation and mitigates the adverse impact of LPS. We find the ADSC’S secreted factors useful for LESC regeneration. ADSC can serve as supporting cells for corneal wound healing. Placing the ADSC in closed carrier can probably be used for temporal wound dressing. The ADSC’S secretome could be used in development of new medicinal products for corneal injures. Acknowledgements We would like to express our deepest appreciation to prof. dr hab. Urszula Mazurek and dr hab. Monika Paul-Samojedny for invaluable methodological support and sharing of laboratory facilities. Authors kindly acknowledge the support from Medical University of Silesia in Katowice (Upper Silesia, Poland) grant no. KNW-1-032/N/9/I and PCN-2-004/N/0/O Competing interests The authors declare that there is no conflict of interest regarding the publication of this article The authors declare that there is no conflict of interest regarding the publication of this article Authors' contributions B.S. and A.S-S. conceived and designed the experiments; B.S. and A.P. performed the experiments; A.S-S. and J.G. analyzed the data; J.G. contributed reagents/materials/analysis tools; B.S., A.S-S. wrote the paper. References Page 11/20 Page 11/20 1. Willoughby, C. E. et al. Anatomy and Physiology of the Human Eye: Effects of Mucopolysaccharidoses Disease on Structure and Function - a Review: Anatomy and Physiology of the Eye. Clin. Experiment. Ophthalmol. 38, 2–11 https://doi.org/10.1111/j.1442-9071.2010.02363.x (2010). 2. Le-Bel, G. et al. Grafting of an Autologous Tissue-Engineered Human Corneal Epithelium to a Patient with Limbal Stem Cell Deficiency (LSCD). Am. J. Ophthalmol. 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The bars represent the means ± standard deviation (SD) of the percentages of Page 16/20 Page 16/20 the control cell viability (100%); ANOVA with the Tukey post hoc test, p<0.05, p<0.01. Figure 2 Cells viability, cytotoxicity and apoptosis of CM, SM, CM_LPS, SM_LPS groups. The activity of the dead- cell proteases (viability and cytotoxicity) and the activity of caspase 3/7 (apoptosis) are shown as relative fluorescence units or the luminescence intensity. The bars represent the means ± standard deviation (SD). T test, p<0.05. the control cell viability (100%); ANOVA with the Tukey post hoc test, p<0.05, p<0.01. Figure 2 the control cell viability (100%); ANOVA with the Tukey post hoc test, p<0.05, *p<0.01. Figure 2 Cells viability, cytotoxicity and apoptosis of CM, SM, CM_LPS, SM_LPS groups. The activity of the dead- cell proteases (viability and cytotoxicity) and the activity of caspase 3/7 (apoptosis) are shown as relative fluorescence units or the luminescence intensity. The bars represent the means ± standard deviation (SD). T test, p<0.05. Page 17/20 Figure 3 Figure 3 Figure 3 Page 17/20 Page 17/20 Left: A. Scratch assay. The results present a microscopic image of the selected repetition for each group (CM, SM, CM_LPS, SM_LPS) at time points (0h, 6h, 12h, 24h ,36h). Scratch area marked with black. C1 = SM, C2 = SM_LPS Right: B. Scratch surface area as fold of time point 0h of examined groups (CM, SM, CM_LPS, SM_LPS) in time (6h, 12h, 24h, 36h). T test, p<0.05. Left: A. Scratch assay. The results present a microscopic image of the selected repetition for each group (CM, SM, CM_LPS, SM_LPS) at time points (0h, 6h, 12h, 24h ,36h). Scratch area marked with black. C1 = SM, C2 = SM_LPS Right: B. Scratch surface area as fold of time point 0h of examined groups (CM, SM, CM_LPS, SM_LPS) in time (6h, 12h, 24h, 36h). T test, p<0.05. CM_LPS, SM_LPS) in time (6h, 12h, 24h, 36h). T test, p<0.05. Figure 4 Heatmap of fold change in Phenotype MicroArrays for mammalian cells, PMM7 and PMM8 – „MicroPlate™ - Hormones & Metabolic Effectors" in examined groups. SM - standard medium, CM - conditioned medium, SM+LPS – standard medium with the addition of 2 µg/ml LPS. Red – highest values, blue – lowest values, yellow – neutral. Interleukins are marked with red frame. Figure 4 Figure 4 Heatmap of fold change in Phenotype MicroArrays for mammalian cells, PMM7 and PMM8 – „MicroPlate™ - Hormones & Metabolic Effectors" in examined groups. SM - standard medium, CM - conditioned medium, SM+LPS – standard medium with the addition of 2 µg/ml LPS. Red – highest values, blue – lowest values, yellow – neutral. Interleukins are marked with red frame. Page 18/20 Figure 5 The mRNA levels of inflammation markers IL-1α, IL-1β, IL-2, IL-6, IL-10, IFNγ. The bars represent the (Me) with the 25th and 75th quartiles and the minimum and maximum of the copy numbers per 1 µg of total RNA. The Kruskal Wallis test with post hoc was applied to assess any differences in the expression of the genes, p<0,05. Figure 5 The mRNA levels of inflammation markers IL-1α, IL-1β, IL-2, IL-6, IL-10, IFNγ. The bars represent the (Me) with the 25th and 75th quartiles and the minimum and maximum of the copy numbers per 1 µg of total RNA. The Kruskal Wallis test with post hoc was applied to assess any differences in the expression of the genes, p<0,05. Page 19/20 Figure 6 Secretion of cytokines by LESC in all examined groups (CM, SM, CM_LPS, SM_LPS) measured by ELISA. ANOVA with the Tukey post hoc test; IL6: p<0,01, p<0,001 Figure 6 Secretion of cytokines by LESC in all examined groups (CM, SM, CM_LPS, SM_LPS) measured by ELISA. ANOVA with the Tukey post hoc test; IL6: p<0,01, ***p<0,001 Page 20/20
https://openalex.org/W2887087557	https://escholarship.org/content/qt1zg9w5x0/qt1zg9w5x0.pdf?t=qe4708	English	null	Inhibition of EGFR Signaling Protects from Mucormycosis	MBio	2,018	cc-by	9,447	UCLA UCLA Previously Published Works Title Inhibition of EGFR Signaling Protects from Mucormycosis Permalink https://escholarship.org/uc/item/1zg9w5x0 Journal mBio, 9(4) ISSN 2161-2129 Authors Watkins, Tonya N Gebremariam, Teclegiorgis Swidergall, Marc et al. Publication Date 2018-09-05 DOI 10.1128/mbio.01384-18 Peer reviewed UCLA UCLA Previously Published Works Title Inhibition of EGFR Signaling Protects from Mucormycosis Permalink https://escholarship.org/uc/item/1zg9w5x0 Journal mBio, 9(4) ISSN 2161-2129 Authors Watkins, Tonya N Gebremariam, Teclegiorgis Swidergall, Marc et al. Publication Date 2018-09-05 DOI 10.1128/mbio.01384-18 Peer reviewed Journal mBio, 9(4) ISSN 2161-2129 Authors Watkins, Tonya N Gebremariam, Teclegiorgis Swidergall, Marc et al. Publication Date 2018-09-05 DOI 10.1128/mbio.01384-18 Peer reviewed Inhibition of EGFR Signaling Protects from Mucormycosis Tonya N. Watkins,a Teclegiorgis Gebremariam,b Marc Swidergall,b Amol C. Shetty,a Karen T. Graf,a Abdullah Alqarihi,b Sondus Alkhazraji,b Abrar I. Alsaadi,b Vonetta L. Edwards,a Scott G. Filler,b,c Ashraf S. Ibrahim,b,c Vincent M. Brunoa,d aInstitute for Genome Sciences, University of Maryland School of Medicine, Baltimore, Maryland, USA bDivision of Infectious Diseases, Los Angeles Biomedical Research Institute at Harbor, UCLA Medical Center, Torrance, California, USA cDavid Geffen School of Medicine at UCLA, Torrance, California, USA dDepartment of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, USA ABSTRACT Mucormycosis is a life-threatening, invasive fungal infection that is caused by various species belonging to the order Mucorales. Rhizopus species are the most common cause of the disease, responsible for approximately 70% of all cases of mucormycosis. During pulmonary mucormycosis, inhaled Rhizopus spores must adhere to and invade airway epithelial cells in order to establish infection. The molecular mechanisms that govern this interaction are poorly understood. We per- formed an unbiased survey of the host transcriptional response during early stages of Rhizopus arrhizus var. delemar (R. delemar) infection in a murine model of pulmo- nary mucormycosis using transcriptome sequencing (RNA-seq). Network analysis re- vealed activation of the host’s epidermal growth factor receptor (EGFR) signaling. Consistent with the RNA-seq results, EGFR became phosphorylated upon in vitro in- fection of human alveolar epithelial cells with several members of the Mucorales, and this phosphorylated, activated form of EGFR colocalized with R. delemar spores. Inhibition of EGFR signaling with cetuximab or geﬁtinib, speciﬁc FDA-approved in- hibitors of EGFR, signiﬁcantly reduced the ability of R. delemar to invade and dam- age airway epithelial cells. Furthermore, geﬁtinib treatment signiﬁcantly prolonged survival of mice with pulmonary mucormycosis, reduced tissue fungal burden, and attenuated the activation of EGFR in response to pulmonary mucormycosis. These results indicate EGFR represents a novel host target to block invasion of alveolar epi- thelial cells by R. delemar, and inhibition of EGFR signaling provides a novel ap- proach for treating mucormycosis by repurposing an FDA-approved drug. IMPORTANCE Mucormycosis is an increasingly common, highly lethal fungal infec- tion with very limited treatment options. Using a combination of in vivo animal models, transcriptomics, cell biology, and pharmacological approaches, we have demonstrated that Mucorales fungi activate EGFR signaling to induce fungal uptake into airway epithelial cells. Inhibition of EGFR signaling with existing FDA-approved drugs signiﬁcantly increased survival following R. arrhizus var. delemar infection in mice. Received 25 June 2018 Accepted 20 July 2018 Published 14 August 2018 Citation Watkins TN, Gebremariam T, Swidergall M, Shetty AC, Graf KT, Alqarihi A, Alkhazraji S, Alsaadi AI, Edwards VL, Filler SG, Ibrahim AS, Bruno VM. 2018. Inhibition of EGFR signaling protects from mucormycosis. mBio 9:e01384-18. https://doi.org/10.1128/mBio .01384-18. Powered by the California Digital Library University of California eScholarship.org RESEARCH ARTICLE crossm RESEARCH ARTICLE Inhibition of EGFR Signaling Protects from Mucormycosis This study enhances our understanding of how Mucorales fungi invade host cells during the establishment of pulmonary mucormycosis and provides a proof-of- concept for the repurposing of FDA-approved drugs that target EGFR function. Received 25 June 2018 Accepted 20 July 2018 Published 14 August 2018 Citation Watkins TN, Gebremariam T, Swidergall M, Shetty AC, Graf KT, Alqarihi A, Alkhazraji S, Alsaadi AI, Edwards VL, Filler SG, Ibrahim AS, Bruno VM. 2018. Inhibition of EGFR signaling protects from mucormycosis. mBio 9:e01384-18. https://doi.org/10.1128/mBio .01384-18. Invited Editor Robert T. Wheeler, University of Maine July/August 2018 Volume 9 Issue 4 e01384-18 KEYWORDS EGFR, geﬁtinib, Rhizopus, mucormycosis KEYWORDS EGFR, geﬁtinib, Rhizopus, mucormycosis M ucormycosis is an invasive fungal infection of humans caused by species of the order Mucorales, subphylum Mucormycotina (1, 2). Rhizopus spp. are the most common etiologic agent of mucormycosis and are responsible for approximately 70% of all cases (1–3). The primary risk factors for mucormycosis include neutropenia, diabetes mellitus resulting in hyperglycemia and diabetic ketoacidosis (DKA), solid M Address correspondence to Vincent M. Bruno, vbruno@som.umaryland.edu. July/August 2018 Volume 9 Issue 4 e01384-18 ® mbio.asm.org 1 mbio.asm.org 1 ® Watkins et al. organ or bone marrow transplantation, treatment with corticosteroids, deferoxamine therapy, trauma and burns (e.g., wounded soldiers in combat), and malignant hema- tological disorders (2, 4). The most common forms of mucormycosis, based on anatomical site, are rhino- orbital/cerebral, pulmonary, cutaneous, gastrointestinal, and disseminated. Rhino-or- bital/cerebral mucormycosis is found almost exclusively in DKA patients, while pulmo- nary disease is mainly found in neutropenic patients (5). Cutaneous necrotizing mucormycosis outbreaks in healthy individuals have also been reported and often follow natural disasters or severe trauma (e.g., infections following the tsunami that devastated Southeast Asia in 2004 and the tornadoes that occurred in Joplin, MO, in June 2011) (6, 7). Since there are no federal requirements to report fungal infections, the true prevalence of mucormycosis is likely to be much higher than currently reported. p There are very few antifungal agents approved by the United States FDA for treating mucormycosis; the ﬁrst is amphotericin B (AmB), which has been used to treat mucor- mycosis for the last 6 decades. AmB has signiﬁcant nephrotoxicity, other adverse effects, and very limited clinical success (2, 8). Although isavuconazole and posacona- zole have been recently approved to treat mucormycosis, neither is considered to be superior to AmB treatment (8, 9). In the absence of surgical removal of the infected focus (for example, excision of the eye in patients with rhinocerebral mucormycosis), antifungal therapy alone is rarely curative. Even when surgical debridement is com- bined with high-dose antifungal therapy, the mortality associated with mucormycosis is 50%. In patients with prolonged neutropenia or disseminated disease, mortality is 90 to 100% (10, 11). The limited treatment options coupled with the high mortality and morbidity rates and the frequently disﬁguring surgical therapy provide a clear mandate to explore alternative approaches to treat this infection. In the case of pulmonary mucormycosis, infection is generally acquired by inhalation of spores that are ubiquitous in nature. KEYWORDS EGFR, geﬁtinib, Rhizopus, mucormycosis As lung epithelial cells are among the ﬁrst host cells that interact with Mucorales spores during pulmonary infection, a molecular understanding of how these cells sense and respond to the pathogen is essential to understanding the pathogenesis of pulmonary mucormycosis. While the invasion of endothelial cells by Rhizopus arrhizus var. delemar (referred to from here on as R. dele- mar) is known to be mediated by the interaction between the fungus-encoded CotH3 and the host-encoded GRP78 (12, 13), the speciﬁc interactions that govern adherence and invasion of lung epithelial cells are poorly understood. In this study, we used transcriptome sequencing (RNA-seq) to examine the host transcriptional response to R. delemar infection in a well-established in vivo murine model of pulmonary mucormycosis. Network analysis of the data set revealed the modulation of host pathways that were not previously linked to the host response to mucormycosis. We provide evidence that the epidermal growth factor receptor (EGFR) pathway is activated by R. delemar and other Mucorales infection and that this activation mediates invasion of lung epithelial cells by these fungi. Importantly, ge- ﬁtinib, an FDA-approved drug that inhibits EGFR, protected mice from mucormycosis. These results suggest that inhibition of EGFR signaling provides a novel therapeutic approach for treating mucormycosis. July/August 2018 Volume 9 Issue 4 e01384-18 RESULTS RNA-seq analysis of a murine model of mucormycosis. We analyzed the host transcriptional response to R. delemar in a DKA murine model of pulmonary mucor- mycosis due to R. delemar infection. At 14 and 24 h postinoculation, animals were sacriﬁced, and the lungs were harvested for extraction of total RNA for subsequent transcriptome analysis using RNA-seq. These time points were chosen because they represent early stages of infection, prior to the onset of massive tissue damage and necrosis, which might complicate interpretation of transcriptome analyses. The early time points also allowed us to focus on the initial response of the lung tissue during adhesion of spores to and subsequent invasion of the airway epithelium. From each of July/August 2018 Volume 9 Issue 4 e01384-18 mbio.asm.org 2 ® EGFR Signaling and Mucormycosis the 12 sequencing libraries, we obtained an average of 96.4 15.8 million reads that mapped to the mouse reference genome (see Table S1 in the supplemental material). The inclusion of a poly(A) enrichment step in the RNA-seq protocol, as predicted, resulted in the detection of transcripts from the infecting fungus. However, a robust analysis of the R. delemar transcriptome was precluded by the lack of sufﬁcient reads that mapped to the R. delemar reference genome (6,488 reads combined from all six infected samples). Therefore, we focused our analysis of these samples on the host response. We have previously demonstrated that infection-induced transcriptome changes can be used to identify signaling pathways that govern the interaction between host and fungal pathogen (14–16). Using the Ingenuity Pathway Analysis (IPA) software (Ingenuity Systems; http://www.ingenuity.com), we performed an upstream regulator analysis on the sets of host genes that were differentially expressed (P 0.05) (see Tables S2 and S3 in the supplemental material) between the infection groups and the appropriate time-matched negative-control groups, which were immunosuppressed but not infected. This approach was validated by our identiﬁcation of two pathways that were already linked to infection by Mucorales: interleukin-22 (IL-22) and IL-17A (Fig. 1A) (17). Our analysis also predicted the activation of signaling pathways that have not been tied to mucormycosis but have been associated with the host response to fungal infection, including colony-stimulating factor 2 (CSF2), extracellular signal-regulated kinases (ERKs), myeloid differentiation primary response 88 (MYD88), and JNK (Jun N-terminal kinase) (18–24) (Fig. 1A). We also noticed a striking temporal dynamic in our data set. RESULTS Speciﬁcally, the majority of the pathways are modulated at 14 h postinoculation (hpi) and returning to normal 24 h postinfection. Furthermore, a smaller subset of pathways was modulated only at 24 h postinfection. Of particular interest was the signiﬁcant overlap between genes that are differen- tially expressed 14 h postinoculation with R. delemar and the known transcriptional targets of the epidermal growth factor receptor (EGFR) signaling pathway (P value of overlap, 9.57 106). Speciﬁcally, R. delemar infection induced the expression of 18 genes that are known to be activated by EGFR signaling (Fig. 1B), providing evidence for the activation of EGFR protein in response to R. delemar infection. Further support of the activation of EGFR signaling was provided by the predicted activation of mir-21 (P value overlap, 8.6 1015 [Fig. 1A]), a microRNA that governs the expression of genes involved in many different biological processes (25–30). EGFR activation enhances the expression of mir-21 in lung epithelial cells (31). Our sequenc- ing approach, which is geared toward the detection of long transcripts, does not allow the examination of microRNAs. However, in our infection model, 28 known repression targets and 5 known activation targets of mir-21 were downregulated or upregulated, respectively, 14 h after inoculation relative to the uninfected control group (see Table S4 in the supplemental material). This downregulation of mir-21-repressed genes is consistent with an EGFR-stimulated increase in mir-21 expression. In the database used to perform the upstream regulator analysis, none of the 33 mir-21 target genes are annotated as EGFR targets, and they are therefore not included in Fig. 1C, so the total number of differentially expressed genes that provide evidence of EGFR pathway activation is 51. EGFR signaling is activated during in vitro infection of airway epithelial cells. We have previously examined the transcriptional response of A549 human alveolar epithelial cells to infection with R. delemar at 6 and 16 h (15). Upstream regulator analysis of this in vitro RNA-seq data set also revealed a signiﬁcant overlap between genes that are differentially expressed following R. delemar infection and the known transcriptional targets of the EGFR signaling pathway (P values of 4.33 102 and 1.43 103 for 6 and 16 h, respectively). Speciﬁcally, R. RESULTS delemar infection induced changes in gene expression of 34 known downstream targets of EGFR signaling in a direction that is consistent with the activation of the EGFR (29 activated and 5 repressed [Fig. 1C]). July/August 2018 Volume 9 Issue 4 e01384-18 mbio.asm.org 3 ® Watkins et al. FIG 1 Host response to R. delemar infection in vivo and in vitro. (A) Mouse upstream regulators that are predicted to be changed in R. delemar-infected lungs in a mouse DKA model of mucormycosis. Red indicates predicted activation (Z score of 2). Teal indicates predicted repression (Z score of 2). Black indicates no predicted effect. (B) Expression of known mouse EGFR targets in lungs 14 h postinoculation of the in vivo DKA model. (C) Expression of known human EGFR targets in A549 cells at 6 and 16 h following inoculation of an in vitro infection. Plotted for panels B and C are the log-transformed reads per kilobase per million (RPKM) values that have been normalized across all samples. Red indicates high gene expression; blue indicates low expression. Each column in panels B and C represents an individual sample from a different mouse. FIG 1 Host response to R. delemar infection in vivo and in vitro. (A) Mouse upstream regulators that are predicted to be changed in R. delemar-infected lungs in a mouse DKA model of mucormycosis. Red indicates predicted activation (Z score of 2). Teal indicates predicted repression (Z score of 2). Black indicates no predicted effect. (B) Expression of known mouse EGFR targets in lungs 14 h postinoculation of the in vivo DKA model. (C) Expression of known human EGFR targets in A549 cells at 6 and 16 h following inoculation of an in vitro infection. Plotted for panels B and C are the log-transformed reads per kilobase per million (RPKM) values that have been normalized across all samples. Red indicates high gene expression; blue indicates low expression. Each column in panels B and C represents an individual sample from a different mouse. FIG 1 Host response to R. delemar infection in vivo and in vitro. (A) Mouse upstream regulators that are predicted to be changed in R. delemar-infected lungs in a mouse DKA model of mucormycosis. Red indicates predicted activation (Z score of 2). Teal indicates predicted repression (Z score of 2). Black indicates no predicted effect. July/August 2018 Volume 9 Issue 4 e01384-18 RESULTS (B) Expression of known mouse EGFR targets in lungs 14 h postinoculation of the in vivo DKA model. (C) Expression of known human EGFR targets in A549 cells at 6 and 16 h following inoculation of an in vitro infection. Plotted for panels B and C are the log-transformed reads per kilobase per million (RPKM) values that have been normalized across all samples. Red indicates high gene expression; blue indicates low expression. Each column in panels B and C represents an individual sample from a different mouse. When EGFR is activated by ligand binding, tyrosine residues at the intracellular carboxy terminus become phosphorylated and the signal is transmitted to a variety of downstream signaling pathways (32). To conﬁrm the EGFR activation by an orthogonal approach, we tested whether R. delemar infection of the A549 pulmonary epithelial cell July/August 2018 Volume 9 Issue 4 e01384-18 mbio.asm.org 4 ® EGFR Signaling and Mucormycosis FIG 2 Phosphorylation and localization of EGFR in A549 cells infected with Mucorales. (A) Representative immunoblot examining tyrosine phosphorylation of EGFR residue Y1068 in response to individual infection with ﬁve different Mucorales fungi. (B) Densitometric analysis of the immunoblots. ctrl, control. FIG 2 Phosphorylation and localization of EGFR in A549 cells infected with Mucorales. (A) Representative immunoblot examining tyrosine phosphorylation of EGFR residue Y1068 in response to individual infection with ﬁve different Mucorales fungi. (B) Densitometric analysis of the immunoblots. ctrl, control. line stimulated the phosphorylation of EGFR by immunoblotting. R. delemar infection induced the phosphorylation of EGFR on tyrosine residue 1068 when examined 3 h postinfection (Fig. 2). The same phosphorylation was also induced following infection with four other Mucorales species—Rhizopus oryzae, Lichtheimia corymbifera, Mucor circinelloides, and Cunninghamella bertholletiae (Fig. 2)—indicating that EGFR activation is not a strain- or species-speciﬁc phenomenon. Consistent with these results, we observed that the phosphorylated (activated) form of EFGR colocalized with R. delemar spores during in vitro infection of A549 cells (Fig. 3). We did not observe colocalization when R. delemar spores were examined in the absence of A549 cells (see Fig. S1 in the supplemental material). Taken together, these results are consistent with a model in which Mucorales interacts with EGFR and activates signaling in airway epithelial cells. EGFR signaling governs the uptake of Mucorales and subsequent damage of airway epithelial cells. The predicted activation of EGFR and mir-21 signaling early during the infection process, the colocalization of activated EGFR with R. RESULTS delemar spores that were germinated for 1 h. , P 0.0001, and , P 0.01, versus control by Wilcoxon rank-sum test. Data are expressed as median interquartile range and represent at least two indepen- dent experiments. FIG 4 Effects of EGFR inhibition on invasion of airway epithelial cells by R. delemar. A549 alveolar epithelial cells were pretreated with vehicle, 25 M geﬁtinib, or 25 g/ml cetuximab for 1 h followed by (top) 3 h of infection in the presence of inhibitor with 2 105 R. delemar spores or (bottom) 24 h of infection (in the presence of inhibitors) with 2 106 R. delemar spores that were germinated for 1 h. , P 0.0001, and *, P 0.01, versus control by Wilcoxon rank-sum test. Data are expressed as median interquartile range and represent at least two indepen- dent experiments. Pretreatment with geﬁtinib also signiﬁcantly reduced R. delemar-induced damage of A549 cells, when assayed by lactate dehydrogenase (LDH) assay (Fig. 4B). To complement our studies with geﬁtinib, which targets the intracellular tyrosine kinase domain of EGFR, we examined the ability of cetuximab to block epithelial cell invasion. Cetuximab is a monoclonal antibody that recognizes the extracellular portion of EGFR and has been shown to block ligand-dependent activation of EGFR signaling (36). When the same host cells were pretreated for 1 h with 25 g/ml cetuximab, endocytosis of R. delemar spores and host cell damage were both signiﬁcantly reduced compared to pretreatment with an equivalent amount of IgG1 control antibody (Fig. 4C and D). Notably, neither geﬁtinib nor cetuximab inhibited R. delemar mycelial growth (see Fig. S2 in the supplemental material). Pretreatment of R. delemar spore prepara- tions with geﬁtinib or cetuximab, followed by rinsing, prior to the infection did not reduce endocytosis or host cell damage (see Fig. S3 in the supplemental material). The concordance between the results of the endocytosis assay and the host cell damage assay is consistent with previous observations that endocytosis of R. delemar is a prerequisite for inducing host cell damage of endothelial cells (37). We also found that pretreatment with geﬁtinib reduced fungus-induced damage of A549 cells following infection with R. oryzae, L. corymbifera, M. circinelloides, and C. bertholletiae (Fig. 5). Collectively, these results indicate that EGFR signaling is required for maximal invasion of and damage to alveolar epithelial cells by Mucorales. July/August 2018 Volume 9 Issue 4 e01384-18 RESULTS delemar, and the involvement of EGFR in invasion by Candida albicans and diverse microbial patho- gens (33–35) compelled us to explore the possibility that EGFR mediates the invasion of airway epithelial cells by R. delemar. Thus, we next tested whether blocking EGFR signaling would protect alveolar epithelial cells from invasion by R. delemar. We used geﬁtinib, a clinically relevant EGFR kinase inhibitor, to study its effect on R. delemar- mediated endocytosis by alveolar epithelial cells and their subsequent damage. When A549 cells were pretreated for 1 h with 25 M geﬁtinib, endocytosis of R. delemar spores was signiﬁcantly reduced compared to pretreatment with vehicle alone (Fig. 4A). FIG 3 Localization of phosphor-EGFR in A549 cells infected with R. delemar. A549 cells were infected for 30 min with 2 105 R. delemar spores that had been germinated for 1 h. Cells were then stained for phospho-EGFR (red) and host cell nuclei using Hoechst (blue). DIC, differential inference contrast. FIG 3 Localization of phosphor-EGFR in A549 cells infected with R. delemar. A549 cells were infected for 30 min with 2 105 R. delemar spores that had been germinated for 1 h. Cells were then stained for phospho-EGFR (red) and host cell nuclei using Hoechst (blue). DIC, differential inference contrast. mbio.asm.org 5 July/August 2018 Volume 9 Issue 4 e01384-18 ® Watkins et al. FIG 4 Effects of EGFR inhibition on invasion of airway epithelial cells by R. delemar. A549 alveolar epithelial cells were pretreated with vehicle, 25 M geﬁtinib, or 25 g/ml cetuximab for 1 h followed by (top) 3 h of infection in the presence of inhibitor with 2 105 R. delemar spores or (bottom) 24 h of infection (in the presence of inhibitors) with 2 106 R. delemar spores that were germinated for 1 h. , P 0.0001, and *, P 0.01, versus control by Wilcoxon rank-sum test. Data are expressed as median interquartile range and represent at least two indepen- dent experiments. FIG 4 Effects of EGFR inhibition on invasion of airway epithelial cells by R. delemar. A549 alveolar epithelial cells were pretreated with vehicle, 25 M geﬁtinib, or 25 g/ml cetuximab for 1 h followed by (top) 3 h of infection in the presence of inhibitor with 2 105 R. delemar spores or (bottom) 24 h of infection (in the presence of inhibitors) with 2 106 R. RESULTS EGFR signaling has also been shown to facilitate invasion of oral epithelial cells by C. albicans which, like R. delemar, enters cells by induced endocytosis. The EGFR- dependent invasion of C. albicans requires the activation of the aryl hydrocarbon receptor (Ahr) and the subsequent activation of Src family kinases, which in turn phosphorylate and activate EGFR (34, 38). To address whether the same mechanism is being employed to facilitate invasion of airway epithelial cells by R. delemar, we measured the effect of an Ahr inhibitor (CH-223191) and an Src inhibitor (Src kinase mbio.asm.org 6 ® EGFR Signaling and Mucormycosis FIG 5 Geﬁtinib inhibits infection-induced damage of airway epithelial cells by four different species of Mucorales. A549 alveolar epithelial cells were pretreated with vehicle or geﬁtinib (25 or 50 M) for 1 to 2 h followed by 24 h of infection (in the presence of inhibitors) with 2 106 (C. bertholletiae and R. oryzae) or 3 106 (L. corymbifera and M. circineollides) spores that were germinated for 1 h , P 0.0001, and *, P 0.01, versus the control by Wilcoxon rank sum test. Data are expressed as median interquartile range and represent at least two independent experiments done in triplicate. FIG 5 Geﬁtinib inhibits infection-induced damage of airway epithelial cells by four different species of Mucorales. A549 alveolar epithelial cells were pretreated with vehicle or geﬁtinib (25 or 50 M) for 1 to 2 h followed by 24 h of infection (in the presence of inhibitors) with 2 106 (C. bertholletiae and R. oryzae) or 3 106 (L. corymbifera and M. circineollides) spores that were germinated for 1 h , P 0.0001, and *, P 0.01, versus the control by Wilcoxon rank sum test. Data are expressed as median interquartile range and represent at least two independent experiments done in triplicate. inhibitor II) on endocytosis. Neither of the inhibitors altered the ability of A549 cells to endocytose R. delemar spores (see Fig. S4 in the supplemental material). These results suggest that the mechanism of activation of EGFR in epithelial cells is distinct from the activation of EGFR by C. albicans. Geﬁtinib treatment increases survival of mice with mucormycosis. We next sought to determine if EGFR signaling governs the establishment and/or progres- sion of mucormycosis in a well-established in vivo murine model of mucormycosis. RESULTS Unfortunately, mice harboring deletions in EGFR die within the ﬁrst 8 days of life (39), thus precluding our ability to test the receptor in a traditional mouse gene deletion experiment. Furthermore, there are no published lung-speciﬁc deletion models for EGFR. Therefore, we infected neutropenic mice intratracheally with R. delemar (40) and treated them with 10 mg/kg of body weight geﬁtinib (41) or vehicle alone (placebo) for 5 consecutive days starting 4 h postinfection. We chose to begin the intervention at 4 h because invasion of lung epithelial cells is an early event in the infection. Placebo-treated mice had a median survival time of 8 days and 85% mortality by day 15 postinfection (Fig. 6A). In contrast, mice treated with geﬁtinib had a median survival time of 21 days, and 55% of the mice remained alive by day 21, when the experiment was terminated, and the surviving mice appearing healthy. Corroborating the effects of geﬁtinib on survival, mice treated with this drug had an ~1-log reduction in their lungs and brains relative to placebo-treated mice (Fig. 6B). Consistent with these results, we found that infec- tion with R. delemar induced marked tyrosine phosphorylation of EGFR in the lung (Fig. 6C) and that treatment of mice with geﬁtinib signiﬁcantly reduced this phosphorylation (Fig. 6C). These results validate our in vitro observations and support the potential of targeting EGFR signaling as a novel therapeutic strategy for mucormycosis by immediately repurposing currently FDA-approved cancer drugs. July/August 2018 Volume 9 Issue 4 e01384-18 DISCUSSION Invasion of the airway epithelium is a crucial, yet poorly understood, step in the initiation of pulmonary mucormycosis. We took an unbiased approach to understand the host side of this interaction by performing RNA-seq analysis of infected lungs harvested during early stages of a well-established clinically relevant murine model of mucormycosis. A network analysis of differential gene expression in these mice re- vealed the enrichment for genes that are known to be downstream of EGFR and its regulatory target, mir-21. Because our analysis was performed on RNA harvested from whole lungs of infected mice, we were unable to determine which cell types responded to the infection with the increased gene expression. The predicted activation of EGFR signaling at 14 h, and not 24 h, could be indicative of a host response to the early stage of infection when R. delemar ﬁrst invades the airway epithelium. Our in vitro analyses mbio.asm.org 7 July/August 2018 Volume 9 Issue 4 e01384-18 ® Watkins et al. FIG 6 Geﬁtinib inhibits EGFR phosphorylation in vivo and protects mice from pulmonary mucormycosis. (A) Survival of neutropenic mice (n 20/group from two independent experiments with similar results) infected intratracheally with R. delemar (average inoculum of 3.8 103 spores per mouse) and treated with vehicle control (placebo) or 10 mg/kg geﬁtinib 4 h postinfection for 5 consecutive days. , P 0.0084 versus placebo-treated mice by log rank test. (B) Tissue fungal burden of lungs and brains of mice (n 10 per group) infected intratracheally with R. delemar (5.6 103 spores per mouse of conﬁrmed inoculum) and treated with vehicle control (placebo) or geﬁtinib. Data are presented as median interquartile range. (C) Twenty-four hours post-intratracheal infection, lungs of geﬁtinib (GEF)-treated or untreated mice were harvested, sectioned, and stained with calcoﬂuor white (CFW) and anti-pY1068 antibody. Scale bars, 20 m. Dashed boxes in the top row of panels indicate the sections shown enlarged in the bottom row. ctrl, control. FIG 6 Geﬁtinib inhibits EGFR phosphorylation in vivo and protects mice from pulmonary mucormycosis. (A) Survival of neutropenic mice (n 20/group from two independent experiments with similar results) infected intratracheally with R. delemar (average inoculum of 3.8 103 spores per mouse) and treated with vehicle control (placebo) or 10 mg/kg geﬁtinib 4 h postinfection for 5 consecutive days. *, P 0.0084 versus placebo-treated mice by log rank test. July/August 2018 Volume 9 Issue 4 e01384-18 DISCUSSION At this point, we do not know the molecular mechanism by which EGFR signaling is being stimulated during R. delemar infection. The colocalization of EGFR with fungal spores is consistent with an interaction between a fungal cell wall component and EGFR. The invasion of A549 cells by an R. delemar strain with attenuated expression of CotH3 (12), the Mucorales ligand to host GRP78 during interaction with human umbilical vein endothelial cells, was not different from A549 cell invasion caused by wild-type R. delemar (Ashraf Ibrahim, unpublished data). Thus, CotH3 protein is not the fungal ligand by which EGFR is activated. Furthermore, the R. delemar genome does not encode a recognizable ortholog of ALS3 or SSA1 and inhibition of Src kinase activity or Ahr does not inhibit R. delemar invasion, suggesting that EGFR stimulation by Mucor- ales occurs via a different mechanism than that by C. albicans. Further experiments are required to determine the molecular nature of the activation. Our in vitro blocking data using EGFR inhibitors did not entirely abrogate the ability of R. delemar to invade and damage alveolar epithelial cells. This strongly indicates that receptors other than EGFR are involved in the interaction of Rhizopus with these host cells. We have previously identiﬁed a host cell receptor, GRP78, required for R. delemar hematogenous dissemination. Speciﬁcally, the host 78-kDa glucose-regulated protein GRP78 interacts with the Mucorales-speciﬁc CotH3 cell wall protein during invasion of human umbilical vein endothelial cells (12). Because of the lack of CotH3 contribution to invasion of A549 cells (Ibrahim, unpublished), it is unlikely that GRP78 acts as a coreceptor with EGFR during interactions with invading Mucorales. Alternatively, GRP78 could contribute to Mucorales invasion of alveolar epithelial cells through interacting with a fungal ligand(s) other than CotH3. These possibilities are the topic of active investigation. Since EGFR plays a central role in the progression of several types of cancer, signiﬁcant effort has been put forth into developing therapies that target EGFR function (46). To this end, geﬁtinib, a small molecular inhibitor of EGFR kinase activity, and cetuximab, a monoclonal antibody speciﬁc for the extracellular domain of EGFR, provide valuable tools to test the role of EGFR in epithelial cell invasion by R. delemar. Indeed, in our mouse model of mucormycosis, treatment with geﬁtinib signiﬁcantly increased survival and reduced tissue fungal burden of target organs. These results are consistent with a model in which R. DISCUSSION (B) Tissue fungal burden of lungs and brains of mice (n 10 per group) infected intratracheally with R. delemar (5.6 103 spores per mouse of conﬁrmed inoculum) and treated with vehicle control (placebo) or geﬁtinib. Data are presented as median interquartile range. (C) Twenty-four hours post-intratracheal infection, lungs of geﬁtinib (GEF)-treated or untreated mice were harvested, sectioned, and stained with calcoﬂuor white (CFW) and anti-pY1068 antibody. Scale bars, 20 m. Dashed boxes in the top row of panels indicate the sections shown enlarged in the bottom row. ctrl, control. of human airway epithelial (A549) cells support this idea. Speciﬁcally, 34 EGFR- responsive genes are modulated during infection with R. delemar, and phospho-EGFR colocalized with endocytosed spores. Furthermore, pharmacological inhibition of EGFR signaling reduced both fungus-induced cytotoxicity and endocytosis of the spores by the A549 cells. Collectively, these results suggest that EGFR signaling mediates the invasion of airway epithelial cells by R. delemar. Hence, EGFR is the ﬁrst host cell receptor to be implicated in the interaction between R. delemar and airway epithelial cells. In general, EGFR signaling can be activated by binding to any one of seven known host-derived ligands, all of which are expressed as precursor proteins that span the plasma membrane and are cleaved by cell surface proteases upon stimulation (42). Upon cleavage, the extracellular domains are released as mature proteins into the extracellular space, where they can bind to and activate EGFR on the surface of the mbio.asm.org 8 July/August 2018 Volume 9 Issue 4 e01384-18 ® EGFR Signaling and Mucormycosis same cells or on adjacent cells (42). In addition, many pathogens, including viruses, bacteria, and fungi, seem to have evolved the ability to exploit EGFR signaling to gain entry into host cells (33), and the mechanism of EGFR activation across these patho- genic microbes varies. For example, in the context of viral infection, hepatitis C virus increases signaling by disrupting EGFR recycling to enhance its surface expression (43, 44). In contrast, the Campylobacter jejuni bacterium induces lipid raft formation, result- ing in clustering and subsequent activation of EGFR (45), while meningitic Escherichia coli activates EGFR signaling by increasing sphingosine 1-phosphate (S1P2)-dependent release of heparin-binding ligand-like epidermal growth factor (HB-EGF) (35). The fungal pathogen C. albicans expresses two surface proteins, Als3p and Ssa1p, that likely bind to and activate EGFR and ErbB2 heterodimers (34). July/August 2018 Volume 9 Issue 4 e01384-18 MATERIALS AND METHODS On day 4, mice were sacriﬁced, and lungs and brains, representing primary and secondary target organs (40), were collected and processed for tissue fungal burden by quantitative PCR (qPCR) (47). Values are expressed as log10 spore equivalents per gram of tissue. All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of the Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center according to the NIH guide- lines for animal housing and care (approval reference no. 21125). Isolation of RNA from lung tissue. Male ICR mice were immunosuppressed and infected as described above. Lungs were harvested 14 or 24 h postinfection and ﬂash frozen in liquid nitrogen prior to extracting total RNA using Tri reagent solution (Ambion). RNA-seq and gene expression analysis. Our deep sequencing analysis was performed in triplicate with three lungs per group isolated from 3 different mice. Sequencing libraries (non-strand-speciﬁc, paired end) were prepared with the TruSeq RNA sample prep kit (Illumina). The total RNA samples were subjected to poly(A) enrichment as part of the TruSeq protocol. One hundred ﬁfty nucleotides of sequence was determined from both ends of each cDNA fragment using the HiSeq platform (Illumina) per the manufacturer’s protocol. Sequencing reads were annotated and aligned to the UCSC mouse reference genome (mm10, GRCm38.75) using TopHat2 (48). The alignment ﬁles from TopHat2 were used to generate read counts for each gene, and a statistical analysis of differential gene expression was performed using the edgeR package from Bioconductor (49). A gene was considered differentially expressed if the P value for differential expression was less than 0.05. To identify modulated signal transduction pathways, we used the upstream regulator analytic of IPA (Ingenuity Systems) to identify signaling proteins that are potentially activated or repressed during the course of infection. This analysis determines the overlap between lists of differentially expressed genes and an extensively curated database of regulator-target gene relationships. It then considers the direction of the gene expression changes to make predictions about activation or repression of speciﬁc pathways. Immunoblot of EGFR phosphorylation in vitro. The A549 type II pneumocyte cell line (American Type Culture Collection) was grown as described previously (50). A549 cells in 24-well tissue culture plates were incubated in F-12K tissue culture medium supplemented with fetal bovine serum to a ﬁnal concentration of 10%. Prior to infection, the A549 cells were serum starved for 120 min. Spores of R. delemar, R. MATERIALS AND METHODS Fungal strains and host cells. R. delemar strain 99-880 (a clinical isolate obtained from a patient with rhino-orbital mucormycosis), R. oryzae strain 99-892, L. corymbifera strain 008-049, M. circinelloides strain NRRL3631, and C. bertholletiae strain 175 were grown on peptone-dextrose agar (PDA) plates for 3 to 5 days at 37°C. Spores were collected in endotoxin-free Dulbecco’s phosphate-buffered saline (DPBS), washed with endotoxin-free DPBS, and counted with a hemocytometer to prepare the ﬁnal inocula. To form germlings, spores were incubated in yeast extract-peptone-dextrose (YPD) with shaking for 1 h at 37°C. Germlings were washed twice with endotoxin-free DPBS. The A549 type II pneumocyte cell line was grown in tissue culture dishes in F-12K medium with L-glutamine plus 10% fetal bovine serum (FBS). Drug. Src kinase inhibitor II (CAS 459848-35-2; Calbiochem) was obtained from Millipore (catalog no. 567806). Murine models of mucormycosis. For the in vivo RNA-seq experiments, diabetic ketoacidosis (DKA) was induced and mice were infected intratracheally with a target inoculum of 2.5 105 fungal spores of R. delemar 99-880 in 25 l as previously described (12). To test the effect of geﬁtinib on mouse survival following infection, male ICR mice (20 to 25 g [from Envigo]) were immunosuppressed by cyclophos- phamide (200 mg/kg administered intraperitoneally [i.p.]) and cortisone acetate (500 mg/kg adminis- tered subcutaneously) given on days 2, 3, and 9 relative to infection. This treatment resulted in 16 days of pancytopenia (40). To control for bacterial infection, immunosuppressed mice received 50 mg/liter enroﬂoxacin (Baytril; Bayer, Leverkusen, Germany) ad libitum on day 3 through day 0, after which the enroﬂoxacin was replaced with daily ceftazidime (5 mg/mouse) treatment administered subcutaneously through day 13 relative to infection. Mice were infected with 2.5 105 spores of R. delemar 99-880 in 25 l PBS given intratracheally as previously described (40). Treatment with geﬁtinib (10 mg/kg dissolved in 10% dimethylacetamide–90% polyethylene glycol 300 [DMA-PEG 300] and administered i.p.) started 4 h postinfection and continued once daily through day 4. Placebo-treated mice received DMA-PEG 300. Survival of mice served as the primary endpoint, with moribund mice humanely euthanized. To determine the effect of treatment on tissue fungal burden, mice were immunosuppressed and infected as described above. Geﬁtinib treatment started 4 h postinfection and continued through day 3. DISCUSSION delemar stimulates EGFR signaling in vivo to facilitate endocytosis of the fungus by airway epithelial cells and raise the exciting possibility of repurposing an FDA-approved drug to potentially control these invasive fungal infections. Since immunocompromised patients are the most susceptible and likely to develop mucormycosis, the observed efﬁcacy of geﬁtinib in neutropenic mice adds to the clinical relevance and the potential for our results to translate to humans as an adjunctive therapy to current antifungal agents. Furthermore, because geﬁtinib targets the host and does not affect the growth or morphology of the fungus, acquisition of resistance is less likely to occur. To summarize, we have identiﬁed host EGFR as a key mediator of early invasion of alveolar epithelial cells by Mucorales. Of great clinical importance is our ﬁnding that inhibition of EGFR function by an FDA-approved drug ameliorates murine mucormy- cosis and is likely to represent a new therapeutic modality as adjunctive therapy to lethal mucormycosis. July/August 2018 Volume 9 Issue 4 e01384-18 mbio.asm.org 9 ® Watkins et al. July/August 2018 Volume 9 Issue 4 e01384-18 mbio.asm.org 10 MATERIALS AND METHODS Twelve-millimeter glass coverslips were seeded with A549 alveolar epithelial cells. Cells were then pretreated for 1 h with geﬁtinib (25 M) or cetuximab (25 g/ml). For controls, host cells will be incubated with DMSO (the solvent used to reconstitute the inhibitor) or 25 g/ml mouse IgG antibody in parallel. Host cells were then infected with 2 105 R. delemar spores. After incubation for 3 h, cells were ﬁxed in 3% paraformaldehyde and stained for 1 h with 1% Uvitex, which speciﬁcally binds to chitin in the fungal cell wall. After being washed with PBS, coverslips were mounted on a glass slide with a drop of ProLong Gold antifade reagent (Molecular Probes) and sealed. The total number of cell-associated organisms (i.e., fungi adhering to the monolayer) per high-powered ﬁeld was determined by phase-contrast microscopy. The same ﬁeld was then examined by epiﬂuores- cence microscopy, and the number of brightly ﬂuorescent, uninternalized fungi was determined. The number of endocytosed organisms was calculated by subtracting the number of ﬂuorescent fungi from the total number of visible fungi. At least 400 organisms were counted per treatment group in at least 15 different ﬁelds per coverslip. Experiments were performed in duplicate or triplicate on at least two separate days. Confocal microscopy. The accumulation of epithelial cell EGFR and pEGFR around R. delemar was visualized using the Zeiss LSM Duo confocal microscopy system. Twelve-millimeter glass coverslips in 12-well dishes were seeded with A549 alveolar epithelial cells and infected with 2 105 R. delemar spores. After incubation at 37°C, cells were washed with HBSS and ﬁxed with 3% paraformaldehyde. Cells were blocked and incubated with 1:500 mouse anti-EGFR (Santa Cruz; no. sc-373746) and 1:500 rabbit anti-pEGFR (Cell Signaling; no. 3777). Coverslips were washed and counterstained with 1:500 Alexa Fluor 546-labeled goat anti-mouse IgG and Alexa Fluor 488-labeled goat anti-rabbit IgG. Host cell nuclei were stained with Hoechst 33342 (Thermo Fisher). After washing, coverslips were mounted on a glass slide with ProLong Gold antifade reagent (Molecular Probes) and viewed by z-stacking using the Zeiss LSM Duo confocal microscopy system. Immunoﬂuorescence of EGFR phosphorylation in vivo. CD-1 mice were immunosuppressed with cyclophosphamide (200 mg/kg) and cortisone acetate (500 mg/kg) on day 2 relative to infection. They were inoculated by intratracheal injection of 1.0 107 R. delemar cells. MATERIALS AND METHODS oryzae, L. corymbifera, M. circinelloides, or C. bertholletiae were incubated in RPMI for 60 min at 37C, washed, and suspended in F-12K medium. A549 cells were infected for 3 h with a multiplicity of infection (MOI) of 5. Next, the cells were rinsed with cold Hanks balanced salt solution (HBSS) containing protease and phosphatase inhibitors and removed from the plate with a cell scraper. After collecting the cells by centrifugation, they were boiled in 2 SDS sample buffer. The lysates were separated by SDS-PAGE, and Y1068 EGFR phosphorylation was detected with a phospho-speciﬁc antibody (Cell Signaling; no. 2234). The blots were then stripped, and total protein levels and -actin were detected by immunoblotting with appropriate antibodies against EGFR (Cell Signaling; no. 4267), and -actin (Cell Signaling; 3700). The immunoblots were developed using enhanced chemiluminescence and imaged with a C400 (Azure Biosystems) digital imager. Measurement of R. delemar-induced host cell damage. R. delemar-induced A549 cell damage was quantiﬁed using the Pierce LDH assay, with slight modiﬁcations to the manufacturer’s protocol. Brieﬂy, A549 cells were grown in 96-well tissue culture plates for 18 to 24 h. Cells were then pretreated for 1 h mbio.asm.org 10 ® EGFR Signaling and Mucormycosis with geﬁtinib (25 M) or cetuximab (25 g/ml) and infected with 2 106 germlings suspended in 150 l F-12K plus 10% FBS. For controls, host cells were incubated with dimethyl sulfoxide (DMSO) (the solvent used to reconstitute the inhibitor) or 25 g/ml of mouse IgG antibody in parallel. After 24 h of incubation at 37°C, 50 l of the cell culture supernatant was collected from uninfected, infected, and fungus-only control wells and transferred to a 96-well plate to assay for LDH activity. Lysis buffer was added to all infected wells, and the mixture was incubated for 45 min at 37°C. After lysis, 50 l of cell culture supernatant was transferred to a 96-well plate and used for the LDH assay kit per the protocol. LDH release was calculated as follows: % cytotoxicity [(experimental release fungal cell spontaneous control host cell spontaneous control)/(host cell maximum control fungal cell maximum control host cell spontaneous control)] 100. LDH is a cytosolic enzyme but will be released into the cell culture medium upon cell membrane damage. The amount of extracellular LDH is proportional to the amount of cell damage. Measurement of host cell endocytosis. MATERIALS AND METHODS After 24 h of infection, the mice were sacriﬁced and the lungs were harvested, snap-frozen in optimal cutting temperature (OCT) compound. Ten-micrometer-thick sections were cut with a cryostat and ﬁxed with cold acetone. Protein phosphorylation was detected as described elsewhere (38, 51). Brieﬂy, the cryosections were rehydrated in PBS and then blocked with 10% bovine serum albumin (BSA). Sections were stained with a phospho- EGFR antibody (Cell Signaling; no. 2234), followed by a secondary antibody. The organisms were stained with calcoﬂuor white (Sigma-Aldrich; no. 18909) and imaged by confocal microscopy. Statistical analyses. In vitro experiments were performed in triplicate on two or three separate days. Data are expressed as the median interquartile range. Treatment groups were compared to controls using the Wilcoxon rank sum test. For the murine studies, survival of mice was analyzed using the log rank test, whereas differences in tissue fungal burden were analyzed by the Wilcoxon rank sum test using GraphPad Prism 6. P values of 0.05 were considered signiﬁcant. Accession number(s). All of the raw sequencing reads from this study have been submitted to the NCBI sequence read archive (SRA) under BioProject accession no. PRJNA429656 (https://www.ncbi.nlm .nih.gov/bioproject/PRJNA429656). The speciﬁc sample accession numbers are presented in Table S1. SUPPLEMENTAL MATERIAL Supplemental material for this article may be found at https://doi.org/10.1128/mBio .01384-18. REFERENCES 15. Chibucos MC, Soliman S, Gebremariam T, Lee H, Daugherty S, Orvis J, Shetty AC, Crabtree J, Hazen TH, Etienne KA, Kumari P, O’Connor TD, Rasko DA, Filler SG, Fraser CM, Lockhart SR, Skory CD, Ibrahim AS, Bruno VM. 2016. An integrated genomic and transcriptomic survey of mucormycosis-causing fungi. Nat Commun 7:12218. https://doi.org/10 .1038/ncomms12218. 1. Ribes JA, Vanover-Sams CL, Baker DJ. 2000. Zygomycetes in human disease. Clin Microbiol Rev 13:236–301. https://doi.org/10.1128/CMR.13 .2.236-301.2000. 2. Spellberg B, Edwards J, Jr, Ibrahim A. 2005. Novel perspectives on mucormycosis: pathophysiology. presentation, and management. Clin Mi- crobiol Rev 18:556–569. https://doi.org/10.1128/CMR.18.3.556-569.2005. 3. Roden MM, Zaoutis TE, Buchanan WL, Knudsen TA, Sarkisova TA, Schaufele RL, Sein M, Sein T, Chiou CC, Chu JH, Kontoyiannis DP, Walsh TJ. 2005. Epidemiology and outcome of zygomycosis: a review of 929 reported cases. 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ACKNOWLEDGMENTS This project was funded in part with federal funds from the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Department of Health and Human Services, under U19AI110820 to V.M.B., S.G.F., and A.S.I., R01AI063503 to A.S.I., R01AI124566 and R01DE022600 to S.G.F., and K99DE026856 to M.S. A.S.I. owns shares in Vitalex Biosciences, a start-up company that is developing immunotherapies and diagnostics for mucormycosis. The remaining authors declare no competing interests. .01384-18. FIG S1, PDF ﬁle, 0.4 MB. FIG S2, PDF ﬁle, 38.1 MB. FIG S3, PDF ﬁle, 0.1 MB. FIG S4, PDF ﬁle, 0.1 MB. TABLE S1, XLSX ﬁle, 0.1 MB. TABLE S2, XLSX ﬁle, 0.2 MB. TABLE S3, XLSX ﬁle, 0.2 MB. TABLE S4, XLSX ﬁle, 0.1 MB. FIG S1, PDF ﬁle, 0.4 MB. FIG S2, PDF ﬁle, 38.1 MB. FIG S3, PDF ﬁle, 0.1 MB. FIG S4, PDF ﬁle, 0.1 MB. TABLE S1, XLSX ﬁle, 0.1 MB. TABLE S2, XLSX ﬁle, 0.2 MB. TABLE S3, XLSX ﬁle, 0.2 MB. 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https://openalex.org/W4309938065	http://repository.essex.ac.uk/34119/1/2022_Kostelka_Singh_Blais_Compulsory_Voting.pdf	English	null	Is compulsory voting a solution to low and declining turnout? Cross-national evidence since 1945	Political science research and methods	2,022	cc-by	11,475	Abstract Despite the substantial body of research on compulsory voting’s (CV) relationship with turnout, much remains unknown about the role of different types of CV rules, their enforcement, and their ability to prevent the secular turnout decline observed around the world. Moreover, existing studies that leverage changes to CV laws are limited to a single country. We assemble rich new data on voter turnout and electoral legislation that, we believe, include the most accurate and extensive cross-national measure of CV to date. We test three theoretically derived hypotheses: that CV enforcement matters for participation; that enforcement’s effect is conditioned by state capacity; and that, only when CV is enforced, will it miti- gate voter turnout’s post-1970 tendency to decline. We find support for each. We also find that the nature of sanctions for non-voting is irrelevant for participation. Keywords: Comparative politics: elections and campaigns; political institutions; political participation and turnout; voting behavior A key and unsurprising correlate of compulsory voting (CV) is elevated voter turnout. Evidence of this dates back nearly a century and comes from a variety of empirical approaches (for reviews, see Birch (2009) and Singh (2021)). Still, much remains unknown about the extent to which dif- ferent types of mandatory rules boost participation and whether states’ ability to enforce penalties might matter. It is also unclear whether CV can prevent long-term declines in participation rates. This is an important question since shifts in turnout can affect electoral outcomes, representation, and political equality (see Blais et al., 2020 for a review). p q y With a focus on both breadth and intra-country changes to CV laws, we assembled a new data set on voter turnout and CV. Our data cover over 1400 elections in 116 countries over the years 1945–2017. In creating our data set, we reviewed contemporary and historical documents, legis- lation, and constitutions to create what we believe is the most accurate and extensive measure of CV to date. There are a total of 26 countries that used CV in at least one election in our data set and 11 countries that experienced a switch to or away from CV within our time period. y We subject three theoretically derived hypotheses to our data. The first predicts that the positive impact of CV on turnout will be biggest where there are enforced legal sanctions for abstention. ORIGINAL ARTICLE ORIGINAL ARTICLE © The Author(s), 2022. Published by Cambridge University Press on behalf of the European Political Science Association. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution and reproduction, provided the original article is properly cited. Is compulsory voting a solution to low and declining turnout? Cross-national evidence since 1945 Filip Kostelka1,2* , Shane P. Singh3 and André Blais4 1Department of Political and Social Sciences, European University Institute, Fiesole, Italy, 2Department of Government, University of Essex, Colchester, UK, 3Department of International Affairs, University of Georgia, Athens, USA and 4Département de science politique, Université de Montréal, Montreal, Canada Corresponding author. Email: filip.kostelka@eui.eu (Received 1 November 2021; revised 16 May 2022; accepted 30 August 2022) Political Science Research and Methods (2022), page 1 of 18 doi:10.1017/psrm.2022.57 Background, theory, and expectations Several dozen previous studies, which we summarize in Table 1, have sought to estimate the impact of CV on turnout across countries. This body of work is nearly unanimous in finding that voter participation tends to be higher where it is legally required. However, prior cross- country studies rely on coarse measures of CV, they rarely take seriously the challenges inherent to demonstrating causation, and they generally ignore intertemporal patterns. Panagopoulos (2008) provides the first rigorous consideration of how qualitative variation in CV laws may affect their influence on turnout, recognizing that utility-maximizing citizens will participate only if the expected benefits exceed the expected costs of doing so. Enforced CV alters the result of this calculation by effectively imposing a “negative cost” of voting (Colomer, 1991). A voter avoids a penalty by participating and thus decides to vote even if the act of voting entails some costs of its own. In line with his model, Panagopoulos finds that CV is ineffectual where there is no enforce- ment but that it robustly increases turnout where non-voters are likely to receive a meaningful punishment. The finding that CV has the biggest impact on turnout where non-voters are most likely to be non-trivially sanctioned is consistent with one earlier (Fornos et al., 2004) and several subsequent (e.g., Birch, 2009; Singh, 2011; Dassonneville and Hooghe, 2017) studies. However, laws may also have an “expressive function” wherein they affect societal norms and shape citizens’ behavior by altering perceptions of what others approve or disapprove (e.g., Sunstein, 1996). As such, cost-benefit calculations about the expected benefits of shirking the law versus the consequences of being caught doing so may not alone predict compliance. Feitosa et al. (2020) find that CV can engender in citizens a sense of civic duty, which is itself a strong motivator of electoral participation (Riker and Ordeshook, 1968). As such, unenforced CV should boost turnout. Still, not all citizens will feel or act upon a sense of duty to vote, and CV may actually reinforce counter-participatory attitudes among those opposed to electoral democracy (Singh, 2018). Therefore, it cannot be taken as a given that “toothless” CV laws will be as effective as the enforced variety. Instead, anticipation of legal sanctions for non-voting will further increase the efficaciousness of mandatory voting rules. Abstract The second puts forth that sanctioned CV is most effectual where the state acts as a credible administrator that will actually implement sanctions for non-voting. Our final hypothesis is that the long-term secular decline in voter turnout observed in much of the democratic world can be mitigated only by enforced sanctions for abstention. Filip Kostelka et al. 2 Using a variety of empirical approaches, we find support for each of our hypotheses. First, we show that CV without enforced sanctions increases turnout by about 7.5–10 points, as a percent- age of registered voters, and that CV with enforced legal sanctions boosts turnout by a sizeable 14.5–18.5 points. These findings do not just confirm the unsurprising positive impact of CV in turnout; they demonstrate that this impact is remarkably large, even where citizens face little prospect of being fined for non-participation. Second, we find that the state’s ability to carry out the administration of fines is a key factor when there are legal penalties for abstention. Thus, compulsory rules are most effectual where there are legal sanctions and the state can credibly identify and punish non-voters. Finally, we find that, while electoral participation has not declined over time in countries with enforced CV sanctions, unenforced CV does not arrest ongoing declines in voter turnout. Thus, unenforced CV can ‘shift the intercept,’ increasing the turnout rate relative to countries with voluntary voting over time, but it does not prevent the secular decline in participation. Background, theory, and expectations As shown by Blais and Achen (2019), cost-benefit calculations shape the turnout decision for those who do not feel an intrinsic duty to vote. For these individuals, enforced CV can shift expected utility in favor of showing up to the polls. In short, in places with CV but no sanctions for abstention, many people participate merely because they feel a duty to obey the law or align with norms. In places with enforced sanctions for non-voting, even those who feel no intrinsic motivation to vote are more likely to turn out to the polls due to the threat of a penalty. Based on this reasoning, we advance our first hypothesis: Political Science Research and Methods 3 Table 1. Cross-national studies that have estimated the effect of compulsory voting on turnout Data Positive Accounts for Escalatory Author(s) (year) level Region effect? levels of CV? effect? Tingsten (1937) Aggregate Europe Yes No − Powell (1980) Aggregate Global Yes No − Crewe (1981) Aggregate Global Yes No − Powell (1982) Aggregate Global Yes No − Powell and Bingham (1986) Aggregate Advanced democracies Yes No − Jackman (1987) Aggregate Advanced democracies Yes No − Colomer (1991) Aggregate Advanced democracies Yes No − Kaempfer and Lowenberg (1993) Aggregate Global No No − Jackman and Miller (1995) Aggregate Advanced democracies Yes No − Oppenhuis (1995) Both Europe Yes No − Franklin (1996) Aggregate Global Yes No − Franklin et al. (1996) Individual Europe Yes No − Katz (1997) Aggregate Global Yes No − Blais and Dobrzynska (1998) Aggregate Global Yes No − Franklin (1999) Individual Europe Yes No − Blais (2000) Aggregate Global Yes No − Franklin (2001) Aggregate Europe Yes No − Pérez-Liñán (2001) Aggregate Latin America Yes No − Franklin (2002) Aggregate Global Yes No − Norris (2002) Aggregate Global No No − Perea (2002) Both Europe Yes No − Siaroff and Merer (2002) Aggregate Europe Yes No − Fornos et al. (2004) Aggregate Latin America Yes Yes Yes Franklin et al. legall Escalator effect? − − − − − − − − − − − − − − − − − − − − − − Yes − − − − − − Yes Yes Yes − Yes Yes − Yes − Yes − Yes − − Yes Yes Yes − theoretic cial turnou nforcemen cement a The studies listed in this table are limited to English-language cross-national studies that have employed compulsory voting as a theoretical independent variable (rather than a control) and meta analyses. “Data level” can be individual (survey data) or aggregate (official turnout statistics). “Accounts for levels of CV?” refers to whether the study uses multiple categories to account for variation in the use of enforcement and sanctions. “Escalatory effect?” refers to whether the study found that CV’s influence is stronger where sanctions and/or enforcement are steeper. Meta-analysis. †Study finds a positive impact of CV in full democracies but not elsewhere. The studies listed in this table are limited to English-language cross-national studies that have employed compulsory voting as a theoretical independent variable (rather than a control) and meta analyses. “Data level” can be individual (survey data) or aggregate (official turnout statistics). “Accounts for levels of CV?” refers to whether the study uses multiple categories to account for variation in the use of enforcement and sanctions. “Escalatory effect?” refers to whether the study found that CV’s influence is stronger where sanctions and/or enforcement are steeper. Meta-analysis. †Study finds a positive impact of CV in full democracies but not elsewhere. The studies listed in this table are limited to English-language cross-national studies that have employed compulsory voting as a theoretical independent variable (rather than a control) and meta analyses. “Data level” can be individual (survey data) or aggregate (official turnout statistics). “Accounts for levels of CV?” refers to whether the study uses multiple categories to account for variation in the use of enforcement and sanctions. “Escalatory effect?” refers to whether the study found that CV’s influence is stronger where sanctions and/or enforcement are steeper. Meta-analysis. †Study finds a positive impact of CV in full democracies but not elsewhere. https://doi.org/10.1017/psrm.2022.57 Background, theory, and expectations (2004) Aggregate Advanced democracies Yes No − Norris (2004) Aggregate Global Mixed† No − Schraufnagel and Sgouraki (2005) Aggregate Latin America Yes No − Geys (2006)* Aggregate Global Yes No − Flickinger and Studlar (2007) Aggregate Europe Yes No − Endersby and Krieckhaus (2008) Aggregate Global Yes No − Panagopoulos (2008) Aggregate Global Yes Yes Yes Birch (2009) Aggregate Global Yes Yes Yes Dettrey and Schwindt-Bayer (2009) Aggregate Global Yes Yes Yes Franklin and Hobolt (2011) Both Europe Yes No − Quintelier et al. (2011) Individual Global Yes Yes Yes Singh (2011) Individual Global Yes Yes Yes Söderlund et al. (2011) Individual Europe Yes No − Rose and Borz (2013) Individual Europe Yes Yes Yes Smets and van Ham (2013)* Individual Global Yes No − Carreras and Castañeda-Angarita (2014) Individual Latin America Yes Yes Yes Gallego (2015) Individual Global Yes No − Singh (2015) Individual Global Yes Yes Yes Cancela and Geys (2016)* Aggregate Global Yes No − Martínez i Coma (2016) Aggregate Global Mixed† No − Dassonneville and Hooghe (2017) Individual Global Yes Yes Yes Kostelka (2017) Aggregate Global Yes Yes Yes Stockemer (2017)* Aggregate Global Yes Yes Yes Frank and Martínez i Coma (Forthcoming)* Aggregate Global Yes No − The studies listed in this table are limited to English-language cross-national studies that have employed compulsory voting as a theoretical independent variable (rather than a control) and meta analyses. “Data level” can be individual (survey data) or aggregate (official turnout statistics). “Accounts for levels of CV?” refers to whether the study uses multiple categories to account for variation in the use of enforcement and sanctions. “Escalatory effect?” refers to whether the study found that CV’s influence is stronger where sanctions and/or enforcement are steeper. Meta-analysis. †Study finds a positive impact of CV in full democracies but not elsewhere. Table 1. Cross-national studies that have estimated the effect of compulsory voting on turnout HYPOTHESIS 1: Compulsory voting increases turnout, especially where there exist legally enforced sanctions for abstention. HYPOTHESIS 1: Compulsory voting increases turnout, especially where there exist legally enforced sanctions for abstention. Even in places with legally enforced sanctions for non-voting, there is variation in the extent to which an abstainer may worry about being penalized. While previous research has acknowledged Filip Kostelka et al. 4 variation in enforcement probability, associated empirical tests typically rely on a subjective ordinal measurement scheme that conflates penalty severity and the likelihood of prosecution. We instead recognize that much of the variance in enforcement likelihood is due to differences in state capacity. p y Where state capacity is weak, nominally enforced CV laws may fail to compel the less dutiful to the polls. Low-capacity states are often unable to routinely collect fines or enforce non-monetary penalties. With regard to executing legal sanctions for abstention, states with weak capacity may falter because the register of enfranchised citizens has gaps or because enforcing agencies are understaffed or overworked (see Malkopoulou, 2015 for a historical review). Citizens who are disinclined to turn out will be aware of state impotence based on their prior interactions with the governing apparatusandmay thusfeelsafeabstaining.Alternatively,where state capacity is strong, it is more likely that authorities will be able to find, contact, and punish abstainers. Here, the negative costs of voting are largest. Thus, for those who lack an intrinsic motivation to obey the legal requirement to vote, the palpable threat of being caught and sanctioned will serve to powerfully tip the result of the expected utility calculation toward participation. From this, we advance our second hypothesis: HYPOTHESIS 2: Legally enforced sanctions for abstention will most sharply increase turnout where state capacity is robust. Data and methods We compiled an original data set of 1421 democratic national elections that were held between 1945 and 2017. This includes first-round presidential and legislative elections for country-years in which Polity IV ≥6 (Marshall, 2017).1 These are time-series cross-section data, and, unless stated otherwise, the following analyses thus apply country fixed effects and country-clustered standard errors.2 The dependent variable in all our analyses is voter turnout, which is measured as percent- age of registered voters that cast a ballot.3 g g The key independent variables for testing Hypothesis 1 are unsanctioned CV (CV Unsanctioned) and sanctioned CV (CV Sanctioned). These binary predictors were carefully coded using both primary (national legislation and constitutions) and secondary sources. The Electronic Appendix lists the sources and provides a brief discussion of major reforms in CV dur- ing the period under study. CV Unsanctioned takes a value of 1 when electoral law stipulates that voting is compulsory, but sanctions for non-compliance, should they exist, are never enforced.4 CV Sanctioned is coded as 1 when two conditions are met. First, there are some legal sanctions for abstention. Second, the state has tried to enforce them (i.e., there are at least some documented cases where some individuals were sanctioned). CV Unsanctioned and CV Sanctioned are mutually exclusive: when one is coded 1, the other is 0. The only exceptions are Switzerland and Austria (until 2004) in which the election legislation varies sub-nationally (Bechtel et al., 2018; Gaebler et al., 2020). For these countries, CV Unsanctioned and CV Sanctioned indicate the share of the population living under the given voting regime.5 g g g g In addition to the FE regression analyses, which leverage within-country variance, we apply two additional methods to test Hypothesis 1 and gauge the effect of CV (and its enforcement) on participation. The ﬁrst consists in inspecting the change in voter turnout between elections that immediately preceded and followed reforms in CV or its enforcement. The second is a two- step calculation of counterfactual voting rates for countries that use CV. In step one, we conduct a generalized least squares random-effects regression of voter turnout in countries that have never used CV.6 In step two, we use the regression coefﬁcients to make out-of-sample predictions of voter turnout in countries where voting is compulsory. HYPOTHESIS 2: Legally enforced sanctions for abstention will most sharply increase turnout where state capacity is robust. Finally, we consider whether, as suggested by Lijphart (1997), CV can prevent the decades- long secular decline in turnout present throughout much of the democratic world. Lijphart implies that electoral compulsion may alter the logic of electoral participation. A legal require- ment to vote increases the psychological and, potentially, material costs of electoral abstention independently of election context and voters’ characteristics. Following Lijphart’s intuition, whatever the causes of the global long-term decline in turnout, these should be less potent in countries with CV. We are skeptical that CV without enforced sanctions can, simply by virtue of being law, induce in the public an enduring participatory norm. Using careful synthetic control and difference- in-differences designs, Bechtel et al. (2018), Ferwerda (2014), and Gaebler et al. (2020) find that CV had no enduring impact on societal turnout norms in Swiss cantons or Austrian states. Employing regression discontinuity models that leverage exogenous age-based eligibilities for CV in Brazil, Dunaiski (2021); Holbein and Rangel (2020) find no evidence that CV engenders civic engagement or a “habit” of voting. Numerous other studies (e.g., Irwin, 1974; Hirczy, 1994; Funk, 2007; Birch, 2009; Barnes and R, 2014; Dassonneville et al., 2017) show that turnout drops sharply after the removal of CV. In Australia and Belgium, which both have deeply entrenched CV regimes, a sizeable portion of survey respondents report that they would stop voting if it were made optional (Hooghe and Pelleriaux, 1998; Mackerras and McAllister, 1999; Selb and Lachat, 2009), and many survey takers are antagonistic toward the voting requirement (Singh, 2021, chp. 4). This further suggests that obligatory voting does not instill a culture of participation. Thus, any initial boost in turnout created by CV is susceptible to deterioration with the passage of time. There must be at least some threat of punishment for abstention if CV is to prevent turnout declines. Even if participatory norms deteriorate, the alteration to the utility calculation induced by enforced sanctions for non-voting should be enough to keep turnout high. That is, people will keep turning out whether or not they are intrinsically motivated to do so for fear of being penalized. In hypothesis form, our final expectation is: HYPOTHESIS 3: Temporal declines in voter turnout are absent only where there exist legally enforced sanctions for abstention. 5 Political Science Research and Methods Political Science Research and Methods 5 1Polity IV excludes countries with population under 0.5 million and, therefore, elections from those countries are not included in our analyses. Our data set mostly draws on printed sources (Nohlen et al., 1999, 2001; Nohlen, 2005; Nohlen and Stöver, 2010), which were complemented with on-line sources for the most recent elections: www.electionguide.org, www.ipu.org, http://africanelections.tripod.com, http://psephos.adam-carr.net, www.electproject.org (all accessed in July and August 2018). 1Polity IV excludes countries with population under 0.5 million and, therefore, elections from those countries are not included in our analyses. Our data set mostly draws on printed sources (Nohlen et al., 1999, 2001; Nohlen, 2005; Nohlen and Stöver, 2010), which were complemented with on-line sources for the most recent elections: www.electionguide.org, www.ipu.org, http://africanelections.tripod.com, http://psephos.adam-carr.net, www.electproject.org (all accessed in July and August 2018). 2The Electronic Appendix reports the results of statistical tests underpinning these modeling choices. 3The United States here is an exception in that, following earlier studies (Franklin, 2004; Marshall and Fisher, 2015), we use voter turnout in terms of the voting-eligible population. 4When the national constitution simply stipulates that voting is a citizen duty, CV Unsanctioned is coded as 0. 5All the key findings in the following analyses are robust to the exclusion of these cases. We calculated the shares using information from Statistics Austria and the Swiss Federal Statistical Office. 6We replace country dummies with continent dummies and apply random effects instead of fixed effects since they allow for generalization. We use a logit transformation of the dependent variable to generate meaningful predictions between 0 and 100 (Baum, 2008): y∗= log( y 1−y). 7Our approach differs from a naive single-step OLS with dummies for countries with CV by modeling unit effects and by the fact that the regression coefficients in step one draw purely on countries that never used CV. Similarly, using an OLS regression in step 1 of our estimation would harm accuracy. In the Electronic Appendix, we describe and present a simulation that demonstrates that our approach (i.e., a comprehensive RE model) yields less biased out-of-sample estimates than naive OLS or less comprehensive RE models. Data and methods The advantage of this approach is that, contrary to the ﬁxed-effects models, the estimate is not exclusively driven by over-time change in those countries that adopted or ceased to use CV but draws on the entire universe of countries that use CV.7 1Polity IV excludes countries with population under 0.5 million and, therefore, elections from those countries are not included in our analyses. Our data set mostly draws on printed sources (Nohlen et al., 1999, 2001; Nohlen, 2005; Nohlen and Stöver, 2010), which were complemented with on-line sources for the most recent elections: www.electionguide.org, www.ipu.org, http://africanelections.tripod.com, http://psephos.adam-carr.net, www.electproject.org (all accessed in July and August 2018). g 2The Electronic Appendix reports the results of statistical tests underpinning these modeling choices. 3The United States here is an exception in that, following earlier studies (Franklin, 2004; Marshall and Fisher, 2015), we use voter turnout in terms of the voting-eligible population. 4When the national constitution simply stipulates that voting is a citizen duty, CV Unsanctioned is coded as 0. 5All the key findings in the following analyses are robust to the exclusion of these cases. We calculated the shares using information from Statistics Austria and the Swiss Federal Statistical Office. 6We replace country dummies with continent dummies and apply random effects instead of fixed effects since they allow for generalization. We use a logit transformation of the dependent variable to generate meaningful predictions between 0 and 100 (Baum, 2008): y∗= log( y 1−y). 7 y 7Our approach differs from a naive single-step OLS with dummies for countries with CV by modeling unit effects and by the fact that the regression coefficients in step one draw purely on countries that never used CV. Similarly, using an OLS regression in step 1 of our estimation would harm accuracy. In the Electronic Appendix, we describe and present a simulation that demonstrates that our approach (i.e., a comprehensive RE model) yields less biased out-of-sample estimates than naive OLS or less comprehensive RE models. Filip Kostelka et al. 6 In order to test Hypothesis 2, we employ two measures of state capacity, a state’s ability to perform its core functions. State capacity may be understood as a multidimensional concept, reﬂecting states’ extractive, coercive, and administrative abilities, and Hanson and Sigman (2021) demonstrate that these dimensions are empirically intertwined. Hanson and Sigman create a new, general-purpose index that distills 21 individual indicators of the three dimensions. Data and methods We use it as our ﬁrst measure of state capacity, captured in the variable State Capacity. As the Hanson and Sigman index is not available for some countries and years, we also employ Neonatal Mortality as a proxy measure of state capacity. This variable indicates neonatal infant mortality per 1000 live births as recorded by the World Bank (2018).8 Earlier research finds that infant mortality “capture[s] a government’s control over its territory and population, as well as its capacity to raise revenue and implement policies” (Bustikova and Corduneanu-Huci, 2017, 288). Hanson and Sigman (2021) find that state capacity is a strong predictor of infant mortality, and in our data set, the two variables are strongly negatively correlated (r = −0.71). If Hypothesis 2 is correct, strong state capacity (or, observably, low infant mortality rates) should thus be associated with higher turnout in countries with enforced CV sanctions but should be unrelated to turnout in other countries. Our analyses also probe whether accounting for the severity of sanctions for abstaining affects the efficacy of CV. We collected country-year specific data on sanctions with a rigorous archival search of domestic legislation. Monetary sanctions may vary from as low as 1.05 Brazilian real (about 0.06 percent of the average monthly salary in Brazil in 2014) to 1000 euros (repeated abstentions in Luxembourg in 2018, about 18 percent of the average monthly salary). The vari- ables Fine and Max Fine indicate the default and maximal monetary sanction for abstention,9 standardized by the country’s GDP per capita (in thousands of 2011 dollars).10 y y We also built a second version of this variable, which expresses the fine as percentage of an average monthly salary.11 Finally, Non-Monetary Sanctions is a binary variable coded as 1 when there is a non-monetary sanction in addition to, or instead of, the monetary fine and if this sanction is not dropped with the payment of the monetary fine. These sanction variables are all coded as 0 for countries that do not enforce CV. Hypothesis 3, which predicts that only enforced sanctions can prevent the decline in voter turnout, is tested with a variable that measures the number of Years Since 1945. This parsimoni- ous operationalization of the negative trend in global voter turnout allows for a straightforward comparison across different voting regimes. Data and methods In most other analyses, where the focus is not on such a comparison, we opt for decade dummies which allow for short-term non-linear time trends. Our analyses control for several predictors of voter turnout identified by earlier research. As the processes of democratization and democratic consolidation may affect participation 9If the default fine is a range, the variable Fine indicates the middle value. The variable Max Fine gives the maximal mon- etary sanction for abstention. If there is a range, it is the top of the range. When there are extra fines for abstaining more than once, it is the highest penalty one could get. The different amounts were converted from the original currencies into current dollars using historical exchange rates (mostly from the World Bank), and subsequently to 2011 dollars using inflation con- version factors (Sahr, 2021). 10For a country with compulsory voting i and election j, the variables are calculated as follows: Fineij = Sanctionij/(GDP per capitaij/1000) (1) Max Fineij = Max Sanctionij/(GDP per capitaij/1000) (2) Fineij = Sanctionij/(GDP per capitaij/1000) (1) (1) Fineij = Sanctionij/(GDP per capitaij/1000) (2) 11The data on salaries were collected from local statistical offices and international organizations such as the Organization for Economic Cooperation and Development (OECD) or the International Labour Office (ILO). Political Science Research and Methods 7 Fig. 1. Turnout change when compulsory voting is removed. Note: CVS and CVU stand for sanctioned and unsanctioned compulsory voting, respectively. ut change when compulsory voting is removed. Note: CVS and CVU stand for sanctioned and unsanctioned oting, respectively. Fig. 1. Turnout change when compulsory voting is removed. Note: CVS and CVU stand for sanctioned and unsanctioned compulsory voting, respectively. (Kostelka, 2017), we also include specific time trends for three groups of new democracies (pre-1974, post-1974, and post-communist).12 All time-invariant factors are controlled with country-level FE. (Kostelka, 2017), we also include specific time trends for three groups of new democracies (pre-1974, post-1974, and post-communist).12 All time-invariant factors are controlled with country-level FE. 12Robustness checks in the Electronic Appendix (see Table EA1) also control for the level of economic development (the natural logarithm of GDP per capita in the 2011 prices, Bolt et al. (2018)). This variable is not available for all country-years and, when included, it is not statistically significant and it does not change the substantive findings. 13The figures thus do not include countries like Czechoslovakia where the pre- and post-reform elections were separated by a non democratic period and countries like Austria and S itzerland here aside from the S iss canton of Schaffhausen CV 12Robustness checks in the Electronic Appendix (see Table EA1) also control for the level of economic development (the natural logarithm of GDP per capita in the 2011 prices, Bolt et al. (2018)). This variable is not available for all country-years and, when included, it is not statistically significant and it does not change the substantive findings. 12Robustness checks in the Electronic Appendix (see Table EA1) also control for the level of economic development (the natural logarithm of GDP per capita in the 2011 prices, Bolt et al. (2018)). This variable is not available for all country-years and, when included, it is not statistically significant and it does not change the substantive findings. 13The figures thus do not include countries like Czechoslovakia where the pre- and post-reform elections were separated by a non-democratic period, and countries like Austria and Switzerland where, aside from the Swiss canton of Schaffhausen, CV was phased out gradually (Bechtel et al., 2018; Gaebler et al., 2020). y g g g 13The figures thus do not include countries like Czechoslovakia where the pre- and post-reform elections wer a non-democratic period, and countries like Austria and Switzerland where, aside from the Swiss canton of Sch was phased out gradually (Bechtel et al., 2018; Gaebler et al., 2020). 14In addition, turnout-enhancing (adoption of CV) reforms exert on average weaker effects than turnout-depressing reforms (removal of CV). Besides potential ceiling effects (when CV is adopted in already high-turnout contexts, such as Cyprus in 1981), this asymmetry presumably reflects the general declining trend in voter turnout, which characterized most of the period under study and which largely reflected generational change (e.g., Tiberj, 2018; Kostelka and Blais, 2021). The trend may have amplified (removal of CV) or cross-pressured (adoption of CV) the reforms’ effects. 14In addition, turnout-enhancing (adoption of CV) reforms exert on average weaker effects than turnout-depressing reforms (removal of CV). Besides potential ceiling effects (when CV is adopted in already high-turnout contexts, such as Cyprus in 1981), this asymmetry presumably reflects the general declining trend in voter turnout, which characterized most of the period under study and which largely reflected generational change (e.g., Tiberj, 2018; Kostelka and Blais, 2021). The trend may have amplified (removal of CV) or cross-pressured (adoption of CV) the reforms’ effects. 15The reasons for this decline may be manifold, including a proliferation of elections in Greece, where four nationwide legislative contests were held between 2012 and 2015. However, had enforced CV been maintained, these declines may not have occurred, or may have been much smaller. 15The reasons for this decline may be manifold, including a proliferation of elections in Greece, where four nationwide legislative contests were held between 2012 and 2015. However, had enforced CV been maintained, these declines may not have occurred, or may have been much smaller. 16Formal sanctions for abstention existed in Italy between 1957 and 1993, but they were not never systematically enforced according to available sources. We thus consider Italy as a case of unsanctioned voting. The robustness checks (Tables EA1 to EA3) show that our main substantive results hold even when Italy is coded as a case of sanctioned compulsory voting. See the Electronic Appendix for these results and more information on Italy’s CV legislation. 17The 95 percent confidence interval ranges from 61.8 to 71.9 percent. 18The 95 percent confidence interval ranges from 67 to 75.8 percent. Test of hypothesis 1 We first inspect the immediate effects of CV reforms on citizen participation. We present three figures that respectively focus on the abolition of CV (Figure 1), the abandonment of enforced sanctions (Figure 2), and the adoption of CV (Figure 3). They display short-term changes in turn- out for countries that adopted one of these reforms in between two democratic elections that were held up to five years (i.e., roughly one legislative term) apart.13 p y g y g p The figures strongly support Hypothesis 1. When voting was made voluntary or the enforce- ment of non-voting sanctions was ended, voter turnout in the subsequent election consistently Filip Kostelka et al. 8 Fig. 2. Turnout change when compulsory voting ceases to be enforced. Note: CVS and CVU stand for sanctioned and unsanctioned compulsory voting, respectively. Fig. 2. Turnout change when compulsory voting ceases to be enforced. Note: CVS and CVU stand for sanctioned and unsanctioned compulsory voting, respectively. drops. Conversely, when voting was made compulsory, this was always followed by an increase in the participation rate. However, there is considerable variation in the magnitude of these boosts and drops, which largely reflects the distinction between enforcement and the lack of thereof.14 Shifts in participa- tion are generally strongest, reaching at least 10 percentage points, when enforced CV is abolished (Chile, the Netherlands, or Venezuela in Figure 1), or when it is adopted (Cyprus in Figure 3). Similarly, Honduras’ 2005 election shows that abandoning enforced sanctions can result in double-digit drops in the voting rate (Figure 2). It is true that, in two other cases, removing enforced sanctions resulted in a very small turnout decline (see Cyprus and Greece in Figure 2). This suggests that, when voting remains formally compulsory, the government’s signal- ing of the new, de facto voluntary, voting regime may remain, perhaps deliberately, muted, and citizens become fully aware of the change only gradually. Such an explanation is corroborated by our data: between the 1990s and 2017, voter turnout progressively dropped by 8.6 and 26.2 points in Cypriot presidential and legislative elections, and by 19.8 points in Greek legislative elections.15 So, despite the limited short-term effect, giving up enforcement presumably did Political Science Research and Methods 9 Fig. 3. Turnout change when compulsory voting is adopted. Note: CVS and CVU stand for sanctioned and unsanctioned compulsory voting, respectively. Fig. 3. Turnout change when compulsory voting is adopted. 16Formal sanctions for abstention existed in Italy between 1957 and 1993, but they were not never systematically enforced according to available sources. We thus consider Italy as a case of unsanctioned voting. The robustness checks (Tables EA1 to EA3) show that our main substantive results hold even when Italy is coded as a case of sanctioned compulsory voting. See the Electronic Appendix for these results and more information on Italy’s CV legislation. 17The 95 percent confidence interval ranges from 61 8 to 71 9 percent pp y 17The 95 percent confidence interval ranges from 61.8 to 71.9 percent. 18The 95 percent confidence interval ranges from 67 to 75.8 percent. Test of hypothesis 1 Note: CVS and CVU stand for sanctioned and unsanctioned compulsory voting, respectively. matter a lot in the long term. By contrast, adopting (Bulgaria and Thailand in Figure 3) or drop- ping (Italy in Figure 1) unsanctioned electoral compulsion was followed by much smaller changes in participation, typically of a few percentage points.16 We display the results of our more rigorous tests of Hypothesis 1 in Table 2, which presents the ﬁxed-effects regression models. These leverage all within-country variation—not only the short-term change—and introduce controls for the other predictors of voter turnout. The base- line analysis (Model 1) indicates that unsanctioned CV increases participation by 9.6 points and, when sanctions are enforced, by 22.7 points. The inclusion of time-variant controls slightly reduces these estimates, but they remain substantively large and retain the roughly 1:2 ratio, reaching 10.1 and 18.6 points respectively. These results provide strong support for Hypothesis 1. g y g y Next, we proceed to the estimation of counterfactual voting rates. Table 2 displays the regres- sion of logit-transformed voter turnout in countries that have never employed CV. The regression coefficients are largely in the expected direction, and we use them to estimate out-of-sample pre- dictions of voter turnout in countries where voting is compulsory. For countries with unsanctioned CV, predicted turnout is 67.1 percent,17 which is 7.6 points lower than the observed average of 74.7 percent. In countries that enforce CV sanctions, predicted turnout (71.6 percent)18 trails observed turnout (86 percent) by 14.4 points. These results, which Filip Kostelka et al. 10 Table 2. Time-series-cross-section analysis Table 2. Time-series-cross-section analysis (1) (2) Baseline Full model CV unsanctioned 9.63 (2.42) 10.13 (2.87)* CV sanctioned 22.72 (3.45)* 18.62 (3.58)* Majority status −0.16 (0.06) Closeness −0.04 (0.03) Concurrent election 7.01 (1.86)* El. system: majoritarian −3.39 (1.99)+ El. system: mixed −3.94 (1.82)* El. system: other 2.03 (2.13) Presidential election −1.49 (0.99) Electorate size (ln) 0.84 (1.39) Pre-1974 democratization 0.13 (0.16) Post-1974 democratization −0.05 (0.11) Post-communist democratization −0.30 (0.12)* 1940s 5.81 (2.03) 1950s 7.51 (1.88)* 1960s 8.18 (1.56)* 1970s 6.64 (1.70)* 1980s 6.47 (1.40)* 1990s 4.56 (1.27)* 2000s 1.47 (0.76)+ Constant 66.68 (0.72)* 63.78 (3.18)* Country FE Yes Yes N 1421 1421 R2 0.13 0.28 Note: Significance levels: +p < 0.1, p < 0.05, p < 0.01, p < 0.001. Standard errors clustered by country in parentheses. Test of hypothesis 1 draw on the entire universe of elections with CV, are largely compatible with the results from Table 2, which leverage within-country changes in CV. Both analyses validate Hypothesis 1, showing that unsanctioned CV increases voter turnout by between 7.5 and 10 points, on average; the positive effect of CV with enforced sanctions, at 14.5–18.5 points, is almost twice as big. draw on the entire universe of elections with CV, are largely compatible with the results from Table 2, which leverage within-country changes in CV. Both analyses validate Hypothesis 1, showing that unsanctioned CV increases voter turnout by between 7.5 and 10 points, on average; the positive effect of CV with enforced sanctions, at 14.5–18.5 points, is almost twice as big. 20The regression coefficient of CV Sanctioned x Neonatal Mortality (−0.42) has to be summed with the re ficient of Neonatal Mortality (−0.00). The resulting p-value is 0.020. 19The interaction means that the regression coefficient of CV Sanctioned x State Capacity (5.3) needs to be the regression coefficient of State Capacity (1.2). The resulting p-value is 0.044. the regression coefficient of State Capacity (1.2). The resulting p value is 0.044. 20The regression coefficient of CV Sanctioned x Neonatal Mortality (−0.42) has to be summed with the regression coef- ficient of Neonatal Mortality (−0.00). The resulting p-value is 0.020. 19The interaction means that the regression coefficient of CV Sanctioned x State Capacity (5.3) needs to be summed with the regression coefficient of State Capacity (1.2). The resulting p-value is 0.044. 20The regression coefficient of CV Sanctioned x Neonatal Mortality (−0.42) has to be summed with the regression coef- ficient of Neonatal Mortality (−0.00). The resulting p-value is 0.020. Test of hypothesis 2 We have found that the effect of enforced sanctions is powerful. However, its magnitude, Hypothesis 2 argues, depends on state capacity. Table 3 builds on Model 2 of Table 2 and tests the effects of state capacity, ﬁnes for abstention, and non-monetary sanctions. Fines and sanctions’ regression coefﬁcients are mostly in the wrong direction, they are never statistically signiﬁcant, and these variables’ inclusion does little to alter the estimated coefﬁcient on the CV sanctioned dummy. There is hence no evidence that sanction severity itself affects turnout. y y By contrast, there is support for the role of state capacity. For countries that sanction electoral abstention, state capacity’s regression coefficient is positive (6.5) and statistically significant (p < 0.05).19 For the proxy measure, Neonatal Mortality, the regression coefficient is expectedly negative (−0.42) and, again, statistically significant (p < 0.02).20 Table EA2 in the Electronic Appendix shows that, when we restrict the analysis to elections with sanctioned CV (N < 200), the association between state capacity and turnout is even more robust (p < 0.001) and unaltered by controlling for GDP per capita. y g p p Figure 4, which draws on Models 1 and 2 in Table 3, illustrates the effect of Sanctioned CV for different levels of state capacity and neonatal mortality. In very strong states, where state capacity Political Science Research and Methods 11 Table 3. Regression model for out-of-sample predictions (1) Majority status −0.005 (0.003) Closeness −0.002 (0.002) Concurrent election 0.361 (0.091)* El. system: majoritarian −0.135 (0.109) El. system: mixed −0.300 (0.095)** El. system: other −0.061 (0.119) Presidential election −0.070 (0.063) Electorate size (ln) −0.069 (0.048) Pre-1974 democratization 0.014 (0.010) Post-1974 democratization −0.003 (0.008) Post-communist democratization −0.012 (0.006)* 1940s 0.335 (0.157)* 1950s 0.307 (0.110) 1960s 0.388 (0.076)* 1970s 0.305 (0.114) 1980s 0.327 (0.095)* 1990s 0.225 (0.072) 2000s 0.038 (0.041) E. Europe and C. Asia −0.592 (0.167)* Latin America −0.865 (0.210)*** MENA −0.274 (0.173) Sub-Saharan Africa −0.558 (0.222)* East Asia −0.119 (0.167) South-East Asia 0.109 (0.225) South Asia −0.438 (0.318) The Pacific −0.117 (0.275) The Carribean −0.350 (0.275) Constant 1.251 (0.188)*** Country RE Yes N 974 R2 0.23 Note: The dependent variable is voter turnout as a logit-transformed proportion. Significance levels: +p < 0.1, p < 0.05, p < 0.01, p < 0.001. Standard errors clustered by country in parentheses. Table 3. Regression model for out-of-sample predictions Table 3. Test of hypothesis 2 Regression model for out-of-sample predictions (1) Majority status −0.005 (0.003) Closeness −0.002 (0.002) Concurrent election 0.361 (0.091)* El. system: majoritarian −0.135 (0.109) El. system: mixed −0.300 (0.095)** El. system: other −0.061 (0.119) Presidential election −0.070 (0.063) Electorate size (ln) −0.069 (0.048) Pre-1974 democratization 0.014 (0.010) Post-1974 democratization −0.003 (0.008) Post-communist democratization −0.012 (0.006)* 1940s 0.335 (0.157)* 1950s 0.307 (0.110) 1960s 0.388 (0.076)* 1970s 0.305 (0.114) 1980s 0.327 (0.095)* 1990s 0.225 (0.072) 2000s 0.038 (0.041) E. Europe and C. Asia −0.592 (0.167)* Latin America −0.865 (0.210)*** MENA −0.274 (0.173) Sub-Saharan Africa −0.558 (0.222)* East Asia −0.119 (0.167) South-East Asia 0.109 (0.225) South Asia −0.438 (0.318) The Pacific −0.117 (0.275) The Carribean −0.350 (0.275) Constant 1.251 (0.188)*** Country RE Yes N 974 R2 0.23 Note: The dependent variable is voter turnout as a logit-transformed proportion. Significance levels: +p < 0.1, p < 0.05, p < 0.01, *p < 0.001. Standard errors clustered by country in parentheses. Note: The dependent variable is voter turnout as a logit-transformed proportion. Significance levels: +p < 0.1, p < 0.05, p < 0.01, p < 0.001. Standard errors clustered by country in parentheses. Note: The dependent variable is voter turnout as a logit-transformed proportion. Significance levels: +p < 0.1, p < 0.05, p < 0.01, p < 0.001. Standard errors clustered by country in parentheses. Fig. 4. Sanctioned compulsory voting and state capacity. Note: average marginal effects with 95 percent confidence inter- vals based on Models 1 and 2 from Table 4. The estimation is limited to the range of values observed in countries with enforced CV. State capacity, as estimated by Hanson and Sigman (2021), thus ranges from 0.52 (Bolivia in 1985) to 2.52 (Belgium in 2014). Neonatal mortality ranges from 1.6 (Luxembourg in 2013) to 46 (Bolivia in 1985). Fig. 4. Sanctioned compulsory voting and state capacity. Note: average marginal effects with 95 percent confidence inter- vals based on Models 1 and 2 from Table 4. The estimation is limited to the range of values observed in countries with enforced CV. State capacity, as estimated by Hanson and Sigman (2021), thus ranges from 0.52 (Bolivia in 1985) to 2.52 (Belgium in 2014). Neonatal mortality ranges from 1.6 (Luxembourg in 2013) to 46 (Bolivia in 1985). Test of hypothesis 2 (3) (4) (5) (6) (7) Fine Fine Maximal fine Maximal fine Other sanctions 21.30 (5.32) 20.00 (4.78)* 19.83 (4.81)* 18.29 (4.80)* 19.29 (4.09)* 10.59 (3.28) 9.82 (3.07) 9.81 (3.07) 9.95 (3.09) 10.28 (2.89)* −0.21 (10.37) −3.16 (3.35) −2.69 (3.57) 0.04 (0.05) −1.25 (2.79) 63.32 (3.17)* 64.03 (3.16)* 64.05 (3.16)* 63.68 (3.05)* 63.34 (3.16)* Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes 1309 1330 1330 1357 1393 0.15 0.16 0.16 0.15 0.17 y country in parentheses. N varies depending on data availability. The same controls as in Tables 2 and 3. Full table Sanctions (3) (4) (5) (6) Fine Fine Maximal fine Maximal fine 21.30 (5.32)* 20.00 (4.78)* 19.83 (4.81)* 18.29 (4.80)* 10.59 (3.28) 9.82 (3.07) 9.81 (3.07) 9.95 (3.09) −0.21 (10.37) −3.16 (3.35) −2.69 (3.57) 0.04 (0.05) 63.32 (3.17)* 64.03 (3.16)* 64.05 (3.16)* 63.68 (3.05)* Yes Yes Yes Yes Yes Yes Yes Yes 1309 1330 1330 1357 0.15 0.16 0.16 0.15 country in parentheses. N varies depending on data availability. The same controls as in T Political Science Research and Methods 13 Table 5. Over-time change by voting regime (1) (2) (3) No CV CV unsanctioned CV sanctioned Year since 1945 −0.19 (0.04)* −0.17 (0.08)+ 0.07 (0.04)+ Constant 74.14 (3.21)* 80.80 (2.44)* 88.67 (4.61)*** Controls Yes Yes Yes Country FE Yes Yes Yes N 1044 130 214 R2 0.18 0.45 0.15 Note: Significance levels: +p < 0.1, p < 0.05, p < 0.01, *p < 0.001. Standard errors clustered by country in parentheses. The same controls as in Tables 2 and 3. Full table displayed in the Electronic Appendix. Table 5. Over-time change by voting regime Note: Significance levels: +p < 0.1, p < 0.05, p < 0.01, p < 0.001. Standard errors clustered by country in parentheses. The same controls as in Tables 2 and 3. Full table displayed in the Electronic Appendix. Note: Significance levels: +p < 0.1, p < 0.05, p < 0.01, *p < 0.001. Standard errors clustered by country in parentheses. The same controls as in Tables 2 and 3. Full table displayed in the Electronic Appendix. Fig. 5. Evolution of voter turnout by voting regime (average observed voting rates). The figure includes only those coun- tries that held democratic elections continuously between 1985 and 2017 and excludes countries that did not remain in one of the three voting categories throughout this time period. Fig. 5. 21The separate analyses for each voting regime inter alia allow for subgroup-specific coefficients on the control variables. In the Electronic Appendix (Table EA3), we present a single-model analysis with interactions between voting regimes and Years Since 1945, which yields similar substantive findings. Test of hypothesis 3 Hypothesis 3 argues that CV can prevent voter turnout from declining only when there are enforced sanctions for abstention. Table 4 presents tests of this hypothesis, displaying three regression models of voter turnout since 1945: for countries without CV, for countries with unsanctioned CV, and for countries with sanctioned CV respectively.21 The regression coefﬁcient on Years Since 1945 shows that, in the ﬁrst group (no CV), participation declined on average by 0.19 points per year since 1945 (p < 0.001). In the second group (unsanctioned CV), the regres- sion coefﬁcient is of similar magnitude (−0.17; p = 0.052). Finally, in the third group (sanctioned CV), the regression coefﬁcient is not negative but slightly positive (0.07; p = 0.09). These results support Hypothesis 3, conﬁrming that enforced CV prevents turnout declines, whereas sanction- less compulsion does not. p Table 5 includes all 1421 elections, and model results thus may be affected by over-time changes in the samples under study (i.e., democratic regimes’ emergence and breakdown). In an additional analysis, we thus trace the evolution of voter turnout in those countries that held democratic elections continuously between 1985 and 2017. The trends displayed in Figure 5 again corroborate Hypothesis 3. Participation dropped by around seven or eight points in coun- tries where CV was not used or did not have enforced sanctions. In countries with sanctioned CV, voter turnout remained strikingly stable and high. Test of hypothesis 2 Evolution of voter turnout by voting regime (average observed voting rates). The figure includes only those coun- tries that held democratic elections continuously between 1985 and 2017 and excludes countries that did not remain in one of the three voting categories throughout this time period. reaches 2.5 on Hanson and Sigman’s latent measure and neonatal mortality is kept below two per thousand births, the estimated effect of sanctioned CV on the turnout rate is between 27 (Graph A) and 22 (Graph B) points. By contrast, in weak states, with state capacity of −0.52 and neonatal mortality exceeding 45, the effect of sanctioned CV lies between 11 (Graph A) and 4 (Graph B) points. Clearly, this analysis supports Hypothesis 2; the impact of CV depends on the state’s cap- acity to predictably enforce non-voting penalties. Filip Kostelka et al. 14 Conclusion This study asked three questions. First, is the positive impact of CV on turnout bigger where there are enforced sanctions for abstention? Second, does the state’s ability to act as a credible admin- istrator that can execute penalties for abstention increase the effect of sanctioned CV on turnout? And, finally, is the long-term secular decline in voter turnout arrested by sanctioned CV? Our results indicate that the answer to all three questions is yes. y We estimate the effect of sanctioned CV to be larger than what has been found in prior com- parative analyses. Our results show that, as a percentage of registered voters, CV with legally enforced sanctions for abstention generates a remarkable 14.5–18.5 points increase in voter turn- out. Birch (2009, pp. 93–94) finds that countries with enforced CV have turnout rates among registered voters about 12 points higher than others (including those with unenforced CV). Panagopoulos (2008) finds that strict enforcement of sanctions is associated with about a 13-point increase in turnout among the total population. Additionally, unlike most previous cross-national studies that have accounted for variation in the severity of CV, we also find that unenforced CV increases turnout. In contrast with Birch (2009, pp. 93–94), who finds no evidence that unenforced CV associates with turnout, and Panagopoulos (2008), who finds that turnout under unenforced or weakly enforced CV is no higher than that in voluntary systems, our analyses show that the mere requirement to vote boosts turnout by 7–10 points as a percentage of registered voters. However, toothless CV will not keep participation high forever. Our findings also show that unenforced CV does not flatten or reverse ongoing declines in voter turnout. This suggests that CV cannot instill an enduring participatory culture into a country simply by virtue of being law. Instead, enforced penalties for abstention are needed to maintain robust levels of elect- oral participation. We believe that our estimates of the effect of CV are credible. Admittedly, we cannot rule out the possibility that countries that adopted CV did so to legitimate or lock in an extant rise in the turnout rate. Nor can we rule out the possibility that CV was removed in countries experiencing a Political Science Research and Methods 15 decline in participation. Conclusion Still, an examination of trends in turnout in pre-intervention periods in our data is not consistent with such patterns, and our review of the historical circumstances sur- rounding the adoption and abolition of CV did not suggest that turnout changes systematically precede a change to or from mandatory voting. We also cannot be sure that other reforms that took place concomitantly with the adoption or removal of CV did not drive changes in turnout, though, again, our review of the legislation introducing or removing CV does not suggest such a pattern. Finally, like any observational design, ours does not eliminate the threat of bias stemming from confounders. Nevertheless, our inclusion of country fixed effects eliminates bias from time- invariant sources, and we do measure and control for many time-variant sources of spuriousness, in addition to modeling time trends. g We also go beyond prior work with our in-depth review of contemporary and historical docu- ments, legislation, and constitutions to create what we believe is the most accurate and extensive measure of CV to date. Our extensive data collection effort also allows us to, for the first time, test the impact of the amount of monetary fines (as adjusted by national economic conditions) on turnout across countries. We find that, while the existence of enforced sanctions matters greatly for turnout, the fine amount has no impact. Moreover, we for the first time identify state capacity as a moderator of the impact of enforced CV. Democratic reformers seeking to reduce abstention often recommend CV (e.g., Dionne and Rapoport, 2022). Our finding that CV can boost voter turnout more than previously thought— and can prevent declines in participation—may make it an even more attractive instrument for electoral reformers interested in strengthening participatory democracy. On the flip side, some see drawbacks associated with elevated turnout. Brennan (2016), for example, argues that low pro- pensity voters rarely choose wisely. If one accepts arguments that high turnout is detrimental, our finding that CV is highly efficacious implies that it could be an especially hazardous tool. g g y p p y Beyond turnout, scholars have recently identified several downstream consequences of a voting requirement for individuals and parties. Supplementary material. The supplementary material for this article can be found at https://doi.org/10.1017/psrm.2022.57 and Replication data Replication Link https://doi.org/10.7910/DVN/7OB0YJ. Conclusion These include increased invalid balloting (e.g., Power and Garand, 2007; Uggla, 2008; Cohen, 2018; Singh, 2019a); election results that are less reflective of ideological preferences (e.g., Selb and Lachat, 2009; Singh, 2016; Dassonneville et al., 2019; Freire and Turgeon, 2020; but see Singh (2022)); more and stronger psychological attachments to pol- itical parties (e.g., Dalton and Weldon, 2007; Singh and Thornton, 2013); and programmatic vote seeking (Singh, 2019b). In each case, the link between CV and the downstream outcome is thought to be, at least in part, mechanized by compelled voting among people who would nor- mally stay home. Because CV may get more such people to the voting booth than previously thought, the putative consequences of CV beyond turnout are even more likely to come to the fore. Supplementary material. 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https://openalex.org/W4211259035	https://www.ssoar.info/ssoar/bitstream/document/66518/1/ssoar-2018-trent_et_al-A_United_Nations_Renaissance_What.pdf	English	null	A United Nations Renaissance	null	2,017	cc-by-sa	73,399	www.ssoar.info www.ssoar.info Zur Verfügung gestellt in Kooperation mit / provided in cooperation with: Verlag Barbara Budrich Terms of use: This document is made available under a CC BY-SA Licence (Attribution-ShareAlike). For more Information see: https://creativecommons.org/licenses/by-sa/4.0 Nutzungsbedingungen: Dieser Text wird unter einer CC BY-SA Lizenz (Namensnennung- Weitergabe unter gleichen Bedingungen) zur Verfügung gestellt. Nähere Auskünfte zu den CC-Lizenzen finden Sie hier: https://creativecommons.org/licenses/by-sa/4.0/deed.de Nutzungsbedingungen: Dieser Text wird unter einer CC BY-SA Lizenz (Namensnennung- Weitergabe unter gleichen Bedingungen) zur Verfügung gestellt. Nähere Auskünfte zu den CC-Lizenzen finden Sie hier: https://creativecommons.org/licenses/by-sa/4.0/deed.de Diese Version ist zitierbar unter / This version is citable under: https://nbn-resolving.org/urn:nbn:de:0168-ssoar-66518-2 John Trent/Laura Schnurr A United Nations Renaissance John Trent/Laura Schnurr A United Nations Renaissance “Politicians, pundits, and publics are moving inward and building walls whereas addressing global threats requires moving outward and breaking down barriers. More than ever, multilateralism is essential for problem-solving, and more than ever we need a fitter-for-purpose United Nations. Trent and Schnurr provide a persuasive and user- friendly introduction for a new generation of change-makers.” Thomas G. Weiss, Presidential Professor of Political Science, The CUNY Graduate Center, Past President International Studies Association Thomas G. Weiss, Presidential Professor of Political Science, The CUNY Graduate Center, Past President International Studies Association “More than a simple forum or arena for confrontation of opposing interests, the United Nations must become an instrument for world governance. This excellent book, from Trent and Schnurr, goes a long way to push forward this idea.” Modesto Seara-Vazquez, Professor of International Organization, and Rector Oaxaca State University System, Mexico “The United Nations remains an essential global institution for advancing the values and practices of cooperation, development, and human rights, but it also needs reform; and especially it needs realistic proposals that give a way forward while still retaining and promoting the ideals of the Charter. This new volume places the UN in historical and contemporary perspective, identifies its critical strengths, challenges, and flaws in a balanced analysis, and suggests clear and constructive arguments and ideas for the changes that are needed.” Alistair Edgar, Professor of Political Science, Wilfrid Laurier University, Executive Director, Academic Council on the United Nations System “The UN is being put to the test in today's jumbled and fractious global system. There is a foreboding over the growing trend among many member states including three of the permanent members of the Security Council towards anti - internationalist sentiments and the undermining of norms of global cooperation. As the title of this book signals, it is time for a UN Renaissance. This will only happen if there emerges a coalition of key member states, civil society groups, international institutions and good advocacy in the media and from the academic world. This book makes clear why this is such an important cause for our time. And its focus on mobilizing young people to the cause is a worthy recommendation.” Lloyd Axworthy, Past President, University of Winnipeg, former Foreign Minister of Canada “Global issues require well-functioning global institutions. The United Nations and it’s agencies are critical global players that are needed more than ever given the increasingly interconnected world. John Trent/Laura Schnurr A United Nations Renaissance The UN is vital in helping to create and support the right operating environment so that the Red Cross and Red Crescent and other organizations are able to fulfill their humanitarian mandates. Trent and Schnurr have written a concise and readable publication that should be read by young people the world over. It is hoped future generations would be encouraged to join the ranks in helping to rejuvenate an essential institution.” George Weber, Secretary-General Emeritus, International Red Cross and Red Crescent Federation; CEO Royal Ottawa Hospital John Trent Laura Schnurr Barbara Budrich Publishers Opladen • Berlin • Toronto 2018 An electronic version of this book is freely available, thanks to the support of libraries working with Knowledge Unlatched. KU is a collaborative initiative designed to make high quality books Open Access for the public good. The Open Access ISBN for this book is 978-3-8474-0860-4. More information about the initiative and links to the Open Access version can be found at www.knowledgeunlatched.org 018 This work is licensed under the Creative Commons Attribution-ShareAlike 4.0. (CC- -SA 4.0) ) ermits use, duplication, adaptation, distribution and reproduction in any medium or format, as g as you share under the same license, 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. © 2018 Dieses Werk ist beim Verlag Barbara Budrich GmbH erschienen und steht unter der Creative Commons Lizenz Attribution-ShareAlike 4.0 International (CC BY-SA 4.0): https://creativecommons.org/licenses/by-sa/4.0/ © 2018 Dieses Werk ist beim Verlag Barbara Budrich GmbH erschienen und steht unter der Creative Commons Lizenz Attribution-ShareAlike 4.0 International (CC BY-SA 4.0): Diese Lizenz erlaubt die Verbreitung, Speicherung, Vervielfältigung und Bearbeitung bei Verwendung der gleichen CC-BY-SA 4.0-Lizenz und unter Angabe der UrheberInnen, Rechte, Änderungen und verwendeten Lizenz. This book is available as a free download from www.barbara-budrich.net (https://doi.org/10.3224/84740711). A paperback version is available at a charge. The page numbers of the open access edition correspond with the paperback edition. This book is available as a free download from www.barbara-budrich.net (https://doi.org/10.3224/84740711). A paperback version is available at a charge. The page numbers of the open access edition correspond with the paperback edition. ISBN 978-3-8474-0711-9 (paperback) eISBN 978-3-8474-0860-4 (PDF) eISBN 978-3-8474-1216-8 (EPUB) DOI 10.3224/84740711 A CIP catalogue record for this book is available from Die Deutsche Bibliothek (The German Library) (http://dnb.d-nb.de) Jacket illustration by Bettina Lehfeldt, Kleinmachnow, Germany – www.lehfeldtgraphic.de Copy Edit: Máiréad Collins, Belfast, UK Typesetting: Anja Borkam, Jena – kontakt@lektorat-borkam.de paper&tinta, Warsaw A CIP catalogue record for this book is available from Die Deutsche Bibliothek (The German Library) (http://dnb.d-nb.de) Jacket illustration by Bettina Lehfeldt, Kleinmachnow, Germany – www.lehfeldtgraphic.de Copy Edit: Máiréad Collins, Belfast, UK Typesetting: Anja Borkam, Jena – kontakt@lektorat-borkam.de paper&tinta, Warsaw g p Copy Edit: Máiréad Collins, Belfast, UK Typesetting: Anja Borkam, Jena – kontakt@lektorat-borkam.de paper&tinta, Warsaw Typesetting: Anja Borkam, Jena – kontakt@lektorat-borkam.de paper&tinta, Warsaw Table of Contents List of Illustrations ......................................................................................... 9 List of Abbreviations ................................................................................... 11 Introduction .................................................................................................. 13 Achieving the unthinkable ......................................................................... 13 The global governance deficit ................................................................... 15 The United Nations’ balance sheet ............................................................ 16 Empowering youth .................................................................................... 19 Objectives of the primer ............................................................................ 21 Chapter 1 – Evolving International Organizations: the UN Past and Present .............................................................................. 22 Early international cooperation efforts ...................................................... 22 The Concert of Europe .......................................................................... 23 A note on the creation of the League of Nations ................................... 26 The League of Nations as an institution ................................................ 27 From the League of Nations to the United Nations .............................. 32 Introducing the United Nations ................................................................. 34 The principal organs of the United Nations ............................................... 35 The Security Council ............................................................................ 35 The General Assembly .......................................................................... 38 The Economic and Social Council ........................................................ 41 Trusteeship Council .............................................................................. 43 The International Court of Justice ......................................................... 43 The Secretariat ...................................................................................... 45 Beyond the organs ..................................................................................... 46 The Secretary-General .......................................................................... 46 UN budgets ........................................................................................... 49 The UN System ..................................................................................... 49 International financial institutions and other international actors ......... 51 Chapter 2 – Peace and Security: Fixing the Security Council ................. 56 The Security Council’s functions and activities ........................................ 58 Security Council strengths .................................................................... 58 Security Council weaknesses ................................................................ 61 5 5 The UN’s peace operations ....................................................................... 64 The Security Council and the future .......................................................... 67 Chapter 3 – Social and Economic Development ........................................ 71 Understanding ‘development’ ................................................................... 71 The first 50 years ....................................................................................... 71 Development in the early years ............................................................. 71 Expanding development efforts ............................................................ 73 The North-South divide ........................................................................ 75 Millennium Development Goals ............................................................... 75 Tallying up the results ........................................................................... 76 Sustainable Development Goals ................................................................ 77 A changed world ................................................................................... 77 More voices at the table ........................................................................ 79 Financing the goals ............................................................................... 81 Looking ahead ........................................................................................... 82 Partnering in a new era of development cooperation ............................ 82 Role for private sector ........................................................................... 84 Role for civil society ............................................................................. 85 Innovation and technology .................................................................... 88 What role for the UN? ........................................................................... 90 Is the UN prepared? ................................................................................... 91 Streamlining the UN development system and ‘Delivering as One’ ........................................................................ 92 Improving business practices ................................................................ 94 Focusing on strengths and priorities ..................................................... 94 ECOSOC reform ................................................................................... 95 Is reform possible? ................................................................................ 97 Chapter 4 – Promoting and Protecting Human Rights ............................ 98 Human rights: one of the UN’s great ideas that too many countries fail to respect ............................................................................................. 98 The fundamental paradox ..................................................................... 98 International human rights law ............................................................. 99 Contradictory interpretations .............................................................. 101 The tremendous cost of violations ...................................................... 102 The United Nations’ Record in Upholding Human Rights ...................... 105 6 From the Commission on Human Rights to the Human Rights Council .............................................................. 106 The Secretary-General and the High Commissioner for Human Rights .................................. 109 Dealing with the worst violations: the International Criminal Court and ad hoc tribunals ........................ 110 Responsibility to Protect and human security ..................................... 114 Migration, refugees and the humanitarian response ............................ 116 What next? ............................................................................................... 120 Reforms: big and small ....................................................................... 120 Can change happen? ........................................................................... 122 Chapter 5 – Workable Global Institutions: How to Get from Here to There? .............................................................. 124 What we have learnt about understanding world institutions .................. 124 Reviewing the literature on revamping the UN ....................................... 126 Nine popular proposals to transform the UN ........................................... 130 1. A more legitimate Security Council ................................................ 131 2. A more balanced and focused General Assembly ........................... 133 3. An Economic, Social and Environmental Council .......................... 134 4. A reconfigured Human Rights Council ........................................... 135 5. Improved staffing and management practices ................................. 136 6. Autonomous emergency services for the UN ................................. 137 7. Financing the UN ............................................................................ 138 8. Principles and criteria for the Responsibility to Protect .................. 139 9. The dispersion and control of global power .................................... 140 Sequencing reform proposals: where to start ........................................... 143 Four steps for how can we help bring about workable global institutions .................................................................................... 144 Bibliography ............................................................................................... 153 Index ............................................................................................................ 161 7 List of Illustrations Box 1 Understanding ‘sovereignty’ .......................................................... 24 Box 2 Strengths and failures of the League of Nations ............................ 31 Box 3 The General Assembly’s six Main Committees ............................. 40 Box 4 Understanding ‘international law’ .................................................. 44 Box 5 Secretaries-General, 1945-present.................................................. 47 Box 6 Other ‘actors’ enter the world stage ............................................... 54 Box 7 The UN’s record of achievements in peace and security ............... 61 Box 8 The Security Council’s challenges, problems and failures ............. 63 Box 9 Putting the environment on the agenda ......................................... 73 Box 10 UN Women .................................................................................... 92 Box 11 Global health crises—Ebola response ............................................ 95 Box 12 Key characteristics of human rights ............................................... 99 Box 13 Categories of human rights .......................................................... 100 Box 14 Select examples of current human rights issues ........................... 104 Box 15 Key terms related to the movement of people .............................. 117 Box 16 Improving the working methods of the Security Council ............ 132 Box 17 Ideas for reforming the Human Rights Council ........................... 135 Box 18 An NGO coalition that made history ............................................ 147 Figure 1 Evolution of international organizations, until 1945 ...................... 22 Figure 2 The United Nations System diagram.............................................. 50 Figure 3 United Nations Development Group members (excluding regional commissions and secretariat bodies) by year established ......................................................................... 74 Figure 4 Millennium Development Goals .................................................... 76 Figure 5 Sustainable Development Goals ..................................................... 78 Figure 6 Key differences between the MDGs and SDGs ............................. 80 Figure 7 Populations of concern (refugees, asylum-seekers, IDPs, returnees, stateless persons) from 1951-2015 .............................. Introduction Welcome to this short, analytical primer on the United Nations as it is and as it could be. It is short, because its first task is limited to only providing essential information about the UN. Analytical, because its second aim is to try to un- derstand how we can think about global institutions. The United Nations is an international organization set up by a treaty between states in 1945 to help them cooperate on peace, development and human rights. Today the word ‘interna- tional’ has been expanded to become ‘global’, signifying that it is no longer limited to states but now includes other actors and activities beyond politics. We are witnessing the birth of global institutions whose task it will be to man- age and govern the increasingly integrated global system. To understand the United Nations, we must understand its historical and global context and ana- lyze its relationships with states, regional organizations, non-governmental or- ganizations (NGOs), multinational corporations, and religious and cultural or- ganizations. We must also study its strengths and weaknesses and its potential for the future. List of Illustrations 118 9 List of Abbreviations CSR Corporate Social Responsibility DAC Development Assistance Committee DESA Department of Economic and Social Affairs ECOSOC Economic and Social Council ECOWAS Economic Community of West African States ESEC Economic, Social and Environmental Council FAO Food and Agriculture Organization FUNDS Future United Nations Development System G20 Group of 20 G77 Group of 77 G7 Group of 7 GA General Assembly GATT General Agreement on Tariffs and Trade GDP Gross Domestic Product GNI Gross National Income IBRD International Bank for Reconstruction and Development ICC International Criminal Court ICISS International Commission on Intervention and State Sovereignty ICJ International Court of Justice ICT Information and Communications Technology IDA International Development Association IDP Internally Displaced Person IFAD International Fund for Agricultural Development ILO International Labour Organization IMF International Monetary Fund IMO International Maritime Organization ITU International Telecommunication Union LGBT Lesbian, Gay, Bisexual and Transgender MDGs Millennium Development Goals MOOC Massive Open Online Course NAM Non-Aligned Movement NATO North Atlantic Treaty Organization NGO Non-Governmental Organization ODA Official Development Assistance OECD Organization for Economic Development and Cooperation OHCHR Office of the United Nations High Commissioner for Human Rights P5 Permanent Five, members of Security Council R2P Responsibility to Protect CSR Corporate Social Responsibility DAC Development Assistance Committee DESA Department of Economic and Social Affairs ECOSOC Economic and Social Council ECOWAS Economic Community of West African States ESEC Economic, Social and Environmental Council FAO Food and Agriculture Organization FUNDS Future United Nations Development System G20 Group of 20 G77 Group of 77 G7 Group of 7 GA General Assembly GATT General Agreement on Tariffs and Trade GDP Gross Domestic Product GNI Gross National Income IBRD International Bank for Reconstruction and Development ICC International Criminal Court ICISS International Commission on Intervention and State Sovereignty ICJ International Court of Justice ICT Information and Communications Technology IDA International Development Association IDP Internally Displaced Person IFAD International Fund for Agricultural Development ILO International Labour Organization IMF International Monetary Fund IMO International Maritime Organization ITU International Telecommunication Union LGBT Lesbian, Gay, Bisexual and Transgender MDGs Millennium Development Goals MOOC Massive Open Online Course NAM Non-Aligned Movement NATO North Atlantic Treaty Organization NGO Non-Governmental Organization ODA Official Development Assistance OECD Organization for Economic Development and Cooperation OHCHR Office of the United Nations High Commissioner for Human Rights P5 Permanent Five, members of Security Council R2P Responsibility to Protect 11 RwP Responsibility while Protecting SC Security Council SDGs Sustainable Development Goals SG Secretary General UNAIDS United Nations Joint Programme on HIV/AIDS UNCTAD United Nations Conference on Trade and Development UNDP United Nations Development Programme UNEF United Nations Environment Fund UNEPS United Nations Emergency Peace Service UNESCO United Nations Educational, Scientific and Cultural Organization UN-HABITAT United Nations Human Settlement Program UNHCR United Nations High Commissioner for Refugees UNICEF United Nations Children’s Fund UNIDO United Nations Industrial Development Organization UNIFEM United Nations Development Fund for Women UNODC United Nations Office on Drugs and Crime UNOPS United Nations Office for Project Services UNRISD United Nations Research Institute for Social Development UNWTO United Nations World Tourism Organization UPR Universal Periodic Review WFP World Food Programme WHO World Health Organization WMO World Meteorological Organization WTO World Trade Organization 12 12 Achieving the unthinkable The world has never been a better place. We live in the most peaceful and prosperous era in human history. More than one billion people have been lifted out of extreme poverty in the past 25 years. From the early 19th century to the mid-20th century, the vast majority of the world’s population lived in extreme poverty (Roser and Ortiz-Ospina, 2017). We have not experienced a war be- tween major powers in decades. The majority of people live in democratic countries, compared to just over 10 per cent of the world population a hundred years ago. Technological advances have rapidly spread across the globe, with more people connected to one another and to information than ever before. Tens of millions of lives have been saved from small pox, polio, measles, ma- laria and tuberculosis, while HIV/AIDS infections and deaths have dropped substantially. More people have access to education and basic health care, and incomes in the developing world are rising. We often forget this as we are constantly fed a stream of bad news from the media and from politicians eager to stoke fear and insecurity. We urgently need perspective. None of this means we should sit back contently, satisfied with the headway we have collectively made. Climate change has exacerbated risks such as water crises, food shortages, social cohesion, livelihoods and security. Terrorism poses a very real threat to our security and stability. Intrastate conflict is 13 devastating for individuals affected while also having regional and global con- sequences. We remain far from an adequate solution to the migration crisis caused by political and economic instability in the Middle East and North Af- rica. We need to respond rapidly when global pandemics occur, as they can spread like wildfire. There is no guarantee that we will avoid another global financial crisis similar to the one experienced in 2008-09. The threats of our time are not like those of past eras that could often be solved by individual states alone or perhaps by a few states within a region. The diverse challenges we face today do share several common characteristics: they are increasingly complex in nature and they transcend national borders. Consider the hundreds of thousands of migrants and refugees who entered Eu- rope in 2016 by crossing the Mediterranean Sea and arriving in Italy, Greece, Spain and Cyprus. The global governance deficit How has the world changed since 1945? Has it changed to a degree which requires us to transform the international institutions that were created at that time? We argue that it indeed has. That our present challenges are as much global as they are national or local is a powerful rationale for improving our institutions, but it is not the only one. The world is far more complex than it was during the post-war period. Some 51 countries came together to form the United Nations in 1945. Today, there are 193 member states. And great power politics have shifted tremendously since that era. Bipolar or unipolar global order has been replaced by one that is multipolar, with all that portends for instability. When the UN was established, state governments were the dominant actors in the global sphere. While it may be too early to declare even the partial de- mise of the state-centric world, power is increasingly shared with other non- state actors, such as NGOs, foundations, multinational corporations, religious communities, regional coalitions or blocs, intergovernmental organizations, and groups of major economies such as the G7 and G20. Economic, social and cultural globalization has meant that we are more con- nected than ever. Greater ease of transportation has facilitated global trade of goods and services. The same is true of the movement of people, resulting in rising migration and international travel. Rapid and complex communications provide new sources of knowledge and instantaneous access to information. Most people’s lives have been affected by globalization in some way, but the extent varies significantly. And the gains from deeper integration have not been evenly spread; there are distinct winners and losers. Social and economic inequalities have reached new heights and capital is ever more concentrated in the hands of a few, with just one per cent of the world’s population controlling more than 50 per cent of the wealth. The global society we live in today is by no means a global community. By contrast, international institutions and their capacity for governance have not changed substantively. Established in 1945, the United Nations was de- signed for a different era. Its institutional structure and culture still reflect this past era, rather than the realities of the 21st century. It has not kept pace with rapid globalization and change. Achieving the unthinkable Or the rapid spread of the Zika virus, which was confirmed to be present in Brazil in 2015 and by September 2016 had reached 48 coun- tries and territories in the Americas and 10 countries in the Pacific, Asia and Africa (PBS Frontline). We know that the so-called Islamic State has devel- oped a global network, to a great extent through social media, that has allowed it to recruit a large number of Western fighters to carry out terrorist attacks in cities like Paris and Brussels, while having branches around the world includ- ing in Yemen, Libya, Afghanistan, Bangladesh and West Africa. Similarly, climate change knows no borders; with rising sea levels, we have seen how carbon emissions in the one part of the world have threatened the very exist- ence of island states on the other side of the globe. These transnational issues require a deeper level of cooperation and coordi- nation between states. They call for strong international laws and norms. Most importantly, they demand effective global institutions to develop and deliver coordinated responses. Yet few would disagree that such institutions remain a distant vision and that in its current state, the United Nations, the only interna- tional organization of its kind, is not up to the task. So, with all this discouraging news, why did we begin this book on a posi- tive note? The answer is simple: given the magnitude of our problems and the barriers we need to overcome, it is useful to remember that we have achieved incredible progress in recent decades—progress that previous generations would likely never have imagined possible. Just as we have surpassed expec- tations in creating peaceful continents and in advances in areas such as health, development and technology, we are equally capable of reforming the United Nations system so it is able to meet present and future challenges. Filling the emerging void in effective global governance will certainly not be easy, but history tells us it is possible. 14 The global governance deficit This stunted institutional development has led to its marginalization, with states looking elsewhere to solve the world’s most pressing challenges. Observing this trend, many fear that the UN will slide into irrelevance unless it adapts to the times. Sadly, at a time when we are in desperate need of greater cooperation and global governance, we are witnessing rising nationalism, xenophobia, and pro- tectionism in many countries. Accelerated globalization and integration, which for decades were assumed to be unstoppable, are being met by a new wave of resistance; leaders and politicians favouring nationalism and isolationism over 15 multilateralism are gaining support. We saw this when voters in the United Kingdom opted to leave the European Union in June 2016, and again less than five months later when voters in the US elected Donald Trump as their next President. The rise of nationalist political movements, on both ends of the po- litical spectrum but particularly on the far right, is undermining international institutions such as the UN. The United Nations’ balance sheet The result is that nearly all of us interact with the UN on a regular basis without recognizing it. Organizations such as the International Postal Un- ion, the International Telecommunications Union, the International Civil Avi- ation Organization, and the International Maritime Organization are critical in a globalized world, yet we tend not to think of the UN each time we fly across a border, buy imported goods, make an international call or mail a postcard to another country. Then there is the fact that the UN has been a stabilizing force contributing to global order for more than seven decades. It is the world’s most important diplomatic forum. It has helped avert another world war, managed nuclear pro- liferation and helped prevent a nuclear weapons war, and reduced and ended internal and international conflicts through numerous peacekeeping operations and political missions. The nature of this work usually does not lend itself to public recognition. This is partly because we simply do not know what wars or conflicts have been averted due to the presence of the UN and its unending diplomacy, negotiations and mediation. Successful prevention rarely makes headlines as the absence of an event is unknowable and causality is difficult to determine. In the end, the UN is often taken for granted and does not get the credit it deserves, especially for its record in fostering peace and security. Nev- ertheless, it is worth remembering that without the UN the world would have to depend on increasingly brittle state-to-state relationships. Even with all its flaws, it remains far better than the alternative. Despite all its achievements, even the most ardent supporter of the United Nations would not claim it is, or is even close to being, a perfect institution. In the peace and security realm, the UN has been dealt multiple blows in recent years following a series of crises where it either failed to act or was bypassed altogether, along with a couple of highly publicized scandals. When the US and a few other states decided unilaterally to invade Iraq in 2003, they set a dangerous precedent in a world where only the Security Council was seen as capable of authorizing military interventions and the use of force. The United Nations’ balance sheet To properly diagnose what is wrong with the United Nations and what possible reforms could improve its ability to govern, we need to study its achievements and failures. As this primer will explore, the UN has had numerous successes in its over 70-year existence. These range from the public achievements attributed to the UN, to the everyday governance that is rarely associated with it, to its effects which cannot easily be measured and rarely make headlines. y y The contribution that the UN has made over its lifetime to creating a more peaceful, just and sustainable world is so immense that it would be impossible to cover everything. It has unmatched legal legitimacy and global convening power and has been indispensable in shaping international law, rules and norms through adopting treaties and other legal instruments. From the Univer- sal Declaration of Human Rights in 1948 to the Treaty on the Nonproliferation of Nuclear Weapons in 1968, it is responsible for a considerable body of inter- national law that guides states’ behaviour. Several other treaties relating to the rights of indigenous peoples, persons with disabilities, children, refugees and other minorities have ensured that specific rights are outlined for individuals or groups that are particularly vulnerable. The UN has made real progress in tackling climate change between the Kyoto Protocol, the UN Framework Con- vention on Climate Change, and the Intergovernmental Panel on Climate Change. Most recently, the Paris Agreement succeeded in getting member states to commit to much-needed emission reductions. Beyond climate change, it has provided leadership on other environmental issues, from curbing ozone layer depletion, to protecting biodiversity and encouraging alternative energy. y p p g y g g gy While the UN cannot take credit for all the progress in socio-economic well- being and health, it has made enormous contributions. Its humanitarian pro- grams deliver vital services to those in need, saving countless lives and im- proving the conditions of many more. It has been instrumental in shaping and implementing a global development agenda, as we witnessed with both the Millennium Development Goals in 2000 and the Sustainable Development Goals in 2015. The UN has done much to promote gender equality, including establishing key international norms and creating UN Women in 2010. 16 Beyond this relatively visible work, most international economic and polit- ical activity takes place fairly seamlessly thanks to a host of UN regulatory institutions. The United Nations’ balance sheet When the UN was not present at the 2015 negotiations on the Iranian nuclear agreement, it sent a powerful signal to the organization, which for decades has been push- ing for nuclear disarmament and non-proliferation. For several years now the Security Council has failed to act in Syria, where a prolonged civil war has resulted in hundreds of thousands of deaths and the displacement of millions. The international community has tried to step in, but Russia and China have used their veto power to halt the attempts. The ability of the Security Council’s five permanent members to veto resolutions helps explain why the UN is not always able to deal effectively with crises such as Syria. The UN’s failure to counter the rising threat of terrorism has further damaged its image as the world’s upholder of peace. On top of all this, reports of sexual abuses by UN 17 peacekeepers in the Democratic Republic of Congo and Central African Re- public along with findings that the peacekeeping force in Haiti was responsible for the cholera outbreak after the 2010 earthquake have together served to tar- nish the reputation of the long-admired blue helmets. On the human rights front, the UN has been inconsistent in its approach to dealing with grave violations and has let politics trump principled action nearly every time. This has severely affected its legitimacy and credibility as a human rights defender. It has proven unable or unwilling to prevent mass atrocities including genocide, crimes against humanity and war crimes, in Rwanda, Dar- fur, the former Yugoslavia, and Syria, among other places. At the same time, it has not been successful in coordinating an international response to the mi- grant and refugee crisis in terms of mobilizing the required resources and get- ting states to accept more people in dire straits. The diffusion of the Responsibility to Protect norm has succeeded in making the principle of state sovereignty and nonintervention conditional rather than absolute. Yet politics and national interests still determine which situations will receive attention and which will be ignored. Meanwhile the International Crim- inal Court—a promising innovation created to prosecute the worst human rights offenders—struggles to remain relevant as some countries have chosen not to join while others are exiting. The UN has had its share of troubles in advancing sustainable development too. The United Nations’ balance sheet The lofty goals set out in the post-2015 development agenda require far more resources than are currently available. At the same time, the complex UN development system made up of numerous organizations and agencies, often with overlapping mandates, has resulted in inefficiencies, duplication, lack of coherence and competition for scarce resources. Its standing as a global health leader has been jeopardized by its slow and inadequate response to pandemics such as the Ebola outbreak in West Africa in 2013-14, where its organizational culture was largely to blame. And the Economic and Social Council, the UN’s principal coordination body for all economic, social and environmental mat- ters, is in urgent need of reform. With other regional and multilateral organizations, such as the World Bank and regional development banks, as well as private, philanthropic actors like the Global Fund and the Gates Foundation, being perceived as more efficient and responsive than UN agencies, the UN risks seeing development funding diverted elsewhere. Finally, as we look ahead it remains unclear whether it is fit to broker and manage 21st century partnerships for development that require deeper collaboration between public, private and civil society actors, while harnessing innovation and technology to enhance its development impact. The urgency of current global challenges alongside the failures of the UN and the growing tendency for states to circumvent it suggest that action is needed now. There are a range of options available, varying from minor tweaks to the existing form and function, to rebuilding the organization from the 18 ground up. If the latter were possible, the potential for a better global institution would be limited only by our collective imagination. But a healthy dose of pragmatism is advisable. We must recognize the hurdles that are to be sur- mounted; perfect cannot be the enemy of good. Nevertheless, the demands on the UN system require more than a series of incremental improvements. The UN’s structure, functions and allocation of re- sources have undergone reform over the years but not to the degree necessary to keep pace with rapid change. The organization today is not ‘fit for purpose’. Yet there is no shortage of proposals for improving the dated institution. Schol- ars, UN officials and other experts are continuously developing workable re- form ideas. Often, there is general consensus around what should be done. The United Nations’ balance sheet Though even in these cases, making change happen is no easy task for a host of reasons. When it involves a slow, bureaucratic and political organization like the United Nations it becomes harder still. Ultimately, no individual actor can do it alone. A concerted effort is needed to transform its institutional defi- cits. This could take the form of a multi-stakeholder coalition between willing states, NGOs, UN officials, independent experts, and other players. Now is the time to mobilize diverse actors, identify common goals and develop and im- plement an agenda for change. Empowering youth We equally cannot achieve the transformation needed without engaging youth. Home to 1.8 billion young people, the world has never in history had such a large youth population as it has today. One quarter of the world’s population is between the ages of 10 and 24. Nine out of ten youth are in developing coun- tries, many of which are experiencing a growing youth bulge while most de- veloped countries tackle issues stemming from an aging population (UNFPA 2014). These young people, who are more informed, engaged and globally connected than ever before, should become the next generation of leaders who will shape our common future. In his address to the 71st UN General Assembly, former US president Barack Obama praised the youth of our time, stating, “I have seen that spirit in our young people, who are more educated and more tolerant, and more inclu- sive and more diverse, and more creative than our generation; who are more empathetic and compassionate towards their fellow human beings than previ- ous generations.” He went on to say that because of young people’s access to information about other peoples and places, they have “an understanding unique in human history that their future is bound with the fates of other human beings on the other side of the world.” Indeed, today’s youth have incredible power to craft a more peaceful, just and sustainable world. The 2016 High-Level Segment of the General 19 Assembly saw an unprecedented number of world leaders acknowledge this, with 59 member states emphasizing the crucial role of youth in their national statements (United Nations Youth Envoy 2016). Assembly saw an unprecedented number of world leaders acknowledge this, with 59 member states emphasizing the crucial role of youth in their national statements (United Nations Youth Envoy 2016). The UN itself has taken note. In his second term as secretary-general, Ban Ki-moon made working with and for young people one of his top priorities. He established the Office of the Secretary-General’s Envoy on Youth and chose Ahmad Alhendawi of Jordan to serve as the first-ever UN Envoy on Youth beginning in 2013. At 28 years old, he was the youngest senior official in the history of the UN. Empowering youth He was mandated to harmonize youth development efforts across the UN system, enhance the UN response to youth needs, advocate for addressing the development needs and rights of young people, and bring the voices of young people to the UN (Youth Envoy website). y g p p ( y ) Despite a series of public statements and gestures recognizing the immense potential of youth, there are too many young people around the globe who live in poverty and countless are being denied the opportunity to pursue their edu- cation and find decent employment. The number of children and adolescents out of school is on the rise, and reached 124 million in 2013. There are over 73 million unemployed youth worldwide. At 13 per cent, the overall youth unem- ployment rate is three times the adult rate, though it is even higher in some regions (in two thirds of European countries the youth unemployment rate ex- ceeded 20 per cent in 2014; the figure is close to 30 per cent in the Middle East and Africa) (ILO 2015). The reality for girls and young women is even more troubling. Secondary school enrolment rates are often lower for girls than boys and only about two out of 130 developing countries have achieved gender par- ity at all levels of education. Unemployment affects young women more than young men in almost all regions, while in North Africa and the Arab States the female youth unemployment rate is almost twice that of young men (ILO 2016). Those in countries affected by conflict are also worse off than most. Meanwhile, many countries are failing to give a voice to their youth; two out of three countries do not consult young people as part of the process of preparing poverty reduction strategies or national development plans (United Nations Youth Envoy 2014). Youth participation in national parliaments is low, with less than 2 per cent of parliamentarians globally under 30 years old (Inter-Parliamentary Union 2016). It is little wonder, then, that, despite their potential, youth often feels disem- powered. Voter turnout among 18-25 year olds continues to be lower than other age groups and a lack of civic engagement among youth is common. But young people cannot afford to watch from the sidelines. They need to press for action and positive change. They need to better the world for themselves and for fu- ture generations. Empowering youth With political rights come responsibilities. More than ever, the world needs its youth to elect good leaders, get involved in politics, expand their understanding of global problems and develop solutions. 20 Chapter 1 – Evolving International Organizations: the UN Past and Present “[He] wondered why men could rarely harness this same sense of oneness toward good ends. Men would sacrifice their own interests, even their own lives, welding themselves together with bonds that far surpassed ordinary life, toward the purpose of killing one another. But when it came to creating beauty and life and love, too often men were left to act alone, their every act weighed against self-interest and simple inertia. If men were as good at creating heaven on earth as they were at creating hell, it would be a very different world.” Rachel Lee (2007: 474) To really understand the United Nations it is not sufficient just to describe its structures, personnel and activities. It is first necessary to explain its begin- nings and the intentions of its founders. And then the hard part begins: we have to weigh its strengths and weaknesses and analyze its components. This is the plan for this chapter. Objectives of the primer This primer on global governance and United Nations reform analyzes the or- ganization in its current form while offering alternatives for the future. It aims to provide the fundamentals to those who are relatively new to the subject. It seeks to be informative and thought-provoking while remaining accessible to a broad range of audiences, varying from students to practitioners. It is designed to:  provide an overview of essential information about the United Nations system including its historical and global context;  provide an overview of essential information about the United Nations system including its historical and global context;  delve into the UN’s record on its three ‘pillars’: human rights, peace and security, and development;  introduce various ideas and proposals for renewing the organization so it can better meet the demands of tomorrow; and  explore the role of norms, values and attitudes as well as diverse actors in building a movement for a UN renaissance.  explore the role of norms, values and attitudes as well as diverse actors in building a movement for a UN renaissance. We take the notion of renaissance to have two meanings. First, it is used to refer to renewal, rebirth, revival or even spring, which leads us to think about change, reform and transformation at the UN. The second sense refers to the essential meaning of the historical renaissance as ‘a return to origins’. Dotted throughout the book are references to the UN’s founding objectives, which in- cluded preventing the scourge of war, getting great powers to cooperate on essential decision making, striving to protect human rights, and ensuring eco- nomic coordination. This book is dedicated to a renewed search for the initial aims of the United Nations: peace, development, cooperation and human rights—and, indeed, much more as the world has continued to evolve. Thus, we use renaissance to call for a transformation of the UN that remains deeply rooted in its original lofty goals. We hope this book prepares and inspires readers to join and expand existing efforts to achieve this renaissance. 21 21 Early international cooperation efforts The United Nations is not the first but the third in a series of international or- ganizations that date back to 1815. To give some historical context to the es- tablishment and workings of the United Nations as it is today, the first part of this chapter describes these organizations and the key events that led to their creation (see Figure 1). Figure 1: Evolution of international organizations, until 1945 22 The Concert of Europe The Concert of Europe The Concert of Europe was founded by the Treaty of Vienna, which put an end to the Napoleonic Wars that had lasted nearly 20 years. In many ways it set the mold for its successors: the League of Nations and the United Nations. Like these last two, the Concert was founded in the aftermath of a devastating war waged by a group of allies to stop one nation from trying to set up an empire to dominate the world. The word ‘concert’ was intended to mean a bringing together of states in a concerted effort to work on common concerns. It was a radical departure from the past. At the time, it was called “a principle of general union, uniting all the states collectively with a federative bond, under the guid- ance of the five principal Powers” (Mazower 2013: 4). Up until the Napoleonic Wars, Europe (as other parts of the world) was ordered by an ever-changing “balance of power” by which each sovereign state attempted to maximize its own interests and stop any state or group of states from obtaining overwhelm- ing dominance. This was the continually shifting basis of foreign policy. Na- poleon’s France had upset this balance. The allied powers wanted to re-estab- lish it on a permanent basis. Thus, the Concert of Europe was like a continuing coalition of the Great Power victors of the Napoleonic Wars (Russia, Prussia, Austria and Great Brit- ain, plus the newly monarchical France). Its role was to convene meetings on a regular basis or upon need and to include other smaller countries to discuss overlapping interests and their efforts to maintain stable European relations. The Concert’s two major functions were to maintain peace between countries and to ensure the internal stability of the established monarchical governments against nationalist, liberal and democratic revolts. Consultation often checked aggressive impulses. It generally achieved its twin goals for more than a half century from 1815 until the Franco-Prussian war in 1870-71. The Concert of Europe sought to manage the affairs of the continent by binding all states to the rules of the international game. Sometimes this could only be achieved by intervening in the affairs of others. In fact, the word ‘in- ternational’ was a relatively new concept that suggested there were ongoing links between states despite their past habit of just wanting to ‘do their own thing’ based on their sovereign independence. This right of sovereign nation- alism harked back to the 1648 Peace of Westphalia—which itself put an end to Europe’s Thirty Years War. Nevertheless, on several occasions after the French Revolution, the Concert did not hesitate to interfere in the internal af- fairs of states to enforce a conservative restoration. For instance, in 1823 Con- cert members invaded Spain to drive a revolutionary government out of Ma- drid. International relations grew slowly but steadily in the 19th century. Between 1840 and 1914, there were nearly 3,000 international gatherings. More than 450 international, private or non-governmental organizations were created and 23 the Concert itself grew to 37 governmental organizations. International NGOs invited themselves to the international meetings, thus becoming new ‘actors’ in international relations and the forerunners of today’s ‘civil society’. Their ‘competitor’ was the historic notion of sovereignty (see Box 1). Box 1: Understanding ‘sovereignty’ Sovereignty grew out of the Peace of Westphalia of 1648 to define the legal identity of a state in international law. Each state is considered to have sovereign equality. There is a corresponding obligation to respect each other’s sovereignty by not intervening in another’s internal affairs. Internally, sovereignty signifies the capacity of the government to make authoritative decisions through exclusive jurisdiction within its territorial boundaries, which it has the right to defend. This is international law and its principles are hotly defended, but, obviously, it is not the international reality. The ‘sovereign equality of states’ is enshrined in Article 2.1 of the UN Charter. The principle of ‘non-intervention’ is to be found in Ar- ticle 2.7. The ‘right of self-defence’ is embodied in Article 51. The UN Secretary-General has discussed the dilemma of the two concepts of so- vereignty, one vested in the state and the second in the people and indi- viduals. The Report of the International Commission on Intervention and State Sovereignty proposed the extension of the concept to include ‘responsibility’ alongside ‘control’, so that state authorities would have the responsibility to protect their citizens and would be responsible to the international community through the UN. This gave rise to the UN’s 2005 resolution on the Responsibility to Protect (R2P). Source: International Commission on Intervention and State Sovereig- nty, 2001: 12-3 Source: International Commission on Intervention and State Sovereig- nty, 2001: 12-3 Right from the beginning of international institutions, there was debate over how much the creation of a set of arrangements to maintain peace and stability could legitimize intervention in the affairs of others—debates which continue with the new UN policy of Responsibility to Protect (R2P). International af- fairs are not just about the arrangement of relationships. They are also about dominant ideas and a desire for power. So, it was said, “The Concert of Europe had not mastered the new art of international government; it was, on the con- trary, a symbol of the very problems—autocratic leadership, bellicosity, an in- comprehension of the values of freedom and the power of social change—that a true internationalism was needed to solve.” (Mazower 2013: 12). In reality, foreign relations in 1815 were really the playthings of sovereign monarchs and their aristocratic ministers who were determined that Europe would not only remain stable but would crush radical democratic tendencies. Box 1: Understanding ‘sovereignty’ One example was the French Foreign Minister, Prince Talleyrand, who used 24 his diplomatic dexterity to insist that France, although defeated, was too central to Europe to be dismembered. These men dominated the 1815 peace treaty, Congress of Vienna, and the Europe to which it gave rise. And they were only men because, with a few exceptions like Queen Elizabeth I, women were not involved in foreign relations until after the Second World War when Eleanor Roosevelt and several colleagues implanted the Universal Charter of Human Rights within the United Nations. We can also recognize that international organizations are not purely polit- ical creations; they also reflect their time and their context. In many ways, pol- itics follows the lead of economics and technology. Thus, the development of international organizations throughout the 1800s was greatly inspired by in- dustrialization, and the expansion of railways and shipping beyond national boundaries. Business sought continent-wide security for markets and invest- ments. Together, science and commerce framed the modern thinking that made possible the ideas of internationalism. Modern forms of trade and transport led to the forming of ‘public international unions’ (the forerunners of today’s ‘spe- cialized agencies’ at the UN) in the fields of transport, communications, weights and measures, statistics, patents, agriculture, labour, science, policing and sanitation. In addition, international movements for workers, women and peace formed world public opinion. If fact, there have been a great number of influences on the development of international organizations. Although we recognize that the impetus for inter- national organization comes out of the chaos of post-war disintegration, theo- rists maintain that, in general, an evolutionary or ‘genetic’ perspective seems best suited for us to understand how they developed from a host of interests (national and international, public and private), as well as internal learning pro- cesses and even the very act of international conferencing (Reinalda: 2001). Evidence suggests that in international organizations, innovation often comes out of lively and continuous debate originating in scientific, technical and hu- manistic communities (Reinalda 2001; Schemeil 2003). Indeed, the conferenc- ing process creates a temporary equality among unequals and encourages the sort of open discussion that often gives rise to innovation (Murphy 1994: 62). Conferencing was also conducive to developing the techniques and the psy- chological aptitudes required for successful multilateral negotiation (Claude 1966: 23). A note on the creation of the League of Nations The context just described was both the process and the inducement for the forming of international institutions from the 1850s to the 1900s. It was also part of the background for the formation of the League of Nations in 1919 following the First World War. Other inducements were the atrocious loss of life, the destruction of countries, the wartime cooperation of the allies, and the need for an on-going organization as a forum to promote peace rather than war. Also of great significance was the spirited leadership of the American Presi- dent, Woodrow Wilson. After the Second World War in 1945 it was much the same story that led to the United Nations except the leadership came from a new American President, Franklin D. Roosevelt. The League of Nations was founded in January 1920 as a result of the Paris Peace Conference and the Treaty of Versailles which terminated the First World War. As the world’s first permanent intergovernmental organization, its principle objective was to maintain world peace via collective security, dis- armament and arbitration. It had an initial 44 state members, which rose to 58 in 1935. Its headquarters were established in Geneva immediately after its founding. As we will see, the various in-depth analyses of the founding of the League and the UN teach us much about the causality, meaning and intentions of in- ternational organizations (see Mazower, Reinalda, Schlesinger, Archer and Trent). The League came about as a result of history, national interests, ideas, and personalities. It was a combination of ‘big bang’ politics responding to the war, and evolutionary development based on historical precedent. When it came to thinking about post-war institutions, the allied participants in the 1919 Paris Peace Conference (dominated by the US, France, Great Britain and Italy, with personalities like Jan Smuts of South Africa as additional players) had in their minds their cooperation during the war but also the internationalism of the past century. In their deliberations, they considered a number of options: resurrecting a new version of the old Concert; a model based on the American Monroe Doctrine which proclaimed US sway over Latin America; the British Empire transformed into a Commonwealth; and simply of a return to balance of power politics. The thinking that predominated was essentially that of Great Britain and the US. Being great powers, neither wanted any new arrangements to tie their hands too much. Box 1: Understanding ‘sovereignty’ Engaged thinkers and practitioners found they could implement their ideas on a world scale and sometimes were called upon to prepare gov- ernment positions. In other cases, NGOs learned that governments can be moved toward cooperation by private or unofficial pressures, when skillfully applied (Lyon 1963: 154). Changes in domestic politics such as new govern- ments and political leaders can constitute windows of opportunity for new ad- vances at the international level (Reinalda 2003: 9). 25 A note on the creation of the League of Nations They wanted to maintain the Anglo-American alliance but in a larger format that did not look too self-interested or racist. The French and the British did not want the League to interfere with their imperial ambitions. Many thought the new arrangements should be as light as possible and be cen- tred on negotiations, international law, mediation and arbitration. For both the League and the UN, the wartime experience of cooperation was crucial as a 26 seedbed for more centralized and systematic world relationships and the im- portance of procedure and precedents. But when push came to shove, it was the negotiations between the leaders of the major, victorious powers which drove the founding of both the League of Nations and the United Nations. In Paris in 1919, U.S. President Woodrow Wilson ended up in protracted negotiations with the French President Georges Clemenceau, British Prime Minister David Lloyd George and Italian Prime Minister Vittorio Orlando—leaders who were equally creatures of their own personalities, philosophies and visions, and influenced by their close advisers. Known as the ‘Big Four’, these leaders had no intentions of relinquishing con- trol of the conference agenda—a tradition that is maintained to this day. They also maintained the earlier tradition of diplomatic secrecy because they thought not to do so would be “a veritable suicide” for their negotiations. But they surrendered to the growing demands of journalists for regular plenary sessions and briefings. Small states were “given something to do” as an adden- dum to the Council and in the powerless Assembly. Following the submission of plans by various leaders, thinkers and associations, Robert Cecil, the British undersecretary of foreign affairs, was able to provide a draft text for the League, based in part on a study of historical precedents (as the American Sec- retary of State later did for the UN). It was discussed, amended and adopted in two months. However, it would be incorrect to think there were no other sig- nificant influences on the peace negotiations. A note on the creation of the League of Nations Among the other major influ- ences were: the blending of morality and realpolitik in the provocative ideas of Jan Smuts, Prime Minister of South Africa; the London intellectual elite in- cluding Leonard Woolf, who had written a suggestive book entitled Interna- tional Government; the demands of small allies, especially those in the nascent British Commonwealth who had helped win the war; and popular associations (now NGOs) such as the League of Nations Association. The League of Nations as an institution To the degree that he wanted a permanent organization, President Wilson of the United States wanted to keep power with the politicians in a forum for quasi-parliamentary deliberations rather than with the lawyers in a sort of super judicial court. He wanted an institution that would evolve with the collective will. He also wanted to protect the territorial integrity of the growing number of nation-states in a universal association of nations capable of acting in the common interest to help prevent war. “The days of the Treaty of Vienna are long past,” as British Prime Minister Lloyd George put it, referring to the birth of the Concert of Europe (Mazower 2013: 127). There were, of course, many contradictions. The general principle of national self-determination remained vague. Britain, the great imperial power, thought that the Empire itself 27 resembled “a league of nations”. Prior to the Paris Peace Conference, many questions remained. How much executive power would a League possess? How far would it be committed to protect established boundaries? Could sanc- tions be automatically triggered? How far would the League go beyond being just a more permanent conference system? However, history shows that once at the Peace Conference, personal diplo- macy took over. A committee chaired by Wilson rapidly approved the British draft agreement. There would be a tripartite division of powers in a parliamen- tary type organization. It consisted of: a Council with Great Power permanent members and four elected, rotating lesser powers in a sort of ‘upper chamber’; a one-member, one-vote Assembly; a relatively weak secretary-general in an administrative rather than a diplomatic post; and, in 1922, a Permanent Court of International Justice. The Council’s permanent members were France, Great Britain, Italy and Japan (the US did not join). Eventually, the Assembly’s 40 members, meeting at the new headquarters in Geneva, represented minimally the various parts of the still-colonized world. Almost all of Africa, Asia and the Middle East were controlled by European imperial powers. The Assembly had no law-making functions and each member could veto any action. In any case, the League lacked any standing forces or mechanisms for enforcing peace. All it could do was to recommend arbitration, sanctions and boycotts. It was even unable to apply sufficient pressures in clear-cut cases of aggression. Nevertheless, it did introduce a democratic dimension of public deliberation and opinion. The League of Nations as an institution Also, the full Assembly only met once a year, so the rest of the time the League’s merit- based civil servants were free to take the initiative. This became a major dimension of the League and its definitive heritage. The bureaucracy was created by Eric Drummond, a mid-level official of the British Foreign Office, who went on to hold the office of Secretary-General for 14 years, longer than anyone either at the League or the UN. Having built a small (650), highly professional international civil service, one with perma- nence and autonomy that favoured expertise over diplomacy, he became one of the architects of modern international organizations. It was divided into functional secretariats that “cemented the connection between internationalism and technical expertise” (Mazower 2013: 148). For instance, German and So- viet officials worked with the League’s technical services before their coun- tries even formally joined. The Secretariat also took advantage of Articles 23 and 24 of the League’s Covenant which gave it wide responsibilities and al- lowed it to become an umbrella organization that gathered under its auspices the 31 international bureaus (also called ‘permanent conferences’ or ‘public international unions’) that had been formed before the First World War. One example of the League’s extraordinary feats as a humanitarian organi- zation was its overseeing of emergency relief camps giving succour to the Rus- sian, Armenian and Greek refugees in the Balkans in the early 1920s. They ran 28 and financed tented encampments and brought in health experts to stop infec- tious diseases. They helped the Greek government get loans to resettle hun- dreds of thousands of refugees. They created the Refugee Settlement Commis- sion to build new settlements, even small towns. This effort by a government organization was entirely new. It had previously been the work of charities. We can easily see how there is an eerie resemblance to events in the Middle East since 2010 and may ask: why is the world still having to deal with the same horrific problems one century later? The League also became recognized for gathering data and issuing volumes of statistics. Its health, transport, and financial and economic sections became indispensable in the interwar years. Strangely enough, many of its personnel came from the United States and much of its technical funding came from the Rockefeller Foundation. The League of Nations as an institution But it was paired with a will to 29 international leadership, exemplified in the interwar years by the private en- dowments which financed support for the League and for institutes, journals, international relations clubs and conferences to train Americans for their role in the world. For its part, the Soviet Union did not even join the League until 1934 and then was the only state to be kicked out after it invaded Finland in 1939. Ger- many, which had not been welcomed at the beginning, joined in 1926 and then withdrew under Hitler’s Nazi regime in 1933. Mussolini’s fascist Italy fol- lowed in 1937 after having invaded Ethiopia. Japan, unhappy with criticism of its occupation of Manchuria, withdrew in 1933. Germany, Italy and Japan, which would become known as the Axis powers, each had a grudge against the dominant colonial system the League represented. They wanted what the French and British had. They wanted territory, colonies and power. For them there was a power imbalance in the world represented by the League. This virtually left Great Britain and France among the Great Powers and they were still too ravaged by their losses during the First World War to devote energy to saving the League. What with its heavy burdens and quarrelling members, the League was not able to stop the slide to a new world war. The battle of ideologies consisted mainly of multiple attacks on the Western theoretical claim about the fairness of international law, equally applied to equal states, as a keystone of the League. For instance, the Western empires did not even subject themselves to minority rights treaties. What Germany, Italy and Japan had in common was their criticism that the League was just a ‘fig leaf’ to keep in place a territorial status quo that favoured Great Britain, France and the US. The same criticism was to be levelled later about the UN. Hegemony, the leadership of one or more states, was the central German con- ception of world rule. Japan too saw the League as protecting the self-interests of the imperialists in Asia. The Soviet Union weighed in with the establishment of the ‘Third Interna- tional’ in 1919 as a direct rival to the League of Nations. Lenin even referred to the League as a “stinking corpse” (Mazower 2013: 177). Soviet diplomats became more measured and conciliatory though, even joining the League in 1934. The League of Nations as an institution It worked hard to restore monetary stability but lost out to the protectionism of the Great Depression. Its members prided them- selves on being impartial and above national interests (unlike the UN) but not above paternalistic interference in member states’ internal affairs. Overall, some two hundred employees of the League’s Secretariat went on to work for the UN and even to be leaders of a number of post-Second World War inter- national organizations—including Jean Monnet, who would become one of the founding fathers of the European Union. That said, the League was battered continuously by horrendous public ca- lamities, the defection of prominent members and the battle of ideologies. In 1918 and 1919, Spanish influenza affected one fifth of the world’s population and killed between 20 and 40 million people, exceeding the deaths during the First World War. More deadly than the Black Plague in the 1300s, it was the worst scourge ever to sweep the world. But the League also had to face con- tinuing war and a refugee crisis in the Middle East, economic collapse in East- ern Europe and a public health disaster including influenza, typhus and mass starvation. In addition, Western leaders were terrified by the spread of com- munism from Russia’s revolution, which was welcomed by many in Western Europe due to economic collapse and massive unemployment. On top of all this, the new League was called upon to administer the territory of the Saar and the Free City of Danzig and look after the plight of the Christians in Turkey and the status of Armenia. By 1929, the world fell into the grip of the Great Depression. The defection of prominent states never ceased. Perhaps the cruellest blow to the League was the vote by the U.S. Senate against joining President Wil- son’s brain-child. The decision reflected a refusal by part of the political elite to become enmeshed in ‘entangling alliances’ and the world’s troubles. Public support for the League was low, and there was resistance in Congress to mak- ing commitments to international organizations that would hamper the freedom of action of the United States. It was a fine example of American isolation- ism—something that still lingers today. Box 2: Strengths and failures of the League of Nations  The quasi-parliamentary model at the League’s core was abused from the start by members who came to Geneva more for theatrics than policy-making. The pre-war ideals of open negotiations and di- plomacy to stimulate world public opinion ended up creating unful- filled expectations.  The quasi-parliamentary model at the League’s core was abused from the start by members who came to Geneva more for theatrics than policy-making. The pre-war ideals of open negotiations and di- plomacy to stimulate world public opinion ended up creating unful- filled expectations. p  The unanimity rule (i.e., each member holding a veto) made deci- sions hard to reach, thus condemning the organization to impotency.  Its irresolution was magnified by its lack of any means of enforce- ment or deterrent such as armed forces or a police force so that even the rules and laws it did proclaim lacked authority.  The League was operating on a shoestring in a period of financial stringency. Its budget of $5 million a year was less than a thirtieth of the UN’s—also considered minuscule in today’s multi-billion-dollar world.  Due to these failures, the League was not able to provide answers to the urgent crises of the day, thus reinforcing public perceptions of its inefficacy.  The League’s technical services provided by its professional civil service took international humanitarian cooperation and the promo- tion of science much further than anyone had imagined possible be- fore the First World War. It offered the promise of democratization and social transformation through technical expertise.  Its technical, intellectual and scientific skills proved the value of an international organization. Even if it was a diplomatic failure, its ex- pertise and international action became a model for the evolutionary growth of cooperative behaviour due in part to its flexibility and multitasking. g  Not all initiatives worked out. For instance, the Institute for Intellec- tual Cooperation had few tangible results as Europe fragmented ide- ologically, and few states ratified the 1937 Treaty for an Interna- tional Criminal Court (60 years before its time)—even the French proposal for a federal union of Europe to combat the Depression was not successful!  The League’s enduring influence was as a vehicle for world leader- ship based on moral principles and the formal equality of sovereign states. The League of Nations as an institution For instance, the Institute for Intellec- tual Cooperation had few tangible results as Europe fragmented ide- ologically, and few states ratified the 1937 Treaty for an Interna- tional Criminal Court (60 years before its time)—even the French proposal for a federal union of Europe to combat the Depression was not successful!  The League’s enduring influence was as a vehicle for world leader- ship based on moral principles and the formal equality of sovereign states. It managed to marry the democratic ideal of a society of na- tions with the reality of Great Power hegemony. The League of Nations as an institution For Moscow, its security came before revolution. For its part, Italy pro- moted a ‘fascist internationalism’ in a fascist European confederation, suppos- edly fighting for the rights of European nation-states. One can see arising out of these affirmations of sovereignty and nationalism the destabilizing potential from which the UN too would suffer. The leading American political scientist, Quincy Wright, worried that, “totalitarianism has unmasked the inadequacy of the philosophical and political foundations of in- ternational law.” (Mazower 2013: 187). Its impartial authority lay in tatters. International law would require a demonstration of shared values and interests, alongside the enforcement capacity. 30 Box 2: Strengths and failures of the League of Nations  The quasi-parliamentary model at the League’s core was abused from the start by members who came to Geneva more for theatrics than policy-making. The pre-war ideals of open negotiations and di- plomacy to stimulate world public opinion ended up creating unful- filled expectations.  The unanimity rule (i.e., each member holding a veto) made deci- sions hard to reach, thus condemning the organization to impotency.  Its irresolution was magnified by its lack of any means of enforce- ment or deterrent such as armed forces or a police force so that even the rules and laws it did proclaim lacked authority.  The League was operating on a shoestring in a period of financial stringency. Its budget of $5 million a year was less than a thirtieth of the UN’s—also considered minuscule in today’s multi-billion-dollar world.  Due to these failures, the League was not able to provide answers to the urgent crises of the day, thus reinforcing public perceptions of its inefficacy.  The League’s technical services provided by its professional civil service took international humanitarian cooperation and the promo- tion of science much further than anyone had imagined possible be- fore the First World War. It offered the promise of democratization and social transformation through technical expertise.  Its technical, intellectual and scientific skills proved the value of an international organization. Even if it was a diplomatic failure, its ex- pertise and international action became a model for the evolutionary growth of cooperative behaviour due in part to its flexibility and multitasking.  Not all initiatives worked out. From the League of Nations to the United Nations One day during a visit to Washington in 1941, Prime Minister Churchill was just getting out of his bath when President Roosevelt rolled his chair in and declared that he had thought of the perfect name for the new organization: the United Nations. Churchill immediately agreed it was a brilliant idea (Mazower 2013: 197). It is little known that, although the UN was not officially founded until the San Francisco Conference in 1945, the Second World War was fought under the auspices of the United Nations Alliance in which 26 allies joined the United States and Great Britain. With their backs to the wall, the war years were in many ways the heydays of liberal multilateralism and institution build- ing. The war encouraged cooperation. In terms of international relations the- ory, we may conclude that building the UN was evidence that multilateralism and robust intergovernmental organizations were viewed as “realist necessi- ties, not liberal window dressing” (Plesch & Weiss 2015: 199). g ( ) In 1942, the UN Information Office went to work and the Food and Agri- culture Organization (FAO) started planning for the eradication of hunger. Aside from the many backroom debates about the nature of the UN, the UN Relief and Rehabilitation Administration was created in 1943 (it then shut down operations in 1947). The International Labour Organization was revived in 1944, the same year that saw the Bretton Woods UN Monetary and Financial Conference that gave birth to the World Bank Group and the International Monetary Fund. The UN War Crimes Commission was a precursor to the Nu- remberg and Tokyo trials. In a strange little twist of fate, one of the remaining utilities of the League of Nations was transferred to the United States in 1941. With the help of the Rockefeller Foundation, 12 key Secretariat members with their files and experience transferred to Princeton, New Jersey to take up resi- dence in the Institute for Advanced Study where they advised on the post-war problems. So, what was behind all this creative action? What did the leaders want out of the new United Nations? Needless to say, they all wanted an organization that was strong enough to promote international peace. In addition, Great Brit- ain wanted to maintain its ‘special relationship’ with the US. Box 2: Strengths and failures of the League of Nations It managed to marry the democratic ideal of a society of na- tions with the reality of Great Power hegemony. 31 We recall all this (along with the League’s strengths and failures outlined in Box 2) now to remind ourselves of the sensitivity of international organizations to the politics of their member states, and to the continuous attacks by ideo- logues, by the media and by an unreasoning public. From the League of Nations to the United Nations From the League of Nations to the United Nations It also believed that having the US and the Soviet Union in the new organization was more important than the exact form it would take. Joseph Stalin, the leader of the Soviet Union, wanted to maintain the alliance until he had time to rebuild his country. U.S. President Roosevelt had learnt from his ‘New Deal’ economic 32 development program that good politics went hand in hand with socio-eco- nomic programs. In the context of the UN, this meant that fostering post-war, international economic and social stability would make a contribution to main- taining peace and security. The massive unemployment following the First World War led to social unrest that was a harbinger of a new world conflict. The Americans thought it was crucial to prevent a renewed post-war economic slump by structuring an international bank and monetary fund to slay the triple- headed dragon of economic nationalism, speculative capital flows and trade barriers. To determine where they were heading, the Americans set up a top- secret ‘Division of Special Research’ as early as 1941 under Leo Pasvolsky, an aide to Secretary of State Cordell Hull, to plan a new permanent world or- ganization. The three major allies (Britain, the Soviet Union and the US) had differing ideas right up to the last minute, even concerning the actual nature of the new organization. Early on, they envisioned an international police force run by the three allies along with China. Another option considered was to set up a ‘World Council’ made up of three regional councils (Europe, the Americas and the Pacific). In the end, they agreed to some post-war structure that could more effectively manage conflicting interests and contain, at minimum, the threat of military and humanitarian disasters (Meacham 2003: 202-29). By the Dumbarton Oaks conference in 1944 and the Yalta conference in 1945, the concept of the United Nations started to gel. The conversation shifted from regional councils and other proposals to planning for an organization that could exert force to try to maintain order, complete with a Security Council (with permanent members) and a General Assembly (ibid: 321). It was decided that the UN would preside over a vast expansion of the social policy initiated by the League Secretariat, and that a voice would be given to the smaller nations under the leadership of the major powers. From the League of Nations to the United Nations From the experience of these three international organizations we should recall the following:  All resulted from efforts to create stability and peace after devastating wars;  They also resulted from the evolution in international relations and the socio-economic context;  All were dominated by the great powers of the period;  Foreign relations are still masterminded by political elites with little democratic control;  The right of international political intervention is still contested; g p ;  All were torn by the contradictory desires for both cooperation and sov- ereign independence, and for equality but also leadership of the strong;  States want the help of international organizations to smooth out their relationships—but these organizations are to be the creatures of the states and must not become too powerful or independent; and  States want the help of international organizations to smooth out their relationships—but these organizations are to be the creatures of the states and must not become too powerful or independent; and p p  International organizations are not purely political creations; they also reflect their unique context.  International organizations are not purely political creations; they also reflect their unique context. From the League of Nations to the United Nations Churchill and Roosevelt sought to build institutions that would prevent the mistakes of the first half of the 20th century from repeating themselves in the second half. Nothing was left to chance as the Americans ramped up the promotion of the United Nations ideal using not only bipartisan political resolutions but adver- tisements, Hollywood showgirls, and musical anthems. On March 1, 1945, just weeks before his death, Roosevelt made a last plea for a global, rather than regional, organization. He told Congress that the UN agreements in Yalta "ought to spell the end of the system of unilateral action, the exclusive alli- ances, the spheres of influence, the balances of power, and all the other expe- dients that have been tried for centuries—and have always failed.” (ibid: 321). What we can see from all this is that states only began thinking about inter- national institutions when they feared their interests or those of the world were in danger. It was major powers and their top leaders who set the rules, explor- ing numerous concepts and taking nothing for granted at the outset. Smaller countries were marginalized in processes that were neither inclusive nor 33 democratic. Concepts and details would be forged by some ‘secretariat’ in the background for debate by the leaders, who in turn would work through the issues and come to conclusions that shaped the future. This particular lesson should give us hope for change. As Plesch and Weiss concluded, “We thus should not act as if today’s international political order were immutable or pre- ordained. The 1940s should give us the courage to formulate ambitious visions about improving future world orders.” (2015: 203). Introducing the United Nations The United Nations was founded at the San Francisco Conference in 1945 by delegates from 50 countries (Poland did not attend the Conference but signed the Declaration later that year, becoming an additional founding member). The overall design of the United Nations had much in common with its predecessor, reflecting how international institutions tend to evolve rather than change rap- idly. Illustrating the continuity between the two organizations, few people even noticed that the United States joined the League after the Second World War, a year before it was legally wound up at a final ceremony in Geneva in 1946. As with the League, the UN is based on a tripartite parliamentary structure including a Security Council (as a sort of Cabinet), a General Assembly or debating chamber with one vote for each state member but with few powers (sort of like legislatures), and an administrative Secretariat and secretary-gen- eral who, once again, is more secretary than general (a prototype of a public service). Likewise, the International Court of Justice in The Hague (replacing 34 the Permanent Court of International Justice) can only accept cases submitted to it by members and its decisions are more recommendations than orders. Also, the UN has no prosecutor, no police and no jail. And once again, although the UN Charter speaks glowingly of fundamental human rights and it gave birth to the Universal Charter of Human Rights, there are no binding obliga- tions that commit members to protect citizens—even if they sign treaties to do so. There are also significant differences between the League and the UN. The very powerful Security Council was designed to take action whenever its mem- bers are in agreement. Vetoes were accorded to the Great Powers (the Perma- nent Five members) to make sure they do not have to leave the UN to protect their interests—which was one of the downfalls of the League and something we must remember when we discuss criticisms of the veto later. No other mem- bers were given veto power with which to block action, so there is no proto- anarchy as in the League. The Security Council is given exclusive jurisdiction over maintaining peace and security. Introducing the United Nations Various chapters of the Charter give the Council gradated powers to enforce the peace, starting with investigations and moving on to negotiations, sanctions and eventually a possible call on all coun- tries to protect the peace of the world. There were also provisions for armed forces and a command system, but when the Cold War started soon after the founding of the UN, these were never acted upon. g p Another new feature was the Economic and Social Council, which was in- tended to spearhead much broader development and cooperation of experts than imagined under the League. As we saw, because of his experience with the New Deal to help overcome the worst aspects of the Great Depression, this had become one of President Roosevelt’s main goals. Thus, the UN combines humanitarian technocracy to promote economic and social development with the powerful potential of the Great Powers to protect the peace whenever they can jointly decide to do so. After more than 70 years, it can be claimed that the UN has kept the major powers together—and talking, not fighting. Some say it was the rebirth of the League, only with more teeth and with the participation of the United States. The principal organs of the United Nations The Security Council The Security Council The Security Council The Security Council is formed of five Permanent Members (the P5) wielding vetoes (the United States, Great Britain, France, Russia and China—the victors in the Second World War) plus, currently, 10 other rotating members elected by the General Assembly for two-year terms. It is the UN’s most powerful 35 forum. The rotating members represent the various regions of the world. Each member has one vote. Procedural matters require nine votes. Substantive mat- ters require nine votes and the absence of a veto. Thus, in theory the rotating members have a negative or blocking vote for stopping things from happen- ing—it is sometimes referred to as the ‘sixth veto’. The presidency of the Council rotates monthly among its members. The Council bears the primary responsibility for the maintenance of inter- national peace and security. Linda Fasulo has provided an excellent word por- trait of its powers: “It has the authority to examine any conflict or dispute that might have international repercussions and to decide matters affecting the fate of governments, establish peacekeeping missions, create tribunals to try per- sons accused of war crimes, apply economic sanctions to misbehaving govern- ments, and in extreme cases declare a nation to be fair game for corrective action by other member states. It is the only UN principal organ whose resolu- tions are binding on member states, which means that governments do not have the option of choosing which of the council’s decisions they will or will not accept and help implement.” (2015: 55). The role of the UN in peacekeeping and peace enforcement (under Charter Chapter VII: ‘Action with regard to threats to peace’) has increased immeas- urably since the first United Nations Emergency Force was deployed for the Suez crisis of 1956. The UN went from modest monitoring and supervision operations in the 1940s, to now supervising more troops in the field (contrib- uted by members) than any country. By 2016, there were 16 UN-led missions in the field for an annual cost of some $8.2 billion (on top of the regular UN budget). They include large civilian and police components. In the post-Cold War period after 1990, the P5 in the Security Council were much more coop- erative and were willing to tackle more numerous and diverse conflicts includ- ing domestic rivalries. The Security Council Often this has led to a need to work cooperatively with regional partners in ‘coalitions of the willing’ from such groups as the North Atlantic Treaty Organization (NATO), the European Union, West African States and the African Union. The Security Council now goes well beyond pure peacemaking to work toward peacebuilding with the monitoring of elec- tions and long-term international support for the development of institutions and finances. There has also been a reinforcement of combatting terrorist threats. Aside from peacekeeping, the Security Council has at its disposal a whole series of measures it can use to try to change the orientation of ‘misbehaving’ states. One of the most dramatic forms of influence is sanctions that can be used to restrict the movement of finances, weapons, trade, travel or diplomatic contacts. As will be discussed in the following chapter, sanctions have become more targeted and efficient over the years in response to criticism and there have been several examples of successful use. 36 The number of formal and informal meetings of the Security Council has fluctuated over the years. For instance, there were 117 in 1988 and 373 in 2012. Under pressure from Canada and other members, the Council has slowly evolved to a more open process beyond the meetings of diplomats of its 15 members. It now invites non-members, UN officials and agencies, and even international NGOs. It also consults with states making major contributions to peacekeeping and other UN operations. The Council has also established some 40 subsidiary bodies to monitor specific places or issues. Over the years, the Security Council’s agenda has broadened to include not only peacekeeping but also humanitarian concerns, the international right to intervene, the plight of war induced refugees, monitoring of elections, post-conflict peacebuilding, ter- rorism and the creation of international criminal tribunals. The question of the veto power afforded to the Permanent Five is highly significant both for the nature of the UN and its future. The major victorious powers insisted on having a veto in the new international organization in order to protect their ‘vital interests’. Other states went along with it to ensure the presence of the major players and make sure they did not leave at the first hiccup. The Security Council However, it was never thought the P5 would use the veto or the threat of a veto to cover their whole foreign policy, as has often been the case, most recently with Syria. y y Russia has long considered Syria to be in its ‘sphere of influence’. China on the other hand has no clear connection with the Middle East. Yet both have used their veto power to block resolutions to halt the Syrian civil war, on the basis that may interfere in their internal affairs or may establish a principle or a pretext for such intervention. In particular, they fear the aggressive overreach of the ‘Western powers’ (the US, Western Europe, and their NATO allies). In 2007, Russia and China accepted that NATO, on behalf of the UN, could use the Responsibility to Protect norm to authorize air support to protect the civil- ians and the rebels against Gaddafi in Libya. Western powers went far beyond this mandate though, by instituting a complete regime change. So, Russia and China feared that even having the Security Council authorize ‘no-fly’ zones over Syria would be the thin-edge-of-the-wedge to permit the complete over- throw of the Assad regime. They considered that stopping such a precedent for intervention in domestic affairs is vital to their interests and worthy of a veto— however hard it is for the rest of the world to understand because it permitted this deadly war to continue for six years. Given this situation, many critics have called for the complete abolition of vetoes in the UN. Meanwhile, those who think the P5 will never let go of their advantage propose that the world work toward a curtailment of the veto so that its use would exclude resolutions dealing with war crimes and be limited strictly to resolutions directly affecting the vital interests of a P5 state. Another basic problem with the current Security Council is that it is not representative of the world to which it is supposed to give leadership. This has 37 led to calls for the expansion of its membership, for which there has already been a precedent in 1965 (one of the few amendments of the UN Charter). However, this too is deeply political and the debate has been ongoing for dec- ades. Various proposals for reforming the Security Council, specifically those dealing with the issue of representation, are discussed in Chapters 2 and 5. The Security Council In sum, the Security Council can be very effective at protecting international peace and security when its members are able to work together. Since the end of the Cold War, it has proved influential and even transformative as it has asserted the UN’s responsibility to intervene, even in internal conflicts. Yet it has crucial shortcomings, including its occasional failure to act, its unrepre- sentative nature and its two-tier membership of permanent and non-permanent members. The General Assembly For instance, the Mal- tese ambassador’s well-known speech in 1967, which called for international regulations relating to the sea and proposing that the seabed and its resources were the “common heritage of mankind”, was just the start of a 15-year long debate that eventually led to the Law of the Sea. Even so, we should note that it is often the bilateral and multilateral discussions that go on outside the formal debates that make the more significant contribution to the peaceful unfolding of world affairs. The Assembly elects a new president each year as well as 21 vice-presidents and the heads of its six Main Committees. Anyone can become involved. Whether countries are small, medium or large, the Assembly tends to give as many delegates as possible some position of responsibility—with attendant recognition and rewards. Yet not all relations are cozy. Power has shifted in the Assembly since 1945 as the regional distribution of states has changed. From the UN’s inception to the present day, the number of African states has increased from 3 to 54, those of Asia have risen from 9 to 53, while those of the West and Latin America have only doubled, from 39 to 85. The GA is riven by all sorts of groupings, mainly regional and the North–South split but also historical (the British Commonwealth) and cultural/linguistic (Spanish, French, Arab, etc.). ) The two major blocs representing the ‘developing countries’ of the ‘South’ are the Non-Aligned Movement (NAM) with its 114 members and the Group of 77 with 133 members—many of them overlapping. The NAM was formed during the Cold War as a home for those who did not simply want to be swept up in the Communist-Capitalist divide. The G77 was founded several years later in 1964 to coordinate the position of developing countries on trade and development and to help them get a better collective grip on international trade and finance. Together they work to defend the General Assembly against what they perceive to be US dominance and attempts by the Great Powers to mar- ginalize them. Some see the NAM as a holdover generally used to oppose the United States. Nonetheless, it is claimed that the Third World majority was hobbled by the fact that its majority inside the Assembly is not paralleled by resources for action outside. The General Assembly The General Assembly (GA) is the main deliberative organ of the United Na- tions. It is composed of one representative from each member state, each of which has one vote. It is sort of like the UN’s legislature where all the general debates are held. The significance of having all 193 member states debating together in one place for three months of the year (September to December)— and on 24-hour call the rest of the time—cannot be over-emphasized. Whether they like it or not, they are learning to apply the rules of international law, diplomacy and even democracy. They practice getting along and listening to one another. They learn to live by the rules of the game—and, indeed, deter- mine what the rules of the game are. They learn to persuade rather than to fight. This is no small matter when you consider the UN has grown from 51 members when it was founded in 1945 to 193 members by 2011 (the year that the last new member, South Sudan, joined). Thus, the GA is a cornerstone institution which demonstrates the UN’s distinctive universality—the very thing from which it derives its legitimacy. That may be why so many heads of state turn up for its annual general debate each September, which symbolizes the UN’s convening power. Together, the above attributes define what is called the prin- ciple of ‘multilateralism’. Nonetheless, we have to recognize that since the 1980s, the Assembly has been largely eclipsed by the more active Security Council and by the fact that major states have turned to institutions or grouping such as the World Bank and the Group of 20 to manage international economic relations. Article 11 of the UN Charter authorizes the General Assembly to “consider” and “discuss” peace and security and “make recommendations” to members, except on issues being considered by the Security Council, to which it must also refer questions of “action”. Article 13 spells out the GA’s main fields of competence. It shall initiate studies and make recommendations for political 38 cooperation, development of international law, and promoting international co- operation in the “economic, social, cultural, educational and health fields”, as well as assist in the realization of “human rights and fundamental freedoms”. Often fundamental developments can take a long time. The General Assembly This highlights the GA’s weakness as a delibera- tive body without effective administrative and coercive capabilities. This is in addition to the fact that the blocs tend to slow the process and bring decisions down to the ‘lowest common denominator’—all of which underlies the ten- dency to want to boot all the action up to the Security Council. h G l A bl ’ d i “ l i ” l bi d The General Assembly’s recommendations or “resolutions” are only bind- ing when they apply to the UN’s internal operations such as budget and mem- bership matters. It elects the rotating members of the Security Council and the 39 Economic and Social Council, and, along with the Security Council, it elects the judges of the International Court of Justice. It appoints the secretary-gen- eral on the recommendation of the Security Council. Economic and Social Council, and, along with the Security Council, it elects the judges of the International Court of Justice. It appoints the secretary-gen- eral on the recommendation of the Security Council. The Assembly’s current agenda runs to some 175 items arrayed across nine broad categories. To get through the agenda, the GA relies on its committee system to examine issues and propose solutions. There is a General Committee, a Credentials Committee and six Main Committees (see Box 3). Box 3: The General Assembly’s six Main Committees First Committee – Disarmament and National Security Second Committee – Economic and Financial Third Committee – Social, Humanitarian and Cultural Fourth Committee – Special Political and Decolonization Fifth Committee – Administrative and Budgetary Sixth Committee – Legal Box 3: The General Assembly’s six Main Committees Although these committees handle discussion of the main issues before the UN, the actual agenda reflects the more day-to-day workings of the world such as sustained economic growth and sustainable development; drug control, crime prevention and combating terrorism; disarmament; and maintenance of international peace and security. As can be imagined, each of these items has many subsections. y One of the main functions of the General Assembly is as a repository for international treaty making. Much international, multilateral activity and inter- national law is treaty based. Treaties are also called covenants. The United Nations itself is essentially a treaty among all its state members (through the Charter). The General Assembly A member state becomes ‘party’ to a treaty by formally ‘consenting to be bound’ by its terms, usually via ‘ratification’ of the treaty by its legisla- ture. Each treaty sets out a minimum number of states that must ratify it for it to become international law by which its signatories are bound. Later, other states may accede to the treaty/convention. An oversight committee is often established as a ‘convention secretariat’ to monitor the implementation of the treaty. The most significant criticism of the General Assembly is that it does not represent the world effectively. With the GA’s ‘one member, one vote’ policy, China gets the same representation as Malta. In 2013, 39 member states had populations under one million and 13 had populations under 100,000. The 65 least populated member states could together block a two-thirds majority vote, despite comprising less than 1 per cent of the total population of all member nations. And, theoretically, the 128 least populated members—which account 40 for less than 8.5 per cent of humanity and collectively pay under 1.3 per cent of the total UN budget—could provide the two-thirds majority required to adopt a resolution (Schwartzberg 2013: 19-20). In other words, the power in the General Assembly is skewed away from the largest states. As Schwartzberg concludes from these figures, “If one is to oppose—quite rightly—the undem- ocratic veto by any one of the five strong nations with permanent membership in the SC, one should also oppose unwarranted exercises of political power by GA coalitions of the very weak.” (ibid: 20). There are also criticisms of the GA’s ineffectual processes. It is accused of debating minutiae and of being unable to reach closure on issues that squander its prestige. Past secretaries-general have noted its need to streamline proce- dures and structures. In fact, most of the largest countries have ‘voted with their feet’ and fled the UN to work through the more restrained membership of the G7 and the G20. To overcome these deficiencies there are proposals for ‘weighted voting’ that would combine a country’s population and economic contributions with its vote as a state (see Schwartzberg 2013). It is certainly clear that reforms are needed to attract the important players to reconsider the United Nations as an effective forum of global decision-making. The Economic and Social Council ECOSOC is the acronym used to refer to the third principal organ of the UN, the Economic and Social Council. Its primary functions are to be a central fo- rum for discussing economic, social and environmental issues, to formulate policy recommendations for states and the UN, and to integrate the three pillars of sustainable development. The UN Charter spells out both its policy and ad- vocacy roles, stating that it may make or initiate studies with respect to inter- national economic, social, cultural, educational, and health matters, as well as human rights; make policy recommendations to the General Assembly; and convene conferences. It is also responsible for coordinating the work of UN agencies (e.g., UNICEF, UNESCO) in these fields and is the intermediary be- tween the GA and the more than 30,000 NGOs doing development work and humanitarian advocacy (including the approximately 4,000 that have consul- tative status with ECOSOC). The Council’s current 54 members, elected by the General Assembly on a geographical basis, serve three year terms. They elect their own president and four vice-presidents every year. Decisions are by majority vote with each member having one vote. It holds preparatory meetings along with relevant NGOs and then holds a four-week substantive session in July, alternating be- tween New York and Geneva. This session includes a high-level segment that cabinet ministers and other officials are meant to attend. The Council is in- tended to coordinate the work of UN programmes and funds, and specialized 41 agencies. The year-round work of ECOSOC is carried out by its subsidiary and related bodies including: eight functional commissions (statistics, population, social development, women, narcotics, crime, science and technology, and for- ests and sustainable development—now a high-level political forum); five re- gional commissions (Africa, Asia-Pacific, Europe, Latin America and Carib- bean, and Western Asia); three standing administrative committees; a Perma- nent Forum on Indigenous Issues; and a number of expert bodies (for instance on tax matters and transport of dangerous goods). ECOSOC has long been criticized for its ineffectiveness. There have been many efforts to reform it over the decades, and yet it is perhaps the organ that has shown the least amount of change. Its difficulties stem from a host of rea- sons. First, its responsibilities and functions often overlap with those of the General Assembly. The Economic and Social Council Although the Charter elevates ECOSOC to the rank of a principal organ, it also clearly places it under the authority of the GA. As we saw above, the Second Committee of the GA deals with economic and finan- cial matters, while the Third Committee’s mandate covers the social, humani- tarian and cultural fields. On top of this duplication, the Assembly has the ad- vantage of being the UN’s foremost plenary debating society where everyone has a voice, while ECOSOC is one-quarter its size. Second, many large countries, particularly Western, industrialized nations, have long sought to limit the UN’s voice when it comes to the international economy. They certainly do not want the UN to be the central forum for de- bating and coordinating economic policy. Nor do they want to be held hostage to listening to the grievances of the scores of small and poor developing coun- tries that dominate the General Assembly and ECOSOC. This sheds light on why, at the creation of the UN, the Great Powers shovelled the serious eco- nomic matters to the independent World Bank and International Monetary Fund where they not only control the agenda but also nominate leaders. This was part of a long process of diverting the UN away from macro-economic policy-making and toward development assistance. More recently, the wealth- iest countries have fled the UN in the direction of the G7 and the G20 to discuss economic matters. Third, in order to promote decentralization, the founders of the UN endowed the 14 Specialized Agencies with their own governing apparatus. Each one re- ports to its own state authorities. The result is that they have never felt com- pelled to want to be ‘coordinated’ by ECOSOC. As if these fundamental prob- lems were not enough, the very ponderous nature of ECOSOC also dooms it to irrelevance. Its 54 members are too numerous to be decisive and too few to have the attraction of the General Assembly. Its processes and meetings are too cumbersome to attract powerful ministers and to deal with emergency is- sues when the UN really needs to be active. ECOSOC has been adorned with such adjectives as ‘fuzzy’, ‘powerless’ and ‘lacking a clear identity’. Proposed reforms have run into the opposition of 42 foreign ministries of powerful countries, the General Assembly or the Security Council. Trusteeship Council There is an additional, little known principal organ of the UN, now in disuse. It is the Trusteeship Council, originally created in 1945 to administer and su- pervise the 80 countries still under colonial jurisdiction while on their path to independence under the guidance of the UN. Palau, a small island group in the Pacific, was the last trust territory before becoming a UN member in 1994. Despite considerable thought and effort by Secretary-General Kofi Annan, a majority could never be rallied behind any of the propositions for an amend- ment to the Charter to change the Council’s vocation (e.g., to be an environ- mental council). Hence, the Trusteeship Council simply suspended operations in 1994 and ceased to exist except on paper. This story demonstrates that the founders of the UN could not foresee every eventuality. It also illustrates how difficult it would be to amend the Charter, even when there is relative consen- sus on non-threatening modifications to better reflect the current reality. De- spite the hurdles that would need to be overcome, the former Trusteeship Council is available to be used for some new purpose. The Economic and Social Council So, ECOSOC continues to limp along helping to advance programs for development and technical matters. It does now meet with the international financial institutions and interacts with the Security Council, but the issues of its ambiguous relations with the General Assembly, the non-binding nature of its decisions and its composition remain to be solved. Chapter 3 will explore some past attempts at reform, as well as ideas put forward by experts to trans- form ECOSOC into a workable institution. Trusteeship Council Box 4: Understanding ‘international law’ In general, international law includes duly ratified international treaties and conventions, decisions of the Security Council, international custom, the general principles of law, and, in a subsidiary manner, previous judi- cial decisions and the teachings of the most notable international law ex- perts. In a broader sense, ‘soft-law’ may include declarations, statements, and plans of action agreed to by signatory countries or international con- ferences. Together they provide the norms of international law, but not the coordinating framework for an international legal regime. The International Law Commission was established by the General Assembly in 1947 to promote the progressive development of international law and its codification. A major part of their work is preparing drafts of aspects of inter- national law and submitting them to the GA. The GA may then convene a con- ference to incorporate the draft into a convention, open to states to become parties if they agree to be bound by its provisions. Some regulate relations be- tween states such as the Conventions on Diplomatic Relations, Consular Rela- tions and the Law of Treaties. There are also a number of extensive bodies of law such as International Trade Law and Environmental Law. One of the world’s most comprehensive instruments is the United Nations Convention on the Law of the Sea, with its 320 articles and nine annexes. It is now universally accepted that all activities and resources in the oceans and seas are governed by the Convention, with its 165 states parties. For instance, there is general acceptance of 12 nautical miles as the limit of a country’s territorial sea and also of an exclusive economic zone and also continental shelf zone up to 200 miles. The Convention has also brought stability to navigation, establishing the right of innocent passage in territorial waters, transit passage in narrow straits, and freedom of navigation in the exclusive economic zone. The ICJ has delivered fewer than 100 judgements on cases running from boundaries and sovereignty to violations of humanitarian law and diplomatic disputes. It has also rendered 27 advisory opinions. Thus, the Court is not ex- actly over-worked. This is in part because only member states can submit dis- putes (no individual or other international actor can bring a case forward) and states must bind themselves in advance to accept the ruling. The International Court of Justice The International Court of Justice (ICJ), founded in 1946 as part of the Charter, is the principal judicial organ of the United Nations. Located in The Hague (the Netherlands), it is the only one of the six principal organs not located in New York. Also known as the ‘World Court’, it is the only court of a universal character with general jurisdiction. Its first role is to settle legal disputes sub- mitted by states in accordance with international law (see Box 4). There are more than 180,000 inter-state treaties in the UN registry and over 500 major multilateral treaties, for which the secretary-general is the repository. ICJ judgements have binding force for the parties concerned and are without ap- peal. Second, the Court gives advisory opinions on legal questions referred by UN organs and agencies. 43 Box 4: Understanding ‘international law’ Box 4: Understanding ‘international law’ The ICJ is not a ‘constitutional court’ for the UN system and has no legal review of decisions. There is also little power of enforcement. The International Court of Justice is now buttressed by the International Criminal Court (ICC), an independent permanent court founded by the Rome Statute in 1998 and entered into force in 2002. By 2013 it had been ratified by 122 states parties. It tries persons accused of ‘international crimes’: genocide, crimes against humanity and war crimes. We include it here for completeness; however, it is not part of the United Nations. Its creation was facilitated by the 44 UN and cooperation between the ICC and the UN is governed by a ‘negotiated relationship agreement’. The Security Council can refer cases to it. The ICC is discussed in further detail in Chapter 4. UN and cooperation between the ICC and the UN is governed by a ‘negotiated relationship agreement’. The Security Council can refer cases to it. The ICC is discussed in further detail in Chapter 4. In its attempts to expand international humanitarian law, the Security Coun- cil also established the International Criminal Tribunals for the former Yugo- slavia (1993) and for Rwanda (1994) and supported the special courts for Si- erra Leone (2002), Cambodia (2006) and Lebanon (2007). These are some- times referred to as ‘hybrid courts’, which cease to exist once all cases have been heard. The Secretariat The Secretariat includes all departments and offices that develop policy and administer the UN. It is the head and the heart of the international organization. Despite continuous accusations of a ‘huge bureaucracy’ from enemies of the UN, the reality is that there are only around 40,000 staff (as of June 2016), comparable to the personnel of some municipalities and indeed less than that of the New York City police and fire departments. Of these, roughly half are located in the field; the remaining half are based out of cities where the Secre- tariat has a presence, mainly New York where it is headquartered but also Ge- neva, Vienna, Nairobi and others. The Secretariat services the other principal organs and administers the programs and policies they establish. The duties carried out by the Secretariat include: peacekeeping administration, mediation of international disputes, organizing humanitarian programs, surveying eco- nomic and social trends, studies on human rights and sustainable development, and promoting international agreements. It also has the task of developing pub- lic information and organizing international conferences. The list of departments and offices within the Secretariat (see Figure 2) shows the immense scope of UN activities. In addition to the diverse respon- sibilities listed above, it must also stay abreast of current affairs, whether re- lated to economic and social affairs, peacekeeping or human rights. Given the ‘globalization of everything’, from financial instability to the movement of people, this is no easy task. To finance its work, the Secretariat’s 2016-2017 budget was approximately $2.4 billion—about half the UN’s total biennium budget for these two years. To secure the human and financial resources to carry out its responsibilities, the secretary-general and his assistants must con- tinually bargain, negotiate and ‘beg, borrow and steal’ with the member states and, to a lesser extent, the corporate sector. It can be said that over time the Secretariat has been as well run as any bureaucracy. In response to multiple criticisms (especially from the Ameri- cans), it has been studied, restructured and seen its budget and personnel re- duced many times. For instance, in 1997-98 Kofi Annan brought together some 45 thirty departments, funds and programmes into four executive groups for De- velopment; Political and Security Affairs; Humanitarian Affairs; and Human Rights—a basic model which is still adhered to. New safeguards on spending and corruption are continually instituted. The Secretariat The real problem with the Secretariat lies in the refusal of nation-states to allow it to be a fully meritocratic international public service as was originally tried with some success under the League of Nations. Everyone, the major powers included, has reasons for wanting to control the secretariat—or not wanting someone else to do so. In 1945 it was intended in Charter Articles 100 and 101 that staff members would be independent of any government. Article 100 enunciated the principle of an independent, international civil service and Article 101 spelled out the overriding values of the “highest standards of effi- ciency, competence and integrity”. But under pressure from the Soviet Union, a ‘gentleman’s agreement’ was reached in London in 1946 and the five perma- nent members were ‘assigned’ major departments of the Secretariat. This was the beginning of the seesaw battle that has continued ever since between those who want a professional and independent public service and those who want to place their nationals in strategic positions. The latter have been winning. Reinforced by demands for ‘geographical distribution’, there has been a grow- ing politicization of the Secretariat over the years and often poor management, lack of accountability and cronyism. Staff members have also complained about poor administrative justice in the Secretariat (Jonah 2007: 165). The Secretary-General “There are limits to what the Secretary-General can do… Multilateral institutions (such as the UN) are conditioned by changing international power configurations, and by conflicts that exist within the broader international system” Edward Newman (2007: 189) Active and skilful secretaries-general such as Dag Hammarskjöld and Kofi An- nan have managed to transform a rather benign position into a “symbol of the United Nation’s ideals and a spokesperson for the interests of the world’s peo- ples” (UN Information Service). The secretary-general is named by the Gen- eral Assembly upon the recommendation of the Security Council for a term of five years. Almost all have completed two terms (see Box 5 for the complete list of past SGs). The description of the position in the Charter is very skimpy. It says little more than, “he shall be the chief administrative officer of the Or- ganization” (UN Charter, Article 97). The UN’s description of the position is a little clearer, stating that the secretary-general “establishes general policies 46 and provides overall guidance to the organization and is in equal parts diplomat and advocate, civil servant and chief executive officer” (Basic Facts about the UN 2014: 17). Tradition has it that the founders expected the SG to be more secretary than general. Nevertheless, the world expects him to ‘speak truth to power’ by telling “the Security Council what it has to know, not what it wants to hear”, as Michael Sheehan, former assistant secretary-general for peace- keeping, put it (Fasulo 2015: 27). In fact, the most significant function of the SG is to make the Council aware of any impending dangers to peace and secu- rity. Box 5: Secretaries-General, 1945-present Trygve Lie (Norway) 1946-1952 Dag Hammarskjöld (Sweden) 1953-1961 U Thant (Burma) 1961-1971 Kurt Waldheim (Austria) 1972-1981 Javier Pérez de Cuellar (Peru) 1982-1991 Boutros Boutros-Ghali (Egypt) 1992-1996 Kofi Annan (Ghana) 1997-2006 Ban Ki-moon (South Korea) 2007-2016 Antonio Guterres (Portugal) 2017- Box 5: Secretaries-General, 1945-present The SG also chairs the Chief Executives Board of the UN system including all the funds, programmes and agencies, and the World Bank and the International Monetary Fund. This is as close as the UN gets to coordination. Traditionally the SGs have rotated among the world’s regions. All have been men, but the calls for a woman head of the UN have become louder. Needless to say, the secretary-general cannot do everything him or herself. The Secretary-General The SG is surrounded by 15 under-secretaries-general who manage the various departments of the Secretariat. Since 1998, the SG has also been seconded by a deputy secretary-general to help with the administrative burden and special activities. To help carry out the secretary-general’s all-important use of his ‘good offices’ (negotiation, mediation, conflict prevention, public relations, arm twisting in public and private, etc.), the SG can name special and personal representatives and envoys, including ‘Messengers of Peace’ and ‘Goodwill Ambassadors’. Each secretary-general also must focus the UN’s activities de- pendent on the international context. For example, Ban Ki-moon’s focus for his second term from 2012 to 2016 included sustainable development, climate change, prevention of natural disasters, armed conflict, human rights abuses, a more secure world, nations in transition, and women and youth. 47 One analyst has summed up very well the complexities of the secretary- general’s position. “Without the traditional levers of power, the Secretary-Gen- eral can nevertheless wield real influence in international politics through the use of moral suasion and his authority as the embodiment of the ‘international community’. However, this is always within the context of an organization controlled by member states which have as their primary concern their national interests.” (Newman 2007: 175). Given the significance of the secretary-general’s position, there have al- ways been debates over the scope of its powers and the mode of nomination. The nomination comes from the Security Council, which essentially means those states which can veto any proposal (the Permanent Five members, though in effect just the largest—the United States, China and Russia). There has been no openness, foresight or planning. Critics have long called for a serious selec- tion process with specified qualifications, an extended search, rules for nomi- nation, a timetable for discussion and a single seven-year term to avoid poli- ticking. Thanks to the 1 for 7 Billion campaign led by the World Federalists Movement and the British United Nations Association, the Security Council finally relented in 2015 and gave the critics half a cake. It was decided that while the method of selection would go according to tradition, there would be an open nomination process for all countries and candidates would make their case to the General Assembly. There were 10 official candidates in the 2016 selection for the next secretary-general. Half the candidates were women, fol- lowing significant pressure for a woman to hold the top post. The Secretary-General The entire process was more transparent than it had been in the past, with public dialogue and televised debates. But the single nomination still came from the Security Coun- cil, and it was done in secret. The candidate finally chosen by the Security Council and elected by the General Assembly was Antonio Guterres. Mr. Guterres served as Prime Min- ister of Portugal (1995-2002) and as the UN High Commissioner for Refugees for two mandates (2005-2015). He was known at the UN for maintaining good relations with member states, civil society and the private sector. He also re- formed and innovated in his Office while minimizing its costs. In his ‘vision statement’ presented to the General Assembly in April 2016, Antonio Guterres summarized his intended priorities for the coming years. He spoke to the need for new approaches to deal with the challenges of our time, which include inequality, exclusion and the changing nature of conflict. He envisioned a UN that is able to ‘connect the dots’ through a holistic approach that effectively links the three pillars of peace and security, sustainable devel- opment and human rights. To achieve this, Guterres highlighted the importance of reforming the UN Development System, mainstreaming both human rights and gender equality across the whole UN system, and implementing the Agenda 2030, the Paris Climate Agreement, and the Addis Ababa Action Agenda. He went on to emphasize the centrality of prevention in ensuring 48 peace and security, and specifically the need for diplomacy, the participation of women and institution building. Reform and innovation, focused on delivery and results, is another key priority outlined. He stated his view that reform is not a onetime action but a permanent attitude, and that efforts should centre on being less bureaucratic and more productive, efficient and field oriented. Fi- nally, he called for strong partnerships with regional organizations, the inter- national financial institutions, civil society and the private sector. UN budgets The United Nations General Assembly approved a two-year UN budget of $5.4 billion for 2016-2017, down 1 per cent from the total spending during the pre- vious two years. The new biennial budget includes a 2 per cent staffing cut, or some 221 posts, and a one-year freeze in staff compensation. This is not the first time there has been a decrease in the UN budget. In fact, the organization is almost always held on a short financial leash by member states. Many critics believe the endemic underfunding of the UN is its most crucial problem. On the other hand, many members, led by the United States, have over the years accused the Secretariat of being a bloated bureaucracy and profligate spender—whether this be a gross exaggeration or not. As in past years, the biennial budget negotiations were marked by a tussle between poor countries seeking to increase the UN development spending, and major developed countries, the biggest budget contributors, trying to rein in the figures as they struggle to reduce expenditures in their own national budg- ets. The so-called core UN budget that was adopted does not include peacekeep- ing, currently running at over $8 billion a year and approved in separate nego- tiations. Noteworthy is the fact that the total peacekeeping budget represents less than half of one per cent of annual world military spending, estimated at $1.7 trillion in 2016, or 2.3 per cent of world gross domestic product (GDP). Nor does the core budget cover the costs of the UN tribunals. Both the peace- keeping and the tribunals are covered by payments assessed by the Assembly, mainly among the wealthier countries. The individual budgets of the major UN agencies and programmes (such as UNICEF, UNHCR, UNDP, UNESCO and WHO) are funded by voluntary contributions from member states. The UN System Technically speaking, the ‘United Nations System’ is formed of the UN family of organizations that includes the principal organs, the United Nations pro- grammes and funds, the specialized agencies and other related organizations (although many of us do use the term UN System more loosely to refer to all 49 Figure 2: The United Nations System diagram Source: United Nations Figure 2: The United Nations System diagram 50 organisms related to the UN). Programmes, funds and offices are subsidiary bodies of the General Assembly. On the other hand, the specialized agencies are independent but are linked to the UN through individual agreements and report to ECOSOC and/or the Assembly. There is also a difference in funding. While all subsist on chronically inadequate voluntary contributions from mem- ber countries, the specialized agencies also receive contributions from the overall UN budget. The International Atomic Energy Agency and the World Trade Organization are considered to be ‘related organizations’ with their own legislative bodies. As we just saw, an attempt to coordinate the UN system is made by the United Nations System Chief Executives Board for Coordination. With its 29 members and chaired by the Secretary-General, it meets twice a year. y The need for better coordination across the system has been an issue for decades. In fact, in their analysis of the lack of institutional coherence of the UN system, Childers and Urquhart concluded that the founders understood from the outset that a loose assemblage of agencies could not provide adequate governance. Recognizing this, the founders had specifically designed linkages that could make a loose collection of organizations work. From the outset it was intended that the UN would be the authoritative hub “able to forge coor- dinated strategies” (1994: 40), while the specialized agencies (by signed legal agreements under Articles 57, 58 and 63 of the Charter) were meant to have their objectives coordinated along with varying degrees of administrative har- monization and coherence. Yet these were never implemented. Moreover, while it was deliberate that specialized agencies would have their own separate legal existence and secretariats (so that politicians and diplomats could not hin- der progress on specialized and technical matters), it was never envisaged that the agencies would grab the degree of separateness that has become one of the system’s chief weaknesses. The UN System As the authors deduce, “There is no intergovernmental assembly or council of the UN system as a whole.” (ibid: 31). Now we see why so many ‘reformers’ are turning to a restructured ECOSOC as the potential new central pillar for economic, social and environmental policy cohesion. International financial institutions and other international actors The two major international financial institutions, the World Bank Group and the International Monetary Fund, are in a class apart. Founded at the Bretton Woods Conference in 1944, they are also known as the Bretton Woods insti- tutions. Although they are completely independent, they are considered to be two of the 15 Specialized Agencies of the United Nations with which they maintain formal relations through the Chief Executives Board for Coordina- tion. However, the founders of the UN specifically separated these two major 51 financial institutions from the rest of the UN, presumably to reduce political interference and provide for shareholder governance as described below. The World Bank Group evolved from the International Bank for Recon- struction and Development (that facilitated post-Second World War recon- struction) to today’s five financial institutions with a mandate to provide fi- nancing and technical assistance for worldwide poverty alleviation. The five institutions are: the International Bank for Reconstruction and Development, the International Development Association, the International Finance Corpo- ration, the Multilateral Investment Guarantee Agency and the International Centre for Settlement of Investment Disputes. p In 2016, the World Bank Group provided $64.2 billion for financial and technical assistance to developing countries around the world, making it one of the largest sources of such financing. This assistance supports a wide array of investments in such areas as education, health, public administration, infra- structure, financial and private sector development, agriculture, and environ- mental and natural resource management. The International Bank for Recon- struction and Development (IBRD) makes loans for development projects to governments of middle income and creditworthy low-income countries; in 2016 it made new commitments of $29.7 billion in 114 operations. The Inter- national Development Association (IDA) provides interest free loans (called credits) and grants to governments of the poorest countries with terms varying from 25 to 38 years. It made new commitments of $16.2 billion in 162 opera- tions in 2016. Together, IBRD and IDA make up “the World Bank” (as op- posed to “the World Bank Group” which consists of all five institutions). The primary purpose of the International Monetary Fund (IMF) is to ensure the stability of the international monetary system—the system of exchange rates and international payments that enables countries to transact with each other. International financial institutions and other international actors It does so in three ways: keeping track of the global economy and the economies of member countries; lending to countries with balance of payments difficulties; and giving practical help to members. For instance, along with Eu- ropean banks, it was the major lender to help Greece out of its troubles in 2014. The Fund's mandate was updated in 2012 to include all macroeconomic and financial sector issues that bear on global stability. Since the global economic crisis in 2009, it has also strengthened its support for low-income countries to respond to changing economic conditions and their increased vulnerabilities. For both the Bank and the IMF, the 189 member countries subscribe funds according to their economic strength. Voting power is linked to the level of subscription. While the voting shares vary by organization, the US, Japan, Ger- many, France and Great Britain control roughly 35 per cent of the votes. Each member country appoints one governor and one alternate to the Boards of Gov- ernors, which meet annually. The governors are usually ministers of finance or governors of a central bank. Traditionally, the President of the World Bank has been American while the Managing Director of the IMF has been European. 52 However, the IMF has moved toward an open, merit based process for selec- tion of the Managing Director in 2011 and the World Bank is likely to follow. However, the IMF has moved toward an open, merit based process for selec- tion of the Managing Director in 2011 and the World Bank is likely to follow. The international financial institutions do more than provide loans and reg- ulate the international economy. They also impose conditions. Starting in the late 1980s, what was called the ‘Washington Consensus’ promoted neo-liberal rules to reduce the role of governments in the economy and increase the role of market forces. The World Bank and IMF were able to promote that view throughout the developing world by attaching policy conditions to the major loans they made for budgetary support. Countries were obliged to cut public spending, eliminate subsidies, and privatize state-owned industries. Later in the 1990s this was described under the banner of ‘good governance’ and in- cluded democratization and transparency. As time went on it was recognized that these policies often spawned more poverty in developing countries and more foreign ownership. Government fiscal restraint sometimes made national financial emergencies worse rather than better. International financial institutions and other international actors Founded in 1995, the World Trade Organization (WTO) is a successor to the post-war General Agreement on Tariffs and Trade (GATT). Over 50 years, the GATT successfully cut tariff protection by more than half among member states, but it had several perceived weaknesses. Formal disputes among mem- bers, for instance, relied on the consent of all parties, including the defendant, to proceed. Countries could simply block proceedings against themselves, though, interestingly, they rarely did. Yet the WTO is a comparatively far more legalistic institution. It also has a broader scope, encompassing issues such as health and safety measures, and intellectual property. Like the GATT, the WTO continues to operate by a consensus voting rule. Combined with its large membership, which counts 164 members as of 2016, this consensus rule has been faulted by many for the institution's current stalemate: its last negotiation round, the Doha Round, has been stalled for over a decade. Yet one aspect of the institution, its dispute settlement function, continues to thrive. Among in- ternational courts, it stands out by its large caseload, with over 500 disputes filed, its high level of compliance with its rulings, and the consistency of its jurisprudence. Insofar as countries continue to liberalize trade under the WTO’s auspices, it is through the continuous clarification of their obligations that occurs with the settlement of formal disputes. Although it is not a formal part of the UN, the WTO maintains regular relations with parts of the UN sys- tem. The rise of other international actors cannot be ignored (see Box 6). Enu- merated her are those that are now generally accepted in the international rela- tions literature but we must keep our eyes open for powerful new partners on the global stage. 53 Box 6: Other ‘actors’ enter the world stage Box 6: Other ‘actors’ enter the world stage Box 6: Other ‘actors’ enter the world stage The term ‘actors’, as it is used here, refers to groups which have a signif- icant enough influence on international politics that they must be taken into consideration both by other players and by analysts. Up until the 1980s and 90s it was generally agreed the only influential actors in inter- national politics were nations states and their creatures like the United Nations and other international organizations. One of the major changes in international politics is the presence of new actors which cannot be ignored. These, in particular, include civil society, in the form of NGOs, and the private sector represented by multinational corporations—but also the media, religious groups, regional actors, and major cities. Civil society, which is considered the “third sector” of society, distinct from government and business, comprises NGOs, social movements, religious insti- tutions, academics, unions and all other non-state organizations and institutions that promote the interests of citizens. A recent count placed the number of NGOs at more than 50,000; most created since the 1980s and 90s. Some NGOs have more resources than even UN agencies. Civil society is most effective when its members come together in what are called ‘campaign coalitions’ to influence major international decisions, such as the creation of the Interna- tional Criminal Court, the ban on landmines, and R2P. It derives its strength, in great part, from having come to represent democratic forces and because it can influence public opinion, the media and corporations at both national and international levels. It is also a source of international expertise. Civil society, which is considered the “third sector” of society, distinct from government and business, comprises NGOs, social movements, religious insti- tutions, academics, unions and all other non-state organizations and institutions that promote the interests of citizens. A recent count placed the number of NGOs at more than 50,000; most created since the 1980s and 90s. Some NGOs have more resources than even UN agencies. Civil society is most effective when its members come together in what are called ‘campaign coalitions’ to influence major international decisions, such as the creation of the Interna- tional Criminal Court, the ban on landmines, and R2P. Chapter 2 – Peace and Security: Fixing the Security Council “Peace cannot be kept by force. It can only be achieved by understanding” Albert Einstein We already outlined the Council’s mission and structures in the last chapter. Now our task is to understand what works and what does not in the Security Council. In the UN, it is rarely all or nothing. We have to avoid the temptation to exaggerate. The Security Council has made great strides since the 1990s. And yet there remain fundamental blockages to its goal of overseeing peace and security in the world. Most of us would like to take a straightforward, rational approach to ana- lyzing a subject like the Security Council. Unfortunately, it does not lend itself well to seeking consistent explanations. It is too fraught with complexity, con- tradictions and anomalies to present purely reasonable conclusions. For in- stance, according to the UN Charter, all members are meant to be equal but the Permanent Five veto-holding members are evidently more equal than the oth- ers. While the Security Council is meant to uphold international peace and security, most of its energy is spent on interventionist peace operations within war-torn countries. The UN is not meant to meddle in the sovereign affairs of independent countries and yet the Security Council regularly does so with im- punity. Despite the Charter, the Council has moved partially from a Westpha- lian sovereignty perspective toward global community responsibility. Another obvious conundrum is that the Security Council is responsible for peace and security and yet not only does it allow the bloodiest conflicts to go on endlessly but it sometimes appears that some of its members aid and abet them. So when we analyze the Security Council we must be at pains to sort out the wheat from the chaff and to seek underlying causes and explanations wherever possible. We also must blend our focus on the Security Council with an attention to the broader world security perspective. Finally, by way of introductory comment, it should be stated that the mean- ing of ‘peace and security’ has evolved over the decades. Originally, it simply meant the ‘national security’ of the nation-state. Later it came to mean the ‘common security’ of the international or global community, which is chiefly the responsibility of the United Nations. More recently, ‘human security’ has been added to focus on individuals—especially civilians in war-torn situa- tions—and the responsibility of governments to them. Box 6: Other ‘actors’ enter the world stage It derives its strength, in great part, from having come to represent democratic forces and because it can influence public opinion, the media and corporations at both national and international levels. It is also a source of international expertise. Similarly, the corporate world has become increasingly influential in inter- national politics, where it is now considered to be an important partner. The relationship reached new heights in 2000 when the UN created the Global Compact to encourage businesses to adopt sustainability principles and report on their implementation. Over 9,000 companies have since joined the initia- tive. Another set of new actors, the G7 and G20 provide ideal settings for eco- nomic influence. The Group of 7, made up of the wealthiest and most powerful Western allies and Japan, wanted a space for policy making that could focus on the political issues of the moment. The Group of 20 is composed of the world’s largest economies. Some consider it a necessary institution to deal with world financial crises and economic planning away from the discordant de- mands of the UN. Others see it as the antithesis of attempts to create universal diplomatic forums. Concentrating on short term issues determined by the most powerful, it is a throwback to the Concert of Europe with no agenda for devel- opment and no secretariat to carry out any long term purposes. An increasingly important actor on the international stage is also the World Economic Forum, meeting annually in Davos, Switzerland. It claims to be committed to 54 improving the state of the world and engages the foremost political, business and other leaders of society to try to shape global, regional and industry agen- das. improving the state of the world and engages the foremost political, business and other leaders of society to try to shape global, regional and industry agen- das. 55 Chapter 2 – Peace and Security: Fixing the Security Council In another sense the term refers to a whole array of global challenges that lead to ‘insecurity’. A first example would be terrorism, which seeks to spread fear and conflict. States have been unable to even agree on a definition of ter- rorism, as one person’s terrorist can be another person’s freedom fighter or 56 even agent of a government. Then there are mass migrations which, as we have seen in Europe, can upset the balance of societies, create frictions, lead to po- litical disputes and even threaten regional integration efforts. The effects of climate change, including rising water levels, extreme weather conditions and greater migration, will only intensify. Pandemics can send whole countries and regions into a tailspin and cause an international blame-game. Cyber attacks are a less deadly but nevertheless destructive form of warfare. Economic shocks spread from one country to another. International crime and mafias de- mand international solutions. At a moral level, the Charter names other forms of security including free- dom from hunger and the right to housing, employment and health. Just where in all this is the Security Council meant to intervene? As we have seen, there are jurisdictional disputes between the Security Council and the General Assembly. They first surfaced in 1950 with the Unit- ing for Peace Resolution 377 under Articles 10 and 11. In the Soviet Union’s absence from the Security Council (which it was temporarily boycotting), the West did an end-run around the Council. Using the General Assembly's power to act for peace and security when the Security Council is incapable of doing so, it created a UN force under American leadership to halt the invasion of the south by North Korea. Today there is another jurisdictional, North-South confrontation over the extension of the Council’s mandate. When the Council thinks it should act, it does. It often does as it pleases because it is thought by some that its relatively restricted format provides the ideal setting to address world crises, whether they relate to peace and security or not. For instance, the Council formally ad- dressed the HIV/AIDS issue in 2000 under the heading of ‘vital security inter- ests’. Later, in 2007, the United Kingdom called on the Council to debate the relationships between energy, climate and security. The Council has also paid considerable attention to the issues of violence against women and women’s contributions to peace. The Security Council’s functions and activities Security Council strengths Security Council strengths It has been said that there has not been a single day since the Second World War when there has not been deadly political conflict somewhere in the world. Often this calls for action by the United Nations. In response, the Security Council has accelerated its pace over the years; it adopted 685 resolutions dur- ing its first 46 years, then 1,650 in the following 26 years (1991-2016). No matter whether it is admired or not, it has become generally accepted that the Security Council must authorize the use of international force—exactly as the Charter says it should. In other words, one source of the Security Council’s authority is the international law arising from the stipulation in the United Na- tions Charter that the use of force by one state against another is limited to situations of self-defence (Art. 51) or circumstances where force is authorized by the Security Council (Chapter VII). Thus, contrary to popular belief, there is no independent justification for “humanitarian intervention” or “responsibil- ity to protect”, both of which are subject to authorization by the Security Coun- cil. Of course, a state or a group of states may still take independent actions on the basis of a purported moral or political legitimacy, but they are not consid- ered legal. In the rare exceptions where UN authority was not obtained, things generally have not ended well (for instance, the NATO ‘humanitarian’ inter- vention in Kosovo in 1999 and the U.S. led invasion of Iraq in 2003). Indeed, this aspect of international law has done much to protect the world since 1945, ensuring that armed conflicts between states remain uncommon. More broadly, as the President of the General Assembly, Mogens Lykketoft, pointed out at the May 2016 High Level Thematic Debate on Peace and Secu- rity, the UN has helped restrain the world’s largest powers, mobilized person- nel and money for peacekeeping, established a clear legal framework on war and human rights, and helped reduce the threat of the world’s most deadly weapons. So, in effect, the Security Council has become a sort of executive body for dealing with world crises. And when the Council acts in harmony it has proven quite effective. For instance, in 2014 it unanimously agreed to place sanctions on six leaders of the Islamic State and Al Nusra in the heat of the Middle East crisis. Chapter 2 – Peace and Security: Fixing the Security Council So now the shoe is on the other foot and many GA members think the Council should stick to armed conflicts. Having noted this jurisdictional dispute between the Assembly and the Council, let us now return to our more specific focus on how well the Security Council handles its central objective of dealing with peace, conflict and secu- rity, before going on to look more specifically at its expanded use of peace operations. 57 57 The Security Council’s functions and activities It has also virtually eradicated the notion of absolute national sover- eignty. Now states can no longer act with impunity with regard to their own population and the Council can interfere in state conflicts that can be said to threaten international security. As David Malone states in his essay on the Se- curity Council, “The Council’s decisions in the post-Cold War era have proved immensely influential, indeed transformative, on a normative level. By assert- ing the UN’s responsibility to intervene, even in internal conflicts—where hu- man rights and the humanitarian interests of populations are severely 58 affected—Council decisions, arising from evolving interpretations of the Char- ter, have deeply affected the meaning of sovereignty.” (2007: 133). y g g y Over the years, the Council has expanded its search for peace, making use of the secretary-general’s ‘good offices’ and engaging in fact-finding missions. It also uses ‘Groups of Friends’, that is to say countries which can be influential and can advise and help intervene in particularly thorny cases when the Coun- cil members, themselves, may not have sufficient contacts. As we explore later in this chapter, the UN has moved beyond peacekeeping to undertake military missions of peace-enforcement as it tackles more numerous and diverse con- flicts such as protecting East Timor in 1999, turning back the Iraqi invasion of Kuwait in 1990, and intervening in the civil wars in Guatemala and El Salva- dor. The SC also empowers regional organizations and ‘coalitions of the will- ing’ to act on its behalf. In recent years, the African Union and the Economic Community of West Africa (ECOWAS) have played leading roles in trying to tame conflicts in Darfur, Sierra Leone, Liberia and Côte d’Ivoire. Sometimes the Security Council acts as one. Sometimes it is just the perma- nent members calling the shots. Sometimes it is just the United States, Russia or China individually pushing their weight around. Other times there are tem- porary alliances among some of the 15 members. However, aside from straight power and national interests, the Council can also be seen from the perspective of leadership. Getting things done among the 193 members of the UN often takes leadership and determination, as well as long term planning and diplo- macy. The Security Council’s functions and activities The United States is often accused of calling the shots but usually its role comes down to drafting resolutions, getting votes and putting things to- gether—in other words, providing leadership. The Council’s difficulty in deal- ing with terrorism is a good example. Although it has adopted many strong, action-oriented resolutions to control terrorism, it is still not able to develop a consensual definition of the meaning of terrorism. Despite this, the leadership of a few led to a heads-of-state summit condemning terrorism in 1992, the pur- suit of the Taliban in Afghanistan after 9/11, and sweeping decisions to combat terrorism financing and safe-havens and to create the Counter Terrorism Com- mittee as a subsidiary body of the SC. The Security Council is relatively more open, active and effective than we give it credit for. It now meets with states contributing to peace missions, NGOs, the business community, and regional organizations. Since the mid- 1990s, the Council’s president has been briefed informally by individual ex- perts, NGOs and observers on special issues. There have been consultations with troop-contributing countries since 1994. Given that the media often focuses on the inability of the UN to act in crises, it is rather astounding to learn that most Security Council resolutions are adopted. For instance, in 2016, there were some 457 meetings resulting in 72 resolutions which were adopted and only three vetoed. The percentage of ve- toes as a portion of the total number of resolutions of the Council dropped from 59 85 per cent during the Cold War to 15 per cent after 1988. In other words, the major business of the Security Council goes on rather smoothly. Nevertheless, what the Council is known for is the relatively small number of resolutions that are vetoed by one or several of the Permanent Members (P5). From 2012 to 2017, Russia and usually China have vetoed resolutions on the Syrian civil war, impeding all consequential actions except for a small amount of humani- tarian aid. One P5 veto, or the threat thereof, is sufficient to stop the Security Council, and hence the UN, from acting. This is even more significant now that, as we have just seen, the Security Council has spread its wings to consider ever more issues such as pandemics, climate change, health and economics. Box 7: The UN’s record of achievement in peace and security  Helping avoid another global war.  Amassing a body of international law, rules and legal norms includ- ing 560 international treaties since 1945, with their resultant norma- tive impacts.  Amassing a body of international law, rules and legal norms includ- ing 560 international treaties since 1945, with their resultant norma- tive impacts.  Putting in place a system of dispute-settlement mechanisms includ- ing the International Court of Justice, the 1982 Law of the Sea, the 2005 Resolution of the Pacific Settlement of Disputes and the Medi- ation Support Office.  Developing a network of 14 multilateral agencies to regulate inter- national daily, vital, practical interaction—everything from telecom- munications to tourism.  Managing the proliferation of weapons of mass destruction and their inspection. inspection.  Imposing sanctions on wayward state behaviour on 26 occasions. p  Imposing sanctions on wayward state behaviour on 26 occasions.  Deploying 54 peace operations and 39 special political missions de- signed to prevent, ameliorate or conclude conflicts.  Deploying 54 peace operations and 39 special political missions de- signed to prevent, ameliorate or conclude conflicts. Source: Rudd 2016: 11-16 The Security Council’s functions and activities  Amassing a body of international law, rules and legal norms includ- ing 560 international treaties since 1945, with their resultant norma- tive impacts.  Putting in place a system of dispute-settlement mechanisms includ- ing the International Court of Justice, the 1982 Law of the Sea, the 2005 Resolution of the Pacific Settlement of Disputes and the Medi- ation Support Office.  Developing a network of 14 multilateral agencies to regulate inter- national daily, vital, practical interaction—everything from telecom- munications to tourism.  Managing the proliferation of weapons of mass destruction and their inspection.  Imposing sanctions on wayward state behaviour on 26 occasions.  Deploying 54 peace operations and 39 special political missions de- signed to prevent, ameliorate or conclude conflicts. Source: Rudd 2016: 11-16 The Security Council’s functions and activities As we have seen earlier, another facet of the Council’s ability to influence the international system is through the use of mandatory sanctions to cut off a state’s access to diplomatic relations, finance, trade and arms. Traditional sanc- tions, which were fairly broad in nature, have been criticized for causing more harm to the population at large than to the economic and/or political elites. Recent sanctions have been more focused, targeted at the travel, banking assets and luxury items of elites so as to have minimal effect on ordinary citizens. It has also become increasingly recognized that the main use of sanctions should be for persuasion rather than punishment. All in all, they have been used with considerable success against the apartheid regime of South Africa and the nu- clear ambitions of Iran, but, so far, seem to have had little effect on the behav- iour of North Korea. In the last decade, the public ‘naming and shaming’ of ‘sanction busters’ has added to the effectiveness of sanctions. The Security Council has many other tasks as well. It nominates the judges of the International Court of Justice, with which it gets along well. The same cannot be said of relations with the International Criminal Court that was founded outside the UN and which the United States and China (as well as India, Israel and others) have refused to join. Though it is worth mentioning that the SC set up, on its own, ad hoc criminal tribunals for the former Yugo- slavia in 1993 and Rwanda in 1994 as well as special courts for Sierra Leone in 2002 and for Cambodia in 2005. The Council is active in sending monitors to national elections when legitimized outcomes are crucial for developing de- mocracy. It has 40 subsidiary bodies dealing mainly with fact-finding missions and implementation of sanctions but also with such continuing problems as counter-terrorism and children in armed conflict. In sum, we may say that the UN Security Council since the 1990s has be- come far more open and partner-oriented and has developed a much larger and more diversified tool kit of means of intervention in peace and security. The cumulative effect has been an impressive list of achievements related to the UN’s first pillar, peace and security (see Box 7). 60 Box 7: The UN’s record of achievement in peace and security  Helping avoid another global war. Security Council weaknesses Despite these achievements, all is not well with the Security Council. In its decisions, national self-interests too often clash with global security concerns. The great hopes people had for the Council as the peace-maker after the Second World War and after the Cold War were barely fulfilled. It did not translate itself into a collective body that was able to engage in the world’s hot spots. There has been insufficient common leadership which has resulted in doubtful institutional legitimacy and international disenchantment. The contentious re- lations with the Non-Aligned Movement and the Group of 77 in the General Assembly only served to deepen the frustrations. The agreement of the P5 to help create the G20, comprising the world’s most financially powerful nations, has further undermined the economic credibility of the United Nations. The Council is noted for being a reactive rather than a preventive body. The failure to include a set of operational criteria for activating the R2P norm symbolized this weakness perfectly. The Great Powers have not seen fit to equip the United Nations with its own emergency peace forces or autonomous finances, thus ensuring that the UN is kept on a leash. Despite these achievements, all is not well with the Security Council. In its decisions, national self-interests too often clash with global security concerns. The great hopes people had for the Council as the peace-maker after the Second Despite these achievements, all is not well with the Security Council. In its decisions, national self-interests too often clash with global security concerns. Despite these achievements, all is not well with the Security Council. In its decisions, national self-interests too often clash with global security concerns. The great hopes people had for the Council as the peace-maker after the Second World War and after the Cold War were barely fulfilled. It did not translate itself into a collective body that was able to engage in the world’s hot spots. Three failures of particular concern deal with nuclear weapons, global de- cision-making and a perception of growing irrelevance of the UN. The first UN resolution in January 1946 called for the elimination of weapons of mass destruction. But old-fashioned power politics took over from the hopes for 61 collective morality. Principles gave way to national interests. The world de- scended into the Cold War dominated by the strategy of Mutual Assured De- struction. Security Council weaknesses Although no nuclear arms have been used since Hiroshima and Na- gasaki at the end of the Second World War, proliferation of nuclear arms and their delivery systems has gone on a pace. Initially limited to the US, France, Great Britain and the Soviet Union (now Russia), the number of nuclear armed states grew to include India, Pakistan, China and North Korea. Many believe Israel possesses nuclear weapons (though it has never admitted to it) and Iran sought to be included on the list. Cognizant of the perils, a Non-Proliferation Treaty was established in 1968. Efforts were made in the 1972 SALT 1 treaty to cap the number of offensive nuclear weapons, but the 1980s saw a renewal of the arms race. Finally, the Comprehensive Test Ban Treaty was adopted in 1996. The underlying idea had been that nuclear states would agree to share peaceful nuclear technology with states that renounced efforts to have their own nuclear weapons, but there is dispute over whether the transfer has actually happened. Aside from these trea- ties was the creation of the International Atomic Energy Agency in Vienna in 1957. As an independent agency, it reports to the GA and SC on its mandate for control, verification, security, and technology transfer. Whenever it has been supported by the Security Council, it has done laudable work. But by the end of 2016, Trump and Putin were still talking about spending billions on modernizing their nuclear arsenals rather than technology transfer or ending poverty. Despite this, in 2017 a majority of 122 countries in the General As- sembly voted for a treaty to ban nuclear weapons. All of the nine countries known or believed to have nuclear weapons boycotted the discussions claiming they disregard the "realities" of international security. The treaty will be open for signatures in late 2017 and will come into force when 50 countries have ratified it. The second failure has been the inability of the Security Council to make decisions at crucial times at which the fate of whole peoples was under threat. The Council has not been able or willing to stop the most horrendous blood- baths of our era is such cases as Bosnia, Rwanda, Zimbabwe, Darfur and Syria. In part, this has been because of the P5 veto or the threat of its use. This is paradoxical. Security Council weaknesses  UN forces responsible for cholera outbreak in Haiti that killed thousands. Source: Rudd 2016: 16-20 Security Council weaknesses The veto was the instrument invented to keep the major powers in the UN. In the modern era, great powers have never felt they had to threaten to quit the UN to defend their interests. But now they do not want to lose the power advantage the UN gives them. Vetoes are a major cause of indecision in the Security Council, even if they have been declining in number. Vetoes are often not used to protect vital inter- ests (e.g., to protect the state’s national territory), but rather to maintain pre- sumed spheres of influence and to protect allies as the US has done for Israel and Russia for Syria. They are also thought to heighten international status and strengthen public opinion at home. In other words, the P5 find plenty of reasons 62 other than their vital interests for using the veto. Thus even without the veto we would still have big power politics in the Security Council and a disruption of global decision-making. That is why France and Mexico are pushing for a limit of the veto (which is possible) rather than its abolition (which is much less likely to happen). Box 8: The Security Council’s challenges, problems and failures  Perceptions of impasse in the Security Council due to the veto and the threat of a veto feed frustrations about the UN’s capacity to act to deal with crises, despite there being only 276 vetoes while 2,296 resolutions have been successfully adopted.  Failure to prevent mass atrocities (war crimes, genocide, crimes against humanity) when the UN was slow or failed to respond—e.g., Cambodia, former Yugoslavia, Rwanda, Darfur, South Sudan, and Syria.  Limited response to global terrorism including state-sponsored ter- rorism.  Continuing repercussions from the invasion of Iraq against the UN’s wishes.  Absence of the UN from negotiations on the Iranian nuclear agree- ment and being used as an afterthought in Afghanistan.  Lack of effective action to resolve the Syrian crisis, standing by as 400,000 have been violently killed and half the population uprooted.  Lack of involvement in the Ukraine crisis.  No UN diplomatic initiative against North Korea’s illegal nuclear program.  Inability to handle the 2015-16 wave of refugees, migrants and asylum-seekers.  Loss of moral authority due to inconsistent response to human rights violations.  Sexual abuse in peacekeeping operations tarnishes reputation of blue helmets. Box 8: The Security Council’s challenges, problems and failures  Perceptions of impasse in the Security Council due to the veto and the threat of a veto feed frustrations about the UN’s capacity to act to deal with crises, despite there being only 276 vetoes while 2,296 resolutions have been successfully adopted. y p  Failure to prevent mass atrocities (war crimes, genocide, crimes against humanity) when the UN was slow or failed to respond—e.g., Cambodia, former Yugoslavia, Rwanda, Darfur, South Sudan, and Syria.  Failure to prevent mass atrocities (war crimes, genocide, crimes against humanity) when the UN was slow or failed to respond—e.g., Cambodia, former Yugoslavia, Rwanda, Darfur, South Sudan, and Syria. y  Limited response to global terrorism including state-sponsored ter- rorism. y  Limited response to global terrorism including state-sponsored ter- rorism.  Continuing repercussions from the invasion of Iraq against the UN’s wishes.  Continuing repercussions from the invasion of Iraq against the UN’s wishes.  Absence of the UN from negotiations on the Iranian nuclear agree- ment and being used as an afterthought in Afghanistan.  Lack of effective action to resolve the Syrian crisis, standing by as 400,000 have been violently killed and half the population uprooted.  Lack of involvement in the Ukraine crisis  Loss of moral authority due to inconsistent response to human rights violations.  Sexual abuse in peacekeeping operations tarnishes reputation of blue helmets.  Sexual abuse in peacekeeping operations tarnishes reputation of blue helmets.  UN forces responsible for cholera outbreak in Haiti that killed thousands. Source: Rudd 2016: 16-20 While these (along with the other failures listed in Box 8) can be considered legitimate limitations or flaws of the Security Council, the last issue relates more to how it is perceived. The word ‘irrelevant’ was first used by President George W. Bush in talking about the UN in 2003, and has become a refrain used by many others since. Of course, we have seen from the achievements of the Security Council that the UN is not in fact irrelevant. The world would be 63 lost without all its contributions. Nevertheless, as regards perceptions, and of- tentimes media coverage, it is another story. Box 8: The Security Council’s challenges, problems and failures We saw this in 2016 when, after the vetoing of another resolution on Syria in the Security Council, Canada was joined by 71 other countries in side-step- ping the Council and bringing the issue directly to the floor of the General Assembly, in the manner of the 1950 Uniting for Peace resolution. This unique and newsworthy endeavour was barely mentioned by major newspapers. How- ever, the next day, the nomination by the UN of Wonder Woman as an honor- ary ambassador was ridiculed in world headlines. It looks like irrelevance—or worse. Unfortunately, the tendency is still for the media to focus on the UN’s shortcomings. However, the consequences reach beyond simply harming public opinion of the UN. When the organization is regarded as being incapable of dealing with the most pressing security concerns, states have and will bypass it, as we saw when the Iran nuclear deal was negotiated in 2015. The UN’s peace operations Historically, analysts have offered many different and sometimes conflicting explanations for the causes of war and the means of fostering peace. In Man, the State, and War, Kenneth Waltz (1954) was able to order these different approaches to causality under three headings: the individual, the state and the international system. Each one corresponds to a further analysis of what the type of causality portends for possible paths to peace. In the first, it is the bes- tial, aggressive nature of mankind which is targeted as the culprit. But, it is asked, if individuals are the cause of war, is it not the public institutions of the states which shape and limit their citizens? Then, it is further proposed that states too have to vie for themselves within the competitive, anarchical inter- national system, leaving individuals and even states few choices in their be- haviour. Waltz concludes that even these very general fields of causality may be further confused by the possibility that the causes of war may come from all three domains and even depend on particular circumstances. Whatever the causes of war, the UN has been active in promoting peace since its inception. Before examining the evolution of UN peace operations, it is worth outlining some key terms related to the full scope of activities under- taken by the UN to maintain peace and security. These are: peacekeeping, peacemaking, peace enforcement and peacebuilding. Secretary-General Boutros Boutros-Ghali delineated these four distinctive but overlapping roles for peace operations in his 1992 Agenda for Peace. Peacekeeping, which broadly involves helping war-torn countries create conditions for lasting peace, is sometimes used in an all-encompassing sense to refer to other activities as well. Peacemaking (under Chapter VI of the Charter) refers to diplomatic 64 action to bring hostile parties to an agreement, including through negotiation, enquiry, mediation, conciliation, arbitration, judicial settlement or with the help of regional agencies. The new methods that have been added to this long list have been the ‘good offices’ of the Secretary-General and his use of special envoys and representatives as mediators. Peace enforcement (under Chapter VII) requires the authorization of the Security Council and involves a range of coercive measures, including the use of military force. Finally, peacebuilding focuses on post-conflict recovery and reconstruction, aiming to build national capacities for conflict management and create the conditions for sustainable peace. The UN’s peace operations Over time, peacekeeping missions have been at once some of the UN’s greatest successes and greatest failures. Among the successes were: the inde- pendence of Namibia; ending the conflict in El Salvador; demobilization, peace and reestablishment of Cambodia; peace, disarmament and elections in Mozambique; independence and a new government in East Timor; and ending the civil war and re-establishing the government in Sierra Leone. Terrible fail- ures were in Somalia, Bosnia, Rwanda and Syria, where the Security Council refused to mobilize sufficient forces to control the situation and civilians were slaughtered by the thousands. A great deal of research has been done to better understand why some mis- sions succeed while other fail. Doyle and Sambanis found that the success of peace settlements depends on three factors: the degree of hostility, local capac- ities left over after the conflict, and the amount of international assistance available. Optimal intervention strategies need to match means to ends. Con- sideration has to be given to local causes, levels of conflict and hostility and factional capacities. A strategy of discrete acts of enforcement must be calcu- lated to fit in with peacemaking (negotiations), peacekeeping (monitoring), and peacebuilding (reconciliation, reconstruction). This juggling act must be con- stituted within the context of military resources and civilian means for the re- building of institutions (2007: 323-348). The UN itself has also initiated efforts to review its peace and security ac- tivities and recommend improvements. A particularly noteworthy study was the Report of the Panel on United Nations Peace Operations, commonly called the Brahimi Report, prepared in 2000 in response to significant UN peacekeep- ing failures in the 1990s. It called for “robust doctrines” and “realistic man- dates” (referring to an explicit mandate for civilian protection and the resources to carry it out), improved headquarters management, and rapid deployment. The Security Council and the secretary-general delivered on all but the last, and many analysts agree that progress made in peacekeeping efforts was in- deed influenced by the report. y Still, as the 2015 report of the High-Level Independent Panel on Peace Op- erations makes clear, there continues to be room for improvement. The report proposes a long list of operations improvements, such as clearer strategic di- rection, improved speed, strengthened partnerships, more purposeful engage- ment with host countries, and addressing sexual abuse which has tarnished the reputation of UN peacekeeping in recent years. The UN’s peace operations Although the first United Nations Emergency Force was deployed for the Suez crisis of 1956, the UN had already gained experience from monitoring and supervision operations as early as the 1940s. The original peacekeeping missions were limited to lightly armed forces acting as mediators and monitors, interceding between conflicting combatants to keep them apart—literally, to ‘keep the peace’. They depended on the principles of consent, neutrality and the non-use of force. Over the years, peace operations have expanded greatly in mission and man- date, going well beyond pure peacemaking, by working toward peacebuilding with the monitoring of elections and long-term international support for the development of institutions and finances in failed states. They may be author- ized to use force and have police forces and administrative specialists oversee- ing the rebuilding of a functioning government and (hopefully) independent civil society. Operations have become multidimensional, aiming to facilitate the political process, protect civilians, promote human rights, support election, restore the rule of law, and assist in the disarmament, demobilization and rein- tegration of former combatants. As it may be imagined, such operations are frightfully complex. What had changed was that from 1900 to 1941, 80 per cent of wars were between states; from 1945 to 1976, 85 per cent of wars were within a single state. These civil conflicts tended to be more all-encompassing and destructive of the economy, civil rights and state institutions. Many became mixed up in illicit trade and terrorism and conflicts threatened to spill over into neighbour- ing countries, thus endangering international peace. The UN was drawn in more and more. For the first time, operations came to combine both war-like enforcement and peace-like negotiations. In the post-Cold War period after 1990, the P5 in the Security Council were more cooperative and willing to tackle more numerous and diverse conflicts including domestic rivalries. By 2016, there were 16 UN-led missions in the field for an annual cost of some $8.2 billion. They include large numbers of civilian and police compo- nents. The Department of Peacekeeping Operations in the Secretariat super- vises more troops in the field—some 120,000—(contributed by members) than any individual country. There is often a need to work cooperatively with 65 regional partners in ‘coalitions of the willing’ from groups such as NATO, the European Union, West African States and the African Union. The UN’s peace operations The report also recognizes that certain political realities limit the UN’s ability to consolidate peace. These in- clude the fact that neither governments nor regional groupings fully entrust the UN with preventive diplomacy, and that the UN continues to be a relatively minor player in the field of international cooperation. This recognition is im- portant when thinking about the future of the UN because it highlights a 66 situation that is the inverse of what it should be if we want to enhance peace and cooperation. State diplomacy, focused on maximizing national interests, is currently winning over global diplomacy—this must change if we are to achieve a renaissance of the United Nations. As this section has shown, the UN has an elaborate structure of prevention and peace operations. However, a number of tendencies have limited success. First, powerful states have sought to avoid assertive secretaries-general there- fore limiting the role SGs can play in international diplomacy. Second, the UN’s ability to prevent conflicts depends on the political will of member states. Unfortunately, the Security Council is oriented toward “the visible, forceful and reactive rather than the invisible, quiet and proactive initiatives required for dispute settlement” (Mani 2007: 318). This relates to perhaps the main chal- lenge with prevention: successful instances are all but invisible, and the more effective it is, the less likely it is to make headlines. A final point is that global trends such as rising economic inequality, political marginalization and de- mocracy movements tend to stimulate conflict, making prevention an onerous task. The Security Council and the future Reforming the Security Council is at once very simple and incredibly complex. There are many good ideas about what to do, but there are so many actors in the pot with too little common political will that nothing gets done. It is said that the Security Council is one of the major world arenas of power and real- politik. Participation provides access, knowledge, power and influence. No wonder there is little consensus on who the players should be and what proce- dures are best. For us to see a little more clearly, we have to start with some basic principles and a set of objectives before going on to look at various re- form proposals. p p The basic principles that we should strive for are legitimacy and effective- ness. It is said that the power of social institutions resides mainly in their legit- imacy. Writing about the legitimacy of the Security Council some years ago, David Caron stated, “For an institution to be considered legitimate it must be recognized as a lawful authority; one that conforms to a particular standard and operates in such a manner that its actions and decisions are seen as legally or morally justified and proper.” (1993, 552). The United Nations is founded on an international treaty that all members have signed, so with this founding au- thority the Security Council may be said to be clearly lawful. As we have seen, its much improved manner of operation has made it more transparent and in- clusive. Yet the organization is much larger now than in 1945, so the Council is no longer representative of the current UN membership. Also, because of its two-tier membership resulting from the veto, as well as the poor record of 67 decision-making, it is no longer considered by many to uphold standards that are morally justified and proper. The second principle is effectiveness. Many question whether the Security Council is properly managing the world’s peace and security and whether it is doing so in a manner that protects human rights and sustainable development. One also wonders just how big a Council could be to operate effectively and make decisions. The Security Council and the future In terms of potential goals for a reform process, UN expert Andy Knight provides useful ideas: “improve the rate of participation from poorly repre- sented categories; improve the geographical representation; improve its dem- ocratic character by limiting the use of the veto to Chapter VII actions and ensuring the Council represents a clear majority of the world’s population; maintain its efficiency by limiting its growth; improve transparency by con- sulting with non-members on actions concerning them, and greater involve- ment of rotating members.” (2002, 33-4). Thus, we have a set of objectives that cover diverse issues of size, fair representation, composition, legitimacy, effi- ciency and the veto. The search for Security Council reform did not start yesterday. Resentment of the P5 members pushed the General Assembly to launch consultations on a reform agenda in 1993. It was called the “Open-ended Working Group on Eq- uitable Representation”. Two decades later it came to be known in the corridors of the UN as the ‘Never-Ending Working Group’. In a 2004 meeting in Baden-Baden, Austria, the secretary-general’s High- Level Panel on Threats, Challenges and Change thought they had found the ideal solution on composition. The Council would be increased to 24 members in three tiers: tier one would include the present P5 (the US, Russia, China, Great Britain and France); tier two would include seven or eight semi-perma- nent members elected on a regional basis for a renewable term of four or five years (Brazil, Germany, India, Japan and South Africa might be in this group); the third tier of rotating members would be elected as at present for a non- renewable two year term. Only the Permanent Five would have a veto. More emphasis would be accorded to giving membership to those who make a real contribution to peace and security. A full review would be made every 15 years. The consensus in the Panel dissolved over a demand by some that all new tier two members be given a veto. The proposal never even got into the final report which included two inferior models catering to special interests. The secretary-general in effect threw up his hands and said members should vote for whatever they liked. This example is given at length because it introduces most of the issues about composition and veto. The Security Council and the future As regards size, it is generally agreed that the 15- member Security Council should be expanded to be more representative of the world. The question is: by how much? More big powers may just add to the current conflict of interests and national prestige, making for more difficult decisions rather than easier. Still, most proposals come in the range of 20 to 68 25—small enough for discussion, big enough to be representative. All sugges- tions propose one to three more non-permanent, rotating seats. At present, the 10 rotating seats are for two-year terms elected by the General Assembly with three going to Africa, two to Western Europe and Oceania, two to Latin Amer- ica and the Caribbean, two to Asia and one to Eastern Europe. Five are replaced each year and the elections are hotly contested. Then we come to the crunch. Who should the new semi-permanent (or per- manent) members of the Security Council be and should they have a veto or not? Surely countries as big as India, Japan, Brazil, South Africa, Indonesia, Nigeria, Germany and Pakistan should not be excluded from being Permanent Members? Alas, it is not so simple. Each of these countries has neighbours who are absolutely set against their elevation to a Permanent Member status. At the 2005 World Summit, Japan, Germany, India and Brazil grouped to- gether to form the G4. They were determined to get permanent seats but ap- peared willing to compromise on the veto. They had considerable support, but their regional rivals, including South Korea, Italy, Pakistan, and Argentina, immediately banded together with other countries like Canada, Mexico, Tur- key and Spain in the ‘Uniting for Consensus’ group to propose there should be 10 new, non-permanent members with possibility for re-election, but no new permanent members. We see here one of the sticking points. Instead of acting like cozy regional friends, most neighbours are age old traditional enemies who do not want to see each other moving ahead. For their part, the African states proposed increasing the Council’s membership from 15 for 26, introducing six new permanent seats with vetoes and five new rotating seats (granting Africa two seats in each category). At the present time the P5 represents four conti- nents but not Africa. “The future depends on what we do in the present.” Mahatma Gandhi The Security Council and the future When all is said and done, even if one were to find a workable model, noth- ing is to say that the current P5 would agree and not use their veto. Also, all these alliances are now a decade old and may have given way to other inten- tions. Nevertheless, they demonstrate to us the complexity of Security Council reform. However, ten years does make a difference. In its 2015 report, the Commis- sion on Global Security, Justice and Governance took a refreshingly different approach to reforming the UN Security Council. Showing that the world had moved on, instead of dwelling on the Council’s composition, the Commission simply states that we must expand its membership and allow immediate re- election of non-permanent members in order to achieve the twin goals of ef- fectiveness and acceptability. When we think about transforming the Security Council, we should remem- ber that if we are to make decisions for the good of the world, we will need something that looks like an ‘executive committee’ or a ‘cabinet’. Perhaps all our present models are too wedded to the present context of ‘nation-state in- ternational relations’. For ‘global governance’, we may need another model 69 based on more state-like institutions with political and democratic foundations. In any case, we can imagine that it is going to take a lot of consultative thinking and perhaps a global movement to break all the impasses we have just seen and to press forward with all the potential reforms. We will return to these possi- bilities in Chapter 5. based on more state-like institutions with political and democratic foundations. In any case, we can imagine that it is going to take a lot of consultative thinking and perhaps a global movement to break all the impasses we have just seen and to press forward with all the potential reforms. We will return to these possi- bilities in Chapter 5. 70 70 Understanding ‘development’ The global understanding of development has changed over the years, as have definitions. Countries now agree that the best path forward is one of sustainable development, which the UN defines as “development that promotes prosperity and economic opportunity, greater social well-being, and protection of the en- vironment”. Sustainable development seeks to achieve these three pillars— economic development, social development and environmental protection—in a balanced manner. The term was originally coined in 1987 by the World Com- mission on Environment and Development in Our Common Future, in what became known as the Brundtland report. The report defines sustainable devel- opment as “development that meets the needs of the present without compro- mising the ability of future generations to meet their own needs”. Thirty years later, this continues to be the most widely used definition. Much of what the UN does falls into the broad category of ‘development’. This includes work related to poverty reduction, food security, climate change, gender equality, housing, education, employment, infrastructure, disaster risk reduction, health and emergency response, water and sanitation, safety and crime prevention, good governance, and early childhood development. Even the areas of human rights and peace and security (the other two pillars of the UN’s work) are now recognized as having important impacts on social and economic development. Their inclusion in the global post-2015 development agenda (Sustainable Development Goals) reflects these linkages. This chapter explores what development meant at the UN’s inception, the period of decolonization, and the introduction of the world’s first global devel- opment agenda to mark the new millennium, as well as what it means in the present era of a second set of global goals. It looks at what lies ahead for the UN and assesses how prepared it really is to achieve the challenging task at hand. Development in the early years Development in the early years Development is among the three founding pillars of the UN system. The UN Charter states that “the United Nations shall promote higher standards of liv- ing, full employment, and conditions of economic and social progress and de- velopment”. Yet the understanding of development when the Charter was 71 drafted was unique to that specific moment in history, and the concept evolved greatly over the years. Consider how the world looked in 1945. The original membership of the UN was 51 countries, of which the vast majority were Eu- ropean, Latin American, North American and the Middle Eastern states. The notions of developed and developing countries, North and South, First World and Third World simply did not exist at that time. Early development efforts of the international system focused on post-World War II reconstruction. In 1948, the US launched the Marshall Plan, a massive aid initiative to rebuild Western Europe following the devastation of the war. Through the Plan, offi- cially known as the European Recovery Program, the US, aided by companion programs in Canada and other countries, channeled more than $12 billion over three years to finance the economic recovery of Europe—a region that, rela- tively speaking, was industrialized and economically advanced. Europe’s swift recovery meant that the international community could soon focus its efforts elsewhere. In the years that followed, the emphasis quickly shifted to developing coun- tries that were gaining independence from their colonial powers. Between 1956 and 1968, UN membership grew from 80 to 126. Nearly all of these additional members were African and Asian countries, newly independent from European colonial powers. This period of rapid decolonization, which began in the late 1940s but accelerated in the two decades that followed, was prompted by the recognition that all peoples had the right to self-determination. In 1960, this basic right was formalized when the UN General Assembly adopted the Dec- laration on the Granting of Independence to Colonial Countries and Peoples, stating that “the subjection of peoples to alien subjugation, domination and exploitation constitutes a denial of fundamental human rights, is contrary to the Charter of the United Nations and is an impediment to the promotion of world peace and cooperation.” It called for immediate steps to be taken in ter- ritories that had not yet attained independence to transfer power to the people to enable them to enjoy independence and freedom. Expanding development efforts The emergence of new international institutions supporting development ef- forts mirrored the shift in thinking. At the time of its inception, the UN was primarily focused on maintaining peace and security. In fact, the words ‘peace’ and ‘security’ appeared in the UN Charter 42 and 148 times respectively, while the notion of ‘development’ appeared only once. The term ‘environment’ was altogether absent from the founding Charter (see Box 9). Development in the early years It was added that lack of political, economic, social or educational readiness should not be used as a reason to delay independence. Thus, a number of states with low levels of de- velopment were born. Two factors drove Western countries to begin providing aid to these devel- oping countries. First, it was acknowledged that moving from an international order based on colonization and imperialism to one of free trade and fair deal- ing would require some degree of intervention. In other words, if post-colonial countries were to become trading partners, they needed assistance to reduce poverty and increase productive capacity. Second, Western countries wanted to prevent Third World countries—those neither belonging to the First World (developed, capitalist countries) nor the Second World (socialist countries of the Eastern Bloc)—from drifting towards communism. This fear motivated the development of the Colombo Plan for Co-operative Economic Development 72 in South and Southeast Asia in 1950, which could be considered the world’s first development cooperation initiative. The Plan was established to combat poverty in Asia, as it was believed that poverty was responsible for fuelling communist political movements in the region. Box 9: Putting the environment on the agenda Environmental issues are one example of how the UN has successfully adapted to changing times. Though the environment was absent from the UN Charter and the initial organizational structure, it was effectively in- tegrated into the existing system later on. At the first United Nations Con- ference on the Human Environment that took place in Stockholm in 1972, the United Nations Environment Program (UNEP) was created as the voice for the environment in the UN system. With UNEP reporting to the GA and ECOSOC, ECOSOC saw environmental issues added to its original mandate of economic and social. Some argue that the environmental realm needs to be given greater prominence and that ECOSOC ought to more formally recognize it, by, for example, rebranding itself as the Economic, Social and Environmen- tal Council (ESEC) as part of a larger set of reforms. It seems unlikely that ECOSOC will get a new name any time soon, but a 2013 reform of the outdated institution mandated it to convene an integration summit to monitor and promote the balanced integration of the three dimensions of sustainable development (economic, social and environmental). Thus, progress is being made. In 1945, the UN had just a handful of development-related agencies. The Eco- nomic and Social Council (ECOSOC) was created to coordinate social and economic activities within the UN system, while the International Labour Or- ganization (ILO), Food and Agriculture Organization (FAO) and United Na- tions Educational, Scientific and Cultural Organization (UNESCO) were spe- cialized agencies that fell under ECOSOC’s mandate. The following year saw the establishment of the United Nations Children’s Fund (UNICEF). However, 73 as preoccupations expanded beyond security issues to areas of development, human rights, humanitarian assistance and the environment, the UN system quickly expanded. During the 1960s and 1970s there was a proliferation of new UN funds, programmes and specialized agencies dealing with poverty and hun- ger, social development, health, women, environment and housing (see Figure 3). Rather than adding to the mandates of existing agencies, new ones were created. Figure 3: United Nations Development Group members (excluding re- gional commissions and Secretariat bodies) by year established Figure 3: United Nations Development Group members (excluding re- gional commissions and Secretariat bodies) by year established The North-South divide Cold War politics saw a political divide between the “East” (the Soviet Union and its allies) and the “West” (North America, Western Europe, Japan, Aus- tralia, and New Zealand). With decolonization, developing countries came to outnumber developed countries and the power dynamics shifted within the United Nations as postcolonial countries pushed to have their voices heard. As we saw in the description of the General Assembly, a group of newly inde- pendent countries joined forces in the 1950s to push back against the two su- perpowers—the United States and the Soviet Union. These countries formed the Non-Aligned Movement (NAM) in 1961, what is now often referred to as the “Global South”, and many of the same countries also organized themselves into the “Group of 77”, to have their economic and social issues heard on the world stage. Despite the breakdown of the East-West divide following the end of the Cold War—to say nothing of the blurring North-South distinction re- sulting from globalization—both the NAM and the G77 continue to live on. As Thomas Weiss has pointed out (2011), the “various constructed roles on the international stage in the global theater are played by actors from the two major troupes, North and South”. This theater, Weiss argues, is counterproduc- tive to generating universal norms and ensuring human security. Though there are some exceptions, countries have generally adhered to artificial roles based on which side they are on, rather than where they may fall on specific issues. Instead, policy debates should reflect issues-based and interest-based alliances and coalitions. Figure 3: United Nations Development Group members (excluding re- gional commissions and Secretariat bodies) by year established Figure 3: United Nations Development Group members (excluding re- gional commissions and Secretariat bodies) by year established Meanwhile, economic issues such as trade, development lending and monetary policy had been delegated to the Bretton Woods institutions: the World Bank, and the International Monetary Fund (IMF) along with the General Agreement on Tariffs and Trade (GATT). The World Bank and the IMF were established as specialized agencies of the UN, while recognizing that they were independ- ent international organizations. As such, the institutions hold observer status at the UN and are able to participate—though not vote—in the General 74 Assembly, the Security Council and ECOSOC. The World Trade Organization (which replaced GATT in 1995), on the other hand, is considered a ‘related organization’ in the UN system, falling under the General Assembly but with no obligation to report to it, and contributing on an ad-hoc basis to the GA and ECOSOC. The relationship between the UN and the Bretton Woods institu- tions has historically been thorny. Many have argued for the UN to have more authority over economic issues, pointing to the so-called democratic deficit of the World Bank, IMF and WTO, while others support the status quo, maintain- ing that these institutions are more effective than the UN in achieving their stated objectives. Figure 4: Millennium Development Goals Figure 4: Millennium Development Goals Figure 4: Millennium Development Goals Source: United Nations The MDGs were praised for their simplicity. Schoolchildren in Nigeria, Laos and Germany alike could memorize and recite them. They also brought atten- tion to previously under-the-radar issues, like Goal 5 on improving maternal health. Maternal health advocates had long fought for greater attention to the issue, and being given equal presence with priorities such as poverty and hun- ger, environmental sustainability, and primary education was an important step in mainstreaming it. However, the MDGs were also criticized for being devel- oped without sufficient consultation with key actors, such as UN member states, civil society organizations, NGOs, think tanks and academia. Instead, they were drawn up by a group of experts in a ‘basement of UN headquarters’. It is likely that this lack of consultation in the design of the goals meant that developing countries had a weakened sense of ownership of them. Figure 4: Millennium Development Goals Source: United Nations Source: United Nations Source: United Nations The MDGs were praised for their simplicity. Schoolchildren in Nigeria, Laos and Germany alike could memorize and recite them. They also brought atten- tion to previously under-the-radar issues, like Goal 5 on improving maternal health. Maternal health advocates had long fought for greater attention to the issue, and being given equal presence with priorities such as poverty and hun- ger, environmental sustainability, and primary education was an important step in mainstreaming it. However, the MDGs were also criticized for being devel- oped without sufficient consultation with key actors, such as UN member states, civil society organizations, NGOs, think tanks and academia. Instead, they were drawn up by a group of experts in a ‘basement of UN headquarters’. It is likely that this lack of consultation in the design of the goals meant that developing countries had a weakened sense of ownership of them. “The 13 years since the millennium have seen the fastest reduction in poverty in human history” 2013 UN Report, A New Global Partnership: Eradicate Poverty and Transform Economies Through Sustainable Development Millennium Development Goals The year 2000 was a milestone for development cooperation. For the first time in history, states came together behind a clearly defined set of goals related to social and economic development. The Millennium Development Goals, also 75 referred to as the MDGs, were eight easy-to-remember, time bound, focused and measurable goals that people worldwide could rally behind (see Figure 4). They were drafted in the late 1990s and came into force through the Millen- nium Declaration, adopted by the UN General Assembly on September 8, 2000. They were intentionally ambitious, with goals such as “halving extreme poverty” and “providing universal primary education” by 2015. Tallying up the results 76 As 2015 approached, the question on everyone’s mind was: how successful were countries in meeting these goals? The most impressive achievements were made on fighting extreme poverty. The target to halve the proportion of people living on less than $1.25 a day by 2015 (with a 1990 baseline) was achieved in 2010, five years early. However, this was not the case in all coun- tries, regions, or population groups, but rather on the aggregate or global level. In South Asia the proportion of people experiencing extreme poverty dropped by 41 per cent, while in Sub-Saharan Africa it fell by a mere 14 per cent. Mean- while huge gains were made in China where the proportion fell from 60 per cent in 1990 to 12 per cent in 2010. Critics noted that China’s rapid economic development, which coincided with the timeline to achieve the MDGs but was due to entirely exogenous factors, skewed the overall picture and drew atten- tion away from countries that fared less well. Significant achievements were made on numerous other goals as well— such as boosting primary school enrolment and improved access to clean wa- ter—even if ambitious targets were not always met. The final assessment of the MDGs highlighted that, despite groundbreaking success, millions of people are being left behind, especially the poorest and most vulnerable populations. It pointed to climate change and environmental degradation as factors under- mining progress achieved, and stated that conflict remains the biggest threat to human development. A changed world Long before 2015 approached, the international community was looking ahead to the post-2015 development agenda. The idea for a new set of goals that would replace the MDGs was formally tabled by Columbia and Guatemala at the Rio+20 Summit in 2012, which was the largest summit in UN history. A three-year process engaging millions of people through dialogues, thematic consultations and national surveys culminated in the 17 Sustainable Develop- ment Goals (SDGs), also known as the Global Goals, being adopted unani- mously by states at the UN Sustainable Development Summit in September 2015 (see Figure 5). The post-2015 consultation process that led to the development of the SDGs sought to address many of the shortfalls of the MDGs, while also adapting to a new era. When consultations were launched in 2013, the world had become more complex and the discourse around development had changed drastically. The MDGs felt outdated. 77 Figure 5: Sustainable Development Goals Figure 5: Sustainable Development Goals Source: United Nations For one thing, the MDGs were meant for developing countries, and funding was to come from developed countries through their official aid contributions, known as Official Development Assistance (ODA). Yet by the time stakehold- ers were discussing the SDGs, it had become apparent that the traditional North-South lines were blurring. South-South cooperation was becoming a new norm and the economic and political power was beginning to shift from developed countries toward emerging economies, in particular the BRICS (Brazil, Russia, India, China and South Africa). It was also recognized that every country, not just those belonging to the Global South, had room for im- provement. Meanwhile development finance was becoming increasingly complex and Source: United Nations For one thing, the MDGs were meant for developing countries, and funding was to come from developed countries through their official aid contributions, known as Official Development Assistance (ODA). Yet by the time stakehold- ers were discussing the SDGs, it had become apparent that the traditional North-South lines were blurring. South-South cooperation was becoming a new norm and the economic and political power was beginning to shift from developed countries toward emerging economies, in particular the BRICS (Brazil, Russia, India, China and South Africa). It was also recognized that every country, not just those belonging to the Global South, had room for im- provement. A changed world For one thing, the MDGs were meant for developing countries, and funding was to come from developed countries through their official aid contributions, known as Official Development Assistance (ODA). Yet by the time stakehold- ers were discussing the SDGs, it had become apparent that the traditional North-South lines were blurring. South-South cooperation was becoming a new norm and the economic and political power was beginning to shift from developed countries toward emerging economies, in particular the BRICS (Brazil, Russia, India, China and South Africa). It was also recognized that every country, not just those belonging to the Global South, had room for im- provement. p Meanwhile, development finance was becoming increasingly complex and ODA was no longer viewed as the only source of funding to achieve the goals. A number of other sources of finances such as domestic resources (taxation), investment and remittances were gaining relevance and in some cases outpac- ing traditional aid. For example, as migrant workers went abroad in search of economic opportunities, remittances—money that foreign workers transfer to an individual, usually a family member, in their home country—came to com- pete with international aid as one of the largest financial inflows to developing countries. In 2000, remittances to developing countries totalled $120 billion (OECD 2006), while by 2015 they had grown to $441 billion (World Bank 2015). Meanwhile, official development aid, or ODA, grew at a much slower pace during the same period, from $80 billion to $147 billion (Compare Your Country). The world had become less state-centric too. Non-state actors such as the private sector and civil society were playing a more central role in promoting 78 global cooperation and development. Sub-national governments (e.g., provin- cial, municipal) were also much more involved. Finally, the global distribution of poverty shifted during the past couple of decades. In 2000, poverty was mainly regarded as an issue for low income countries, with the greatest portion of world’s poor in these countries. In 1990, there were 1.6 billion poor people in low income countries, representing 95 per cent of the world’s poor. Yet by 2012 when the SDGs were being developed, many of these low income countries had graduated to middle-income status and the number of poor people in low income countries had dropped to 0.3 billion (FUNDS 2016). It was thus argued that inequality and redistribution in all countries needed to be given greater weight. A changed world The SDGs reflected this by having a goal devoted to reducing inequalities. Several other issues addressed by the SDGs but not the MDGs include good governance, respecting human rights, climate change, and peace and security as fundamental to development. These additions are mostly a reflection of emerging challenges and an evolution in thinking about development issues. For instance, climate change is a global threat that became widely recognized only after the MDGs. The same can be said about the linkages between devel- opment outcomes and peaceful and just societies. The other reason for certain additions was pressure from stakeholders to include key issues that the MDGs neglected (e.g., secondary/post-secondary education, governance, and energy). More voices at the table Quite unlike the process to establish the MDGs, the post-2015 development planning favoured a participatory, consensus-based approach that included years of consultations with tens of millions of people. Consultations consisted of three separate streams. First, UN country teams in coordination with other key stakeholders facilitated 88 national consultations in countries where the UN provides assistance. Second, UN agencies worked with national govern- ments and other stakeholders to conduct 11 thematic consultations. Both of these channels used numerous methods of outreach, including face-to-face meetings, online mechanisms and door-to-door surveys. Lastly, views from the public were sought online through the MYWorld survey. The survey asks in- dividuals to choose six issues that matter most to them and their families from a list of 16 (e.g., affordable and nutritious food, better transport and roads, po- litical freedoms). This survey continues to collect votes and in March 2017 had over 9.7 million votes. According to Amina Mohammed, special adviser to the UN Secretary Gen- eral on post-2015 development planning, about 70 per cent of those consulted were under 30 years of age and at least 8 million were contacted through social media. 79 With regards to the process, Ms. Mohammad states: “[Developing the MDGs] was just a bunch of technocrats sitting round a table to produce a set of goals based on what UN agencies had been doing over the years. There was no attempt to be transformative. For me, the SDG process has been one of the most intellectually and politically and technically challenging endeavours I have ever engaged in.” A summary of how the SDGs differed from the MDGs is provided in Figure 6. Figure 6: Key differences between the MDGs and the SDGs MDGs SDGs Timeframe 2000-2015 2015-2030 Scope 8 goals 18 targets 61 indicators 17 goals 169 targets 230 indicators Target Developing countries only Universal Process UN-led with little input from external stakeholders or the public Consultative, participatory, concrete role for civil society organizations Figure 6: Key differences between the MDGs and the SDGs Despite the inclusive and consultative approach taken to their development, the SDGs have not been without criticism. Following the announcement of the new goals, many voiced discontent with their broad scope, calling the 17 goals and 169 targets “dizzying” and “worse than useless”. Economist William East- erly suggested SDG stood for “senseless, dreamy, garbled”. Financing the goals A legitimate concern is the high cost of achieving the lofty goals coupled with a lack of adequate financing. It is estimated that meeting the goals would cost $2-3 trillion a year over 15 years, which is roughly 4 per cent of world GDP. Research by Development Finance International suggests that low- and mid- dle-income countries could fund the SDGs if three key sources of public fi- nance were raised: doubling developing country tax revenues; doubling aid flows from developed countries; and raising $500 billion a year from innova- tive financing (e.g., through introducing taxes or levies on carbon emissions and/or financial transactions and allocating these new monies to achieving the SDGs) (UNRISD 2015). ) ( ) Currently, none of these financing sources seem likely. Doubling develop- ing country tax revenue would require an increase equivalent to 10 per cent of GDP (ibid). Meanwhile, doubling aid flows would require OECD Develop- ment Assistance Committee (DAC) members—the world’s major donor coun- tries—to meet their pledge to spend 0.7 per cent of their national income on aid, despite never having met this target since it was agreed upon in 1970. In 2014, the average across DAC members was 0.03 per cent, with only 5 of 29 countries meeting or exceeding the target (Denmark, Luxembourg, Norway, Sweden and United Kingdom) (OECD). Ideas for innovative financing ap- proaches, such as a global financial transaction tax from which revenues would go to achieving development goals, have been proposed for years but have yet to be implemented. The UNDP has also launched an SDG Fund, which, similar to the MDG Fund, is an official funding mechanism in support of the post- 2015 agenda. Currently, the Fund’s total budget is approximately $60 million (SDG Fund website). ( ) In reality, public funding alone cannot be relied on to meet the financing needs of the SDGs. Blended finance, a concept that seeks to blend public and private capital to finance development goals, has become a new norm. Mobi- lizing private capital for development-related purposes such as education, health, green technology or microfinance in developing or emerging markets is a promising though still nascent approach to supplementing traditional fund- ing sources. Often referred to as impact investing or social impact investment, capital is typically supplied by financial institutions, investment firms, foun- dations’ endowments, high net worth individuals, pension funds and individual or retail investors. More voices at the table Many argued they are aspirational and unrealistic, while lacking the precision and clarity needed to be measurable. That these two realities go hand in hand should not be a surprise—the trade-off between the number of parties involved in negotiations and the usefulness of the outcome is an oft-cited challenge in international re- lations. The process vs. product trade-off is seen across the UN system. It is most pronounced in comparing the Security Council and the General Assem- bly: the former is known for being an exclusive club of member states but more effective decision making and less watered-down products, while the latter for greater participation but an inability to produce meaningful outcomes. 80 Financing the goals Financing the goals Financing the goals Financial return on investment ranges from below market (“impact first”) to market rate (“finance first”). 81 Looking ahead The new development agenda has been set and implementation is already un- derway. Attention has shifted to how national governments and other stake- holders will measure progress on the goals and how they will report on that progress. The review process is multi-level and complex. The High-Level Po- litical Forum on Sustainable Development, which convenes annually under the auspices of ECOSOC, is the central platform for follow-up and review of the SDGs. States report to each other at the regional level and regions report to the global level. Each year, the High-Level Forum focuses on a different overall theme and zeros in on a few of the 17 goals. Member states are encouraged to prepare Voluntary National Reviews on successes and challenges related to implementing the agenda. Every four years, the UN Secretariat will prepare a Global Sustainable Development Report covering overall trends, and heads of state will meet to review progress. National governments are thus busy incorporating the SDGs into their na- tional development plans, strategies and budgets, and considering how they will raise awareness and mobilize action among diverse actors. They are not the only ones getting to work. Consultations on the SDGs revealed a strong desire from other actors to be involved not only in the design of the goals, but also in future implementation. What role will the UN play in this next phase and what role will be played by civil society, businesses, and other key stake- holders? Is the UN development system fit to tackle current global challenges in partnership with other sectors? How can it be improved? Partnering in a new era of development cooperation The traditional understanding of ‘development’, a simple characterization of developed-developing, donor-recipient relationships that emerged in the 1960s, is giving way to a new era of universality and global partnerships. The universal nature of the SDGs makes clear that the greatest challenges of our time are not problems of the global South, but global problems. One example is the inclusion of climate change action in the goals. Evidence has emerged that Western, industrialized countries are most responsible for climate change, while its harmful effects have disproportionately affected developing countries that have done little to contribute to it. The goals also recognize that developed countries have a particular duty to ensure sustainable consumption and produc- tion, given their sizeable consumption levels relative to poorer states. Reducing income inequality within countries is another example. The level of inequality in the United States—an exemplary country according to indicators related to maternal health, education, preventing and combating disease, extreme poverty and hunger, etc.—exceeds that of many developing countries. Similarly, 82 despite having a much higher Gross National Income (GNI) than most African countries, South Africa is one of the most unequal countries in Africa, and indeed in the world. Alongside an understanding that current challenges extend beyond develop- ing countries, there is a general acceptance that these broader, more complex problems require new, innovative approaches as well as enhanced collabora- tion between sectors. Old ways of thinking coupled with a reliance on devel- opment aid will not suffice. “Lasting solutions to global problems no longer lie in the hands of governments alone. The United Nations of the 21st century must think in terms of networks and coalitions.” (UNSG 2013). The role of multi-sector partnerships in UN Secretary-General Ban Ki-Moon’s special initiatives launched over the past years is telling of the UN’s commit- ment to collaboration:  Climate Resilience Initiative, launched in 2015, seeks to strengthen climate resilience of world’s most vulnerable countries and people by “bringing together private sector organizations, governments, UN agencies, research institutions and other stakeholders to scale up trans- formative solutions.” (UN 2015).  Global Education First Initiative, launched in 2012: “a partnership comprised of a range of actors and institutions including governments, UN agencies and multilateral organizations, civil society organisations, and the private sector.” (UNESCO GEFI). Partnering in a new era of development cooperation p ( )  Every Woman Every Child, launched in 2010: “an unprecedented global movement that mobilizes and intensifies international and na- tional action by governments, multilaterals, the private sector and civil society to address the major health challenges facing women, children and adolescents around the world.” (Every Woman Every Child web- site).  Global Pulse, launched in 2009: “Global Pulse partners with experts from UN agencies, governments, academia, and the private sector to research, develop, and mainstream approaches for applying real-time digital data to 21st century development challenges.” (Global Pulse website).  Global Pulse, launched in 2009: “Global Pulse partners with experts from UN agencies, governments, academia, and the private sector to research, develop, and mainstream approaches for applying real-time digital data to 21st century development challenges.” (Global Pulse website). Analysis of two related UN documents, The Future We Want (the outcome document of the 2012 Rio+20 Conference at which the idea for SDGs was officially tabled) and We the Peoples (the report proposing the MDGs, released ahead of the 2000 Millennium Summit), demonstrates that the emphasis on partnerships is relatively new. The report published in 2012 cited the concept of partnerships three times more often than the 2000 report did. The term stake- holder(s) was used 26 times in the 2012 report, and only once in the slightly longer report from 2000. Another key document, the UN Secretary-General’s 2014 synthesis report The Road to Dignity by 2030 includes partnerships as 83 one of six “essential elements for delivering on the SDGs”. This report men- tions partnerships twice as many times as the The Future We Want published just two years earlier (Volt 2015). ECOSOC has placed greater emphasis on leveraging partnerships in support of the international development agenda since 2008 when it began convening an annual Partnership Forum, bringing together governments and stakeholders, particularly from business and foundations, to discuss ways to engage and col- laborate to achieve the development goals. At the 2016 Forum, ECOSOC Pres- ident Oh Joon stressed that governments, the UN system, civil society, the pri- vate sector, the philanthropic community and academia must break down tra- ditional silos for more and better cross-sectoral decision-making and solutions (ECOSOC 2016). Role for private sector “Putting social and environmental purposes in the driving seat of business is the only way to ensure an equitable and sustainable economy for the 21st century.” Professor Muhammad Yunus (British Council and SEUK 2015) “Putting social and environmental purposes in the driving seat of business is the only way to ensure an equitable and sustainable economy for the 21st century.” Professor Muhammad Yunus (British Council and SEUK 2015) The private sector is a critical element in the 21st century notion of partnerships for development. Beyond being a potential source of capital to finance the in- ternational development agenda, in itself quite significant, the business world brings expertise, innovation, rigour and efficiency. Businesses and corpora- tions focused on providing technology, products and services to the poor can have huge impacts on poverty alleviation. Think about a company that pro- duces low-cost medical supplies or affordable home water-filters to those at the bottom of the pyramid. Or microfinance institutions, like Grameen Bank or BRAC, that offer micro-loans to the poor who lack access to traditional fi- nancial services and would not qualify for a bank loan due to a lack of collat- eral. Even growing ventures like Lucky Iron Fish, a social enterprise working to reduce iron deficiency in Cambodia and worldwide through a reusable iron fish that can be added to any pot while cooking. The ingenuity and drive of a new class of social entrepreneurs that seek to solve complex social challenges through business is changing the way we think about addressing social issues. In fact, there is a spectrum of private sector actors that can support the UN’s goals. These range from for-profit social enterprises to socially responsible businesses, to corporations looking to engage in corporate social responsibility (CSR) work and purely profit-oriented companies. When incentives align, there is enormous potential for corporations to contribute to meeting the UN’s needs while achieving their own social responsibility goals. The UN Global Compact, a voluntary initiative based on business leaders’ commitments to adopt sustainable and socially responsible policies, seeks to tap into the boom- ing CSR trend. Self-described as the world’s largest voluntary corporate 84 sustainability initiative, it had the participation of over 8,400 companies from 162 countries as of April 2016. This reflects an acknowledgement that global trade, investment and business activity can be powerful drivers of economic transformation and social change. Role for private sector One just needs to compare the $134.8 billion in ODA in 2013 to worldwide trade in goods and services, worth $23 trillion in 2014, to see the potential of a more social impact-oriented private sector (British Council and SEUK 2015). If economic globalization were harnessed for international development, the result would be transformational. Even corporations not driven by the desire for social change have a place at the table. Public-private partnerships have traditionally been essential to de- velopment projects, particularly those related to public infrastructure, transpor- tation, and information technology (e.g., internet access). Recently, interest has broadened to other areas such as affordable or social housing, water and edu- cation. The new attention paid to the opportunities associated with private sector involvement in development outcomes has led to concerns around the risks posed. Many have pointed to the need to ensure transparency and accountabil- ity, which is now acknowledged in nearly every UN speech that stresses the potential of private sector partnerships. Accountability is an issue as corpora- tions often have an incentive to keep information confidential in order to pro- tect any competitive advantages. As well, corporations are accountable to their shareholders, not stakeholders, and have a fiduciary responsibility to maximize profits. Inclusion of all relevant stakeholders is not a given. In fact, despite the UN framing public-private partnerships as a way to enhance inclusion of di- verse actors, evidence suggests that work in this area remains to be done. Sev- eral studies have shown that such partnerships have failed to achieve inclusive participation, particularly of certain groups such as farmers, workers and trade union, indigenous peoples, women, youth or children. If business interests are put ahead of the UN’s interest to advance the internationally agreed develop- ment agenda, the UN will risk losing its legitimacy and credibility in the eyes of the people. Ultimately, what is required for these partnerships to be successful is for there to be proper due diligence screening, clear partnership principles and guidelines, and monitoring and evaluation mechanisms to ensure that expected results are achieved. Role for civil society Amidst all the promise of leveraging the private sector as a force for good, civil society has been sidelined and its contribution undervalued. That corporations are relatively new actors in this space is partially responsible for the hype. As well, the financial resources they offer are welcome at a time when developed 85 countries are facing recession and cutting their ODA budgets accordingly. Yet civil society, made up primarily of NGOs but also social movements and net- works, has been pivotal to advancing the UN development agenda over the past decades. At the founding conference of the United Nations in San Francisco in 1945, around 1,500 participants from NGOs joined the 5,000 government delegates, members of the media and other officials and staff in attendance. NGO partic- ipation at UN world conferences and summits has risen dramatically since. Over 17,000 NGO participants took part in the 1992 Earth Summit in Rio, and in 2012 at Rio+20 the number reached more than 40,000. At the same time, the number of NGOs that enjoy consultative status with ECOSOC has risen from 41 in 1946 to 4,189 today, reflecting both the expanding number of NGOs globally and the greater influence they hold in the UN development arena. NGOs and civil society organizations are uniquely placed to support gov- ernments and the UN in achieving social and economic development, gender equality, environmental sustainability and human rights, as they typically share the same vision and ideals. They achieve impact through knowledge, expertise, lobbying, advocacy, research, priority setting, media outreach and monitoring. They have succeeded in raising public awareness of issues, shaping public opinion, norm setting, influencing international deliberations and negotiations, and pressuring states and the corporate world to be more responsible and ac- countable. During the climate negotiations in Paris in 2015, civil society move- ments and self-organizing networks were crucial in getting states to take action against climate change. Their immense contribution to the creation of the SDGs helped ensure an inclusive decision-making process and goals that ulti- mately reflected the world’s most pressing needs. Through campaigning and political pressure, NGO coalitions played a cen- tral role in the adoption of international bans on landmines, ending apartheid, and establishing the International Criminal Court, among many other achieve- ments. Role for civil society With the internet’s ability to rapidly mobilize global supporters around an issue or concern, NGOs that make effective use of new technologies have been able to reach the public like never before. Avaaz, the world’s largest online activist network, has over 44 million members worldwide. Launched in 2007, its model of internet organizing engages citizens around the world to take action on pressing global, regional and national issues through signing petitions, funding media campaigns and direct lobbying, and organizing “of- fline” protests and events. Though technology is enabling civil society organizations to achieve un- precedented scale, NGOs have also increased their impact through partnering or forming coalitions with other NGOs. In global decision-making processes such coalitions are favoured as they are seen to represent agreement and con- sensus around specific issues. Yet this pressure for a single voice poses a chal- lenge for NGOs, which are as diverse as the problems they were created to 86 solve and the constituents’ views they give voice to. This is especially true for NGOs from the South, which tend to be less established and powerful than their Northern peers. Reaching consensus and getting others on board has his- torically been easier for Northern NGOs than Southern ones, which can mean that voices from the North may be more likely to be heard (Browne and Weiss 2014: 200). This is problematic as NGOs in the South have distinct priorities and interests. UN leaders have for decades proclaimed that NGOs and civil society are vital to human progress. Former Secretary General Boutros Boutros-Ghali called NGOs “an indispensable part of the legitimacy” of the United Nations. Similarly, Kofi Annan referred to NGOs as “the conscience of humanity” and Ban Ki-Moon said that “civil society has never been more important or needed” (UNSG 2013). Yet despite these declarations, civil society participa- tion in global governance at the UN is not as strong as it could be and NGOs are too often perceived as a nuisance. The following chapter on human rights explores this challenge in greater depth, as NGOs have less influence in the current Human Rights Council than in its predecessor, the Commission on Hu- man Rights. The hostility towards NGOs within the walls of the UN mirrors the fraught relationship between civil society and national governments. Mobilizing civil society Mobilizing civil society One outcome of the internet’s penetration in developed and developing coun- tries alike has been a new form of civic engagement. Self-organizing networks and online social movements have succeeded in mobilizing previously unfath- omable numbers of people to support causes, pressure governments and cor- porations, contribute resources, and coordinate action. Social media has been instrumental in enabling the shift from state-based institutions and forums di- recting change towards a model based on leadership from civil society and other non-state actors. Leading up to the 2015 UN Climate Change Conference in Paris (COP21), individuals and civil society groups expressed their discon- tent with the inability of states to take action on climate change. Six months before the Conference, over 20 million people had joined the Climate Reality Project and other self-organizing networks pushing for action against climate change (Tapscott 2014). Ultimately, states agreed on a far more ambitious deal than ever before, restoring some degree of faith in the ability to states to act, though the considerable pressure from civil society was undoubtedly a crucial factor contributing to COP21 success. Role for civil society In many countries—from those in the global South to the North, from authoritar- ian regimes to developed democracies—NGOs are increasingly being targeted by governments. Turkey is just one example; its government shut down hun- dreds of NGOs following the failed coup attempt in July 2016, and interna- tional aid organizations helping Syrian refugees in Turkey are experiencing hostility and expulsion (Sanchez 2017). A number of reforms have been proposed to strengthen civil society’s in- volvement and influence in UN processes. Key recommendations include: es- tablishing permanent, more inclusive structures and mechanisms for civil so- ciety participation in institutional decision-making; allocating more funds for relations with civil society organizations; cultivating positive attitudes towards civil society; and increasing the UN’s capacity to engage with civil society through staffing decisions, training and guidance. As well, the UN should ac- tively encourage the participation of underrepresented NGOs, with a particular focus on empowering NGOs from the global South that may struggle to attract levels of international attention similar to their Northern peers. Going forward, civil society and NGOs will be essential to effective global partnerships for development. States recognize that their success will remain limited without the support of civil society, while the business community struggles with accountability. Civil society cannot be marginalized by global actors looking to new solutions to solve the world’s most pressing challenges. Their place at the table can contribute to more effective global governance by ensuring a diversity of views and interests. 87 Innovation and technology The digital revolution and the spread of mobile technology have transformed international development efforts. The internet and social media have fostered the swift mobilization of people and resources in an unprecedented way. New technology and new applications of existing technology have made possible significant improvements in the developing world across diverse sectors such as banking, education, health and agriculture. Access to technology is not yet universal, but uptake is rising exponentially. Innovation for development is the latest trend. While the focus here is on the potential of tools such as social media, mobile technology, crowdsourcing, big data and open data, the concept is much broader. It refers to doing things differently, with an emphasis on achieving better outcomes through evidence- based decision making, experimentation to determine what works, scaling suc- cessful models and adopting a human-centered design approach. Fundraising Social and environmental causes have embraced the internet for its fundraising potential. A particularly striking example was the ALS Association’s Ice Bucket Challenge, which dared people to post videos of having ice water dumped on themselves, and challenging others to do the same. It exploded in July and August 2014 and in less than three weeks the ALS Association—a non-profit health organization that few had heard of before the challenge—had 88 raised $15.6 million. The crowdfunding industry, of which social impact initi- atives make up a rising proportion, grew from $16 billion in 2014 to an esti- mated $34 billion in 2015, surpassing the venture capital industry which in- vests an average of $30 billion each year (Barnett 2015). Online platforms for microfinance lending have also gained momentum in recent years. Kiva, the most prominent platform, has nearly 2.5 million users and has lent over $846 billion to individuals in 84 countries through its field partners. Sectors: banking, commerce, disaster response, education Sectors: banking, commerce, disaster response, education Technology has had a tremendous effect on the banking and commerce world, reducing transaction costs and facilitating participation of marginalized popu- lations in global trade. Kenya’s mobile money-transfer scheme, M-Pesa, al- lows people to make microfinance loan repayments, pay bills and send remit- tances to family members back home using their mobile phones, saving time and money. The cashless nature of electronic transfers also helps reduce theft. In April 2016, M-Pesa had about 19 million active users in Kenya, more than two thirds of its adult population, and over 6 million subscribers outside of the country in Africa, Asia and Europe (Ochieng 2016). East Africa is now re- garded as a global leader in mobile banking. Meanwhile, the growth of e-com- merce has removed barriers for entrepreneurs to participate in global markets and sell their goods or services to businesses and consumers around the world. In the field of disaster response, crowdsourced data and geographic infor- mation system mapping have drastically improved abilities to respond to nat- ural disasters and other crises in a timely and effective manner. Following the devastating 2015 earthquake in Nepal, Kathmandu Living Labs developed cri- sis map QuakeMap to better match relief efforts with the needs of affected people and communities. Similarly, HaitiData makes public geospacial maps of Haiti, containing a wealth of information and data to support policy makers in post-earthquake reconstruction. These technologies have applications in other areas as well, such as climate change adaptation and mitigation. Online learning and massive open online courses, referred to as MOOCs, are innovations disrupting and creating opportunities in higher education. MOOCs first entered the scene in 2008, but gained worldwide attention and praise (along with some criticism) in 2012. Several top US universities, includ- ing Stanford University, Princeton, MIT, and University of Berkeley, intro- duced MOOCs at that time, and hopeful observers envisaged vast numbers of students in developing countries benefitting from the highest quality of educa- tion. The hype was not entirely overblown; enrolment began to spread from the West to the rest, emerging countries such as India and Brazil saw home- grown MOOCs take off, and higher education has indeed become more acces- sible and affordable for everyone. Sectors: banking, commerce, disaster response, education The spread of MOOCs in the developing world is not without challenges, namely that many lack the basic level of 89 education and literacy required to benefit and advantages of face-to-face inter- action with professors are lost, but potential is great. There are numerous other sectors, such as health and agriculture, where in- formation and communications technology (ICT) has been altering the status quo. In terms of making progress on the SDGs, effectively utilizing new tech- nology will be key. As this chapter has explored, social media outreach was instrumental in ensuring that the SDGs reflected the diverse voices from around the globe. Moving forward, data collection through mobile devices and crowdsourcing will be essential to monitoring achievements and progress on the goals. A main caveat to the great promise that technological advancements hold for accelerating global change is that information and communication technol- ogy infrastructure is still lacking in many parts of the world, particularly in rural communities. While access to modern technology varies greatly within countries and according to certain demographics, the digital divide is particu- larly stark between developed and developing countries. Another risk may be referred to as the “dark side of ICT”, when new technology and social media platforms are harnessed to advance global threats, such as terrorism, human trafficking and cybercrime. In recent years we have sadly witnessed that ICT can just as easily be used to reverse social and economic progress as it can be to accelerate it. What role for the UN? The changed development landscape has already begun to put pressure on the UN development system. With a multitude of new actors involved, a shift in the nature of poverty, and a reduced portion of ODA being channelled through the UN (a mere 16 per cent of ODA to least developed countries came from the UN in 2012, down from 25 per cent in 1990), the UN needs to consider what its role in development will be moving forward. Overall, its development function appears to be losing relative importance when compared to other UN functions such as peace and security, human rights, and humanitarian aid. With numerous previously low-income countries having graduated to middle-in- come status, countries have greater capacity to solve their own challenges, and technical assistance is less needed outside least developed countries and fragile states. Yet a different type of support will be sought. The UN’s greatest strength lies in its ability to convene diverse stakeholders and set global standards and norms. It succeeded in consulting a broad range of actors to establish a post-2015 development agenda by and for ‘the people’. The challenge ahead will be implementation. Given the centrality of multi- stakeholder partnerships to the UN’s plan for implementing the SDGs, the UN will need to ensure that efforts of diverse development actors are aligned with 90 the objectives of the international development agenda. As the primary body for coordinating sustainable development, ECOSOC should play a leadership role in setting global norms. It could start by establishing the principles and guidelines that govern the partnerships. To ensure a convergence between UN and business interests, it could also ask companies engaged in partnerships with the UN to commit to the UN Global Compact Principles. Actions need to be based on the right intentions, but they also need to be coordinated. To enhance coordination, UN agencies and programmes could be assigned as Task Managers for each SDG. A meta-partnership could then be created for each target which would oversee the contributions of various parties towards the targets and report back to the appropriate Task Managers (Dodds 2015: 13). The UN will also be looked upon for leadership in overseeing SDG monitoring and review, which will take place at the national, regional, and global levels. Is the UN prepared? There is little question that the task at hand is a mighty one. In a post-2015 era, the UN is expected to lead in implementing the sweeping and ambitious new development agenda, manage multi-stakeholder partnerships, adapt to a rap- idly evolving development landscape, embrace technology and innovation, and measure and evaluate progress. Yet the UN development system (which refers to the 30+ organizations in the UN system mandated to work in areas related to social-economic develop- ment) struggles to remain relevant. The system is fragmented and exists in si- los, there is significant overlap in the work of organizations with similar man- dates, software and business practices used are as numerous as organizations, and competition for limited resources is rife. Moreover, ECOSOC, the coordi- nating body, is known for being ineffective and cumbersome. Meanwhile, the World Bank, which is a specialized agency of the UN, and regional development banks (e.g., the African Development Bank, the Asian Development Bank), which are not part of the UN system but coordinate closely with it, are seen by many experts as the true leaders in development, producing superior research and policy, and operating more efficiently. The UN development system risks being marginalized by other actors if it contin- ues to put off much-needed reform. Ban Ki-Moon made reference to the shortcomings of the UN development system on multiple occasions, and called for swift action. He reinforced that sentiment in a 2015 statement to the Security Council, stating that “the 2030 Agenda calls on us to move from silos to synergy, to move from fragmentation to partnership.” This recognition that the UN development system requires re- form to operate more efficiently and effectively is not new. Numerous attempts 91 at reform have been made, including efforts to streamline the entire system, coordinate the work of multiple UN programs in a given country, and improve how ECOSOC operates. The three key reform initiatives have been the 1969 capacity study which sought to address fragmentation of the development sys- tem, Kofi Annan’s resident coordinator initiative in 1997, and the 2006 High- Level Panel on UN System-wide Coherence. Yet results have been limited and observers continue to stress the urgent need for more ambitious reforms such as the creation of UN Women (see Box 10). Streamlining the UN development system and ‘Delivering as One’ In the UN’s early days, its development work was carried out by a few special- ized agencies. During the 1960s and 1970s there was a proliferation of organ- izations in the UN development system, which today boasts 30 organizations plus research and training institutions. These organizations are headquartered in 16 countries, with over 1000 field offices dotted around the globe. The sys- tem has become extremely complex and inefficiencies abound. Some of the challenges associated with having so many players in the system include du- plication in policy advice and on-the-ground service delivery, lack of coher- ence and consistency across the UN system, and competition for funds among UN agencies. For instance, the Future UN Development System, or ‘FUNDS’ Project, an NGO devoted to accelerating change in the UN development sys- tem, points out that there are 16 UN organizations carrying out water and en- vironmental projects. Box 10: UN Women Box 10: UN Women In July 2010, the General Assembly decided to establish UN Women, a UN entity for gender equality and the empowerment of women by merg- ing four existing entities dedicated to women: the Office of the Special Adviser on Gender issues and Advancement of Women, the Division for the Advancement of Women of the Secretariat, the UN Development Fund for Women (UNIFEM) and the International Research and Training Institute for the Advancement of Women. In this rare move of merging entities with overlapping mandates, the UN demonstrated that solutions to an increasingly fragmented and unco- ordinated system are possible. The need for a more robust overall approach has been understood for years. In 2006, Secretary-General Kofi Annan launched the ambitious Delivering as One initiative, following recommendations by the UN Secretary-General’s High-Level Panel on UN System-wide Coherence, a group that had been 92 exploring options for strengthening and modernizing the UN system. The ob- jective was for the UN system to “deliver as one” at the country level. Most developing countries have numerous UN development organizations present (e.g., UNDP, UNICEF, FAO, WFP), each with their own office, budget, pro- gramme and so on. Through the “Delivering as One” approach, each country sought to improve service delivery and collaborate as one UN team based on One Programme, One Leader, One Budget and One Office (the four overarch- ing principles set out by the High-Level Panel). It was believed that such a unified approach to delivery would reduce duplication, fragmentation and competition for funds, while enhancing capacity for strategic approaches (United Nations 2012: 8). When it was launched, the governments of eight countries jumped on board, volunteering to become pilots: Albania, Cape Verde, Mozambique, Pakistan, Rwanda, Tanzania, Uruguay, and Vietnam. These pilot countries were encour- aged to innovate and experiment with new approaches to collaboration based on the four ‘Ones’. During implementation two additional concepts—One Voice and One Fund—were added. The pilot phase concluded with an inde- pendent evaluation conducted in 2012 of achievements across the six Ones. Conclusions were mixed. The most positive finding was a strong increase in national ownership and leadership, whereby UN programming in pilot coun- tries was being driven by their own needs and priorities. The greatest challenge proved to be achieving efficiency gains and reduced transaction costs, mainly due to the persistence of overlapping or incompatible reporting requirements and regulations of organizations. Box 10: UN Women This failure revealed the barriers to experi- mentation that pilot countries face, pointing to the need for more systemic changes at headquarters level. As of 2016, 52 countries have formally adopted the Delivering as One ap- proach. Most are low and middle income countries, with a handful of upper middle and high income countries as well. To date, progress has remained slow and the UN has not demonstrated leadership in accelerating the process of achieving a unified approach at the country level. This view was reflected in the results of a 2014 survey conducted by FUNDS that asked over 3,200 people from across sectors who interact regularly with the UN which changes in the UN Development System should be implemented by 2025. Many respondents wanted to see changes that are consistent with the Delivering as One” agenda, with the top suggestions including fewer organizations, a single country pro- gramme, a single head of the UN Development System and single country rep- resentatives for the system. 93 Improving business practices The UN system is large and unwieldy, not to mention notoriously inefficient. The top ideas for reform identified by experts in the above-mentioned 2014 FUNDS survey were in fact related to technology, administration and infor- mation management. Over 90 per cent of respondents agreed or strongly agreed with suggestions to cut costs and improve efficiency through greater use of technology; develop a single UN gateway to all UN research and publi- cations; and use a common, system-wide technology platform for administra- tion. Currently, virtually every UN organization and agency uses its own enter- prise resource planning software to manage its business activities, service de- livery, technology and human resources. Harmonizing business practices across the system and adopting a single platform for all administration would significantly reduce costs and increase the likelihood of delivering through a single office at the country level (which continues to be improbable with the multitude of platforms). Box 11: Global health crises—Ebola response Box 11: Global health crises—Ebola response The Ebola outbreak in West Africa in 2014 and 2015 was among the largest health emergencies the world has faced in recent years. During the two years, the uncontrollable spread of the deadly virus caused more than 11,300 deaths, most of which were in Liberia, Sierra Leone and Gui- nea. The World Health Organization was widely criticized for its delayed and inadequate response. An independent panel selected to assess the WHO response to the epidemic charged the agency’s culture and politics with its poor response, calling for a transformation of WHO’s organiza- tional culture and delivery approach, as well as changes on the part of member states. A key finding of the Panel report was that WHO has a “technical, nor- mative culture, not one that is accustomed to dealing with such large- scale, long-term and multi-country emergency responses occurring at the same time”. Lacking a culture of rapid decision-making, WHO “tends to adopt a reactive, rather than proactive approach to emergencies”. This contributed to WHO’s unnecessary deferral in declaring a public health emergency. Another factor behind the delay was that it did not wish to challenge national governments of affected countries who initially de- nied the extent of the outbreak due to fears of economic and trade impli- cations. Even when it did recognize the extent of the crisis, a lack of fun- ding for emergency response posed real challenges. At present, assessed contributions account for less than 25 per cent of WHO’s biennial pro- gram budget; the remainder comes from voluntary funds from donors. While donors do provide a considerable amount of money for emergen- cies, WHO has no core funds for emergency response (the outbreak and crisis response budget line). p g ) If WHO is to increase its effectiveness in dealing with future pandem- ics and improve its governance of the entire global health system, it needs to learn from past mistakes and make the reforms necessary for it to be a true leader. Source: World Health Organization 2015 Focusing on strengths and priorities There is little doubt that some organizations and agencies within the UN de- velopment system are far more successful at achieving their stated objective than others. Thus any claims about the effectiveness of the UN development system as a whole fail to account for variation by organization and by sector. For example, the fields where the UN development system is seen to excel are health, education and gender. Similarly, the organizations that are viewed as most effective are WHO, UNICEF, UNAIDS, WFP and UNDP. It is worth noting though that while the UN is generally seen to be a leader in health, its reputation is stronger for achieving long-term impacts than for dealing with pandemics (see Box 11). The areas in which the UN is perceived to be least relevant are crime prevention, drug control, and financial stability (FUNDS 2014). In assessing where to allocate energy and resources, the UN should fo- cus on areas where it has a comparative advantage. In addition to assigning resources based on relative strengths, the UN should also critically assess how its current programmes and funds fit with the 2030 Agenda for Sustainable Development, and make adjustments as necessary. There are a number of priorities on the agenda where the UN currently lacks expertise and has not allocated sufficient resources, such as urban design and sustainable cities, ocean health, and clean energy. New UN programs may need to be established, while others should be combined or done away with alto- gether. 94 ECOSOC reform As we saw in the first chapter, ECOSOC is the principal body for coordination, policy review, dialogue and recommendations on economic, social and envi- ronmental issues. Established by the UN Charter in 1946, ECOSOC coordi- nates the work of many UN agencies and bodies, and acts as a forum to discuss the issues falling within its broad mandate. The Council’s membership has 95 expanded over the years, from 18 in 1946 to the current 54 states, elected by the General Assembly for overlapping three-year terms. ECOSOC has received more than its fair share of criticism. Prominent dip- lomats, politicians, policymakers and researchers alike have called it a weak and irrelevant institution. Various reasons for this have been put forth, includ- ing: an overly expansive agenda that overlaps with that of the General Assem- bly; a lack of authority or “teeth”; a disregard for results and outcomes; and an unrealistic workload with too many bodies reporting to it. Critics point to a need for ECOSOC to be less bureaucratic, more action oriented and policy relevant. While many complain about its ineffectiveness and limited ability to exer- cise authority, there is little consensus around what to do about it. Indeed, some experts have argued that membership is too large and should be reduced, while others have called for a slightly expanded membership. Some feel that certain minor tweaks would do the trick, yet others make a compelling case for a com- plete overhaul of the institution. Ambitious proposals envision ECOSOC as a more powerful body, capable of making binding decisions rather than merely recommendations. Many observers support the idea of elevating ECOSOC’s status to that of the Security Council, which, despite its challenges with repre- sentation and fairness, is highlighted as a model in terms of its decision-making authority. Global economic policy coordination features in many reform pro- posals as well. In the socio-economic development space, the “institutional di- vision of labour” is such that the UN leads on peace and social concerns; the IMF on finance and macroeconomic management; the World Bank on devel- opment lending; and the WTO on trade. Certain proponents of a strengthened ECOSOC have argued that the Bretton Woods institutions and the WTO be made more democratic, reintegrated into the United Nations system and sub- jected to ECOSOC’s overall policy coordination. Under pressure, ECOSOC has attempted to revive itself through several re- forms over the years. ECOSOC reform The early 1990s saw several tweaks, including replacing biannual sessions with one more substantive annual session that had three seg- ments: a high-level segment, a coordination segment, and an operational seg- ment. Member states began sending higher level officials to attend, reflecting renewed interest in ECOSOC’s work. In 2006, the General Assembly adopted another resolution (61/16) on strengthening ECOSOC. It mandated ECOSOC to review progress made in implementing the outcomes of UN conferences and summits, including the MDGs. It also gave the Council a greater role in mon- itoring trends and progress in international development cooperation and of- fering policy guidance and options to improve effectiveness. In 2013, ECOSOC again underwent reforms, this time with the aim of mak- ing it a more issues-oriented body capable of overseeing implementation of the 2030 development agenda. Among other changes, the Council was mandated to provide greater leadership and guidance on substantive priorities by 96 convening a separate segment on integrating the economic, social and environ- mental dimensions of sustainable development. In the same vein, ECOSOC began hosting an annual partnership forum in 2008 to explore how multi-sector partnerships can advance global development, as well as an annual youth fo- rum in 2012 to promote youth engagement and the inclusion of key priorities for youth in drafting and implementing the development agenda. Relative to most reform proposals put forth by experts, these adjustments are fairly minor, and will lead to incremental rather than transformative change. Still, efforts to reform show a willingness on the part of the UN to respond to criticism. However, the absence of a broad consensus around fun- damental change demonstrates how complex the issues are. Is reform possible? Is reform possible? There is a general sense of optimism around the UN’s standing, with 86 per cent of programme country governments considering it to be a more relevant partner than two years ago (United Nations Secretariat 2015). And no matter how difficult reform may be, many also believe that it is possible. In the 2014 FUNDS survey of individuals from private sector, UN organizations or agen- cies, national governments/public sector, academia or NGOs who are familiar with the UN’s work, over three quarters said they felt that the UN development system was capable or strongly capable of significant reform. This is encour- aging, given the pressure on the UN to deliver on the ambitious 2030 develop- ment agenda. ECOSOC and all UN entities active in the development space still have much work to do to prove they are up for the task. 97 97 The fundamental paradox The protection and promotion of human rights is among the most controversial issues at the United Nations. In a world where states (as opposed to other levels of government or non-state actors) are the central unit, it is they that must pro- tect the rights of their citizens. Yet it is states that are the primary violators. Attempts by the international community to intervene in a given country in order to protect the rights of individuals or groups are often met with accusa- tions of interference with the principle of sovereignty and nonintervention. In- deed, the prevalence of state sovereignty as the guiding norm in international relations remains the greatest obstacle to realizing universal human rights. Human rights are intended to be universal legal guarantees protecting indi- viduals and groups against actions which interfere with fundamental freedoms and human dignity. They are considered to be inherent to all human beings, whatever our nationality, place of residence, sex, national or ethnic origin, col- our, religion, language, or any other status. The defining characteristics of hu- man rights are that they are: universal, internationally recognized, legally pro- tected, equal and non-discriminatory, inalienable, interdependent and indivisi- ble (see Box 12). Human rights featured prominently in the UN Charter. The Charter begins with a series of pledges: to save succeeding generations from the scourge of war, to maintain compliance with international law, to promote social progress and to “reaffirm faith in fundamental human rights, in the dignity and worth of the human person, in equal rights of men and women and of nations large and small”. This strong commitment to human rights and the dignity of each indi- vidual came after the devastating loss of tens of millions of lives and human abuses during the Second World War. But the notion that individuals have basic rights precedes the UN Charter, and even early international cooperation efforts. The UK Bill of Rights was adopted in 1689, the US Bill of Rights which came into effect in 1791, and France’s Declaration of the Rights of Man and of Citizen, adopted just after the revolution and establishment of the first French Republic in 1789, are early examples of human rights protection. Historians will trace the origins of human rights ever further back, beginning with the Cyrus Cylinder, an ancient record dating back to 539 B.C. Box 12: Key characteristics of human rights Universal – The principle of universality has become the cornerstone of international human rights law. It has been emphasized in numerous hu- man rights conventions, declarations, and resolutions, beginning with the Universal Declaration of Human Rights in 1948. All states have ratified at least one of the core human rights treaties and 80 per cent of states have ratified four or more. Interdependent and indivisible – Human rights can be grouped into civil and political rights, economic social and cultural rights, and collec- tive rights. Certain rights cannot be sacrificed in favour of other rights as all of these rights are interdependent, indivisible and interrelated. Inalienable – Rights should not be taken away, except in specific situa- tions determined by law and solely for the purpose of securing recogni- tion and respect for the rights of others and of meeting the just require- ments of the general welfare, morality and public order in a democratic society. Equal and non-discriminatory – Present in all the major human rights treaties, this principle prohibits discrimination on the basis of sex, race, nationality, place of residence, national or ethnic origin, colour, religion, language or any other status. The fundamental paradox The rights set out in national bills and declarations 98 pertained to civil and political rights and freedoms, and not to what we refer to today as economic, social and cultural rights. Box 12: Key characteristics of human rights Universal – The principle of universality has become the cornerstone of international human rights law. It has been emphasized in numerous hu- man rights conventions, declarations, and resolutions, beginning with the Universal Declaration of Human Rights in 1948. All states have ratified at least one of the core human rights treaties and 80 per cent of states have ratified four or more. Interdependent and indivisible – Human rights can be grouped into civil and political rights, economic social and cultural rights, and collec- tive rights. Certain rights cannot be sacrificed in favour of other rights as all of these rights are interdependent, indivisible and interrelated. Inalienable – Rights should not be taken away, except in specific situa- tions determined by law and solely for the purpose of securing recogni- tion and respect for the rights of others and of meeting the just require- ments of the general welfare, morality and public order in a democratic society. Equal and non-discriminatory – Present in all the major human rights treaties, this principle prohibits discrimination on the basis of sex, race, nationality, place of residence, national or ethnic origin, colour, religion, language or any other status. International human rights law A series of human rights treaties, conventions and declarations adopted since 1945 have given legal form to inherent human rights. Together, these contrib- ute to a strong body of international human rights law. The most significant thus far has been the Universal Declaration of Human Rights, adopted by the General Assembly on December 10th 1948 (which has since been designated Human Rights Day). It was the first international attempt to explicitly spell out basic human rights for all individuals, as the UN Charter did not in fact define human rights. The rights covered can be categorized into various categories: civil, political, economic, social and cultural rights (see Box 13). The Declara- tion is regarded today as having established the fundamental norms of human rights that we, individually and collectively, should respect and protect. In 1966, the International Covenant on Civil and Political Rights (along with its two Optional Protocols) and the International Covenant on Economic, So- cial and Cultural Rights were adopted by the General Assembly, though they 99 only came into force ten years later, in 1976. The two Covenants were in fact completed in 1956 but due to disagreements over definitions, extent and mean- ing as well as concerns over potential limitations on state sovereignty, it took ten years before they were formally adopted. Even today, not all states have ratified these treaties, including great powers—China is not party to the former, while the US still refuses to become party to the latter. The two Covenants along with the Universal Declaration of Human Rights together form the In- ternational Bill of Human Rights. By becoming parties to these and other in- ternational treaties, states are, in principle, legally obligated to respect, protect and promote human rights. This means that states cannot interfere with the enjoyment of human rights, they must protect individuals and groups against abuses, and they must take positive action to facilitate the enjoyment of basic rights. Box 13: Categories of human rights Civil rights – Both civil and political rights protect individuals’ freedom from infringement by governments, social organizations, and private in- dividuals. Civil rights include life, liberty and security; privacy; protec- tion from discrimination; and freedoms of thought, speech, religion, press, assembly and movement. Political rights – Rights of individuals to participate in politics and civil society such as freedom of association, the right to assemble and the right to vote. Political rights also include procedural fairness in law, including right to a fair trial, due process and legal remedy. Social, economic and cultural rights – Rights to adequate food, ade- quate housing, education, health, social security, water and sanitation, work, enjoy one’s culture, practice of religion and use of one’s language. There are a total of nine core international human rights instruments. These include the two Covenants discussed above, and an additional seven conven- tions regarding racial discrimination, discrimination against women, the rights of the child, the rights of persons with disabilities, the rights of migrant work- ers, enforced disappearance and torture. Beyond the International Bill of Rights and the core human rights treaties, the General Assembly has adopted over 100 other international treaties and declarations addressing a host of is- sues such as indigenous peoples, marriage, slavery and war crimes. A number of other human rights instruments exist at the regional and national level, which serve to compliment international norms. At the regional level, treaties and other instruments are adopted to reflect human rights concerns specific to a particular region and provide special protection. For instance, the African Charter on Human and Peoples’ Rights was strongly inspired by the Universal 100 Declaration of Human Rights and international treaties, but also demonstrates the African conception of “rights” and the emphasis on individuals’ responsi- bilities. At the national level, when a government ratifies international human rights treaties, it commits to develop national legislation compatible with its treaty obligations and duties. Typically states will do so through adopting con- stitutions and other laws that formally protect human rights. Contradictory interpretations Despite universal human rights norms elaborated in key treaties as well as in the Universal Declaration of Human Rights, a single and consistent interpreta- tion of these rights remains a distant aspiration. There has traditionally been a North-South divide over the prominence given to various groups of human rights. Western, democratic countries have long stressed the importance of civil and political rights, which deal with lib- erty and participation in civil and political life. These are sometimes referred to as “negative” rights as the state must refrain from any action that would interfere with individuals’ rights and freedoms. Conversely, many countries in the global South have pushed more for social, economic and cultural rights, which are considered “positive” rights as they require the state or other public authority to take positive steps to ensure access to basics such as food, shelter and education. Since negative rights came first, they are called “first-genera- tion”, while positive rights which followed afterward are called “second-gen- eration”. A third generation later emerged, which focuses on collective or group rights rather than individual ones. The right to self-determination and the protection of groups of persons, such as indigenous peoples, are examples of these “solidarity” rights. In many countries, particularly in Africa and Asia, the emphasis of collective rights over individual rights is reflective of ideolo- gies and values that historically do not regard the individual as the central unit. States often perceive a trade-off between these different human rights, ar- guing that certain rights need to be pursued immediately while others can re- main on the back burner until the timing is right. Developing countries have argued that the right to development should take priority over other human rights, and that they should be able to neglect certain human rights, namely political rights, as they catch up to the West. Yet this, of course, runs counter to the core human rights principle of interdependence. Stressing the indivisi- bility of all human rights, the UN High Commissioner on Human Rights re- cently stated “If States pick and choose which rights they will uphold, the en- tire structure is undermined.” (UN OHCHR 2016). Cultural relativists have long challenged the notion of universality, asserting that it is a means for the West to impose its values and norms on other coun- tries. Contradictory interpretations While there is a strong case for cultural relativism and the usefulness of 101 considering contextual factors, relativists have seen their cause seized upon by those seeking to justify violations. The latest variant of this has been the em- phasis by many countries on the importance of “traditional values”. In 2012, the UN Human Rights Council adopted a resolution on “promoting human rights and fundamental freedoms through a better understanding of traditional values of humankind”. While it sounds benign, or perhaps even productive, customs and traditions are often cited as justifications by human rights abusers. Consider the practices of child and forced marriages, female genital mutilation, so-called “honour killings”, and marital rape that persist in many countries to- day. Or the countless lesbian, gay, bisexual and transgender (LGBT) people globally who are denied the same rights that others in society enjoy. Traditions have not always been kind to everyone, and in many cases they have indeed impeded people’s rights. The 2012 resolution, which was preceded by two others in 2009 and 2011 requesting further study of traditional values, set a dangerous precedent by cre- ating a potential means through which countries can justify discrimination, harmful practices and other rights violations. Spearheaded by Russia, it had the support of a number of known human rights violators, such as China, Libya and Uganda. Meanwhile those that voted against were mostly Western coun- tries with relatively good human rights records. Former Secretary-General Ban Ki-moon expressed disapproval with this trend on several occasions. At the 2013 International Conference on Human Rights, Sexual Orientation and Gen- der Identity he stated “I respect culture, tradition and religion—but they can never justify the denial of basic rights.” (United Nations 2013). These varying perspectives reveal that despite a supposedly universal un- derstanding of human rights, what we witness in practice is continuous debate and negotiation around how basic rights are defended. The tremendous cost of violations Despite a robust legal and normative framework surrounding human rights, there are numerous cases of human rights violations around the world each and every day. Individuals and groups are denied basic rights to which they are entitled, often by states that have signed and/or ratified human rights treaties. The current situation in Syria is a particularly appalling. The government has carried out indiscriminate airstrikes in which it has bombed markets, schools, hospitals and clinics, killing thousands of civilians (UNHRC 2017). It has detained people arbitrarily, including many activists, human rights defend- ers, journalists and humanitarian workers. In early 2017, Amnesty Interna- tional exposed the Syrian government’s secret campaign of mass hangings and extermination, in which as many as 13,000 people, most of whom were civil- ians believed to be opposed to the government, were hanged at Saydnaya 102 Prison between 2011 and 2015 (Amnesty International 2017). The Syrian gov- ernment is believed to be responsible for killing scores of civilians in numerous chemical attacks against its own people, including those in August 2013 and April 2017 which attracted international attention and condemnation. Not sur- prisingly, Syria continues to refuse to grant the UN access. This case is far from isolated. When the UN tries to investigate alleged abuses, states are often uncooperative and claim interference with sovereignty. In his opening remarks at the Human Rights Council session in September 2016, the UN High Commissioner on Human Rights, Zeid Ra’ah Al Hussein, expressed grave concern over attempts by countries to block human rights scrutiny. He stated that “monitoring activities, and advocacy intended to help better protect the people of your countries, are refuted as somehow violating the principle of state sovereignty—or even the UN Charter”, adding that cred- ible statements by the Office of the High Commissioner for Human Rights are often dismissed as ‘biased’, ‘irresponsible’, ‘misleading’ or based on ‘false’ premises by countries. A number of countries were singled out for preventing the UN from conducting independent and impartial investigations into alleged abuses, including Syria, Venezuela, Turkey, Ethiopia, Mozambique, Gambia, India, China, Nepal, Iran and the United States. Unfortunately, this lack of willingness by many states to cooperate with UN human rights observers is correlated with a large number of violations globally. Some examples of human rights issues in the world today are outlined in Box 14. The tremendous cost of violations It would be wrong to assume that human rights violations only occur in non- Western countries, particularly undemocratic regimes. The example of the United States’ use of torture on prisoners illustrates that abuses can certainly occur in Western, liberal democracies. Following the 9/11 attacks and in- creased concern with the threat of terrorism, the United States used “stress and duress” interrogation techniques on prisoners in Afghanistan and in Guantá- namo Bay, Cuba, and subjected detainees in Iraq to torture and cruel treatment. The United States Senate report on the CIA Detention and Interrogation Pro- gram released in 2014 concluded that “the program caused immeasurable dam- age to the United States’ public standing, as well as to the United States’ longstanding global leadership on human rights in general and the prevention of torture in particular.” (Senate Select Committee on Intelligence, 16). The 2015 Universal Periodic Review of the US’ human rights record similarly sug- gested tremendous room for improvement. 103 103 Box 14: Select examples of current human rights issues Box 14: Select examples of current human rights issues Lesbian, Gay, Bisexual and Transgender (LGBT) rights – There are 76 countries around the world (mostly in Africa and Asia) that have anti- gay laws, a clear violation of basic human rights. A number of these are passing or attempting to pass even more draconian bills. In 2013, an Anti- Homosexuality Act was passed in Uganda that broadened the criminali- zation of same-sex relations including life imprisonment (the original bill proposed the death penalty). Meanwhile Indonesia, where homosexuality is not illegal, made international headlines in early 2016 after a series of attacks against members of the LGBT community, including by promi- nent public figures. Freedom of religion – Sadly, religious minorities are often the victims of violent attacks. The Muslim minority in the majority Buddhist Burma and Hindu and Christian minorities in the predominantly Muslim Pakis- tan are but a couple of examples from a long list of cases where indivi- duals and groups have come under attack based on their religion. In Saudi Arabia, conversion from Islam to another religion is punishable by death and the government provides no legal recognition or protection for free- dom of religion. Freedom of speech and press – Too many countries today are guilty of denying people basic freedoms of expression. The United Nations’ Record in Upholding Human Rights The United Nations’ Record in Upholding Human Rights The organizational structure of UN organs, offices, agencies, treaty bodies and committees charged with ensuring that states apply human rights norms is complex and dizzying. Though not an exhaustive list, some of the main ones are: the Security Council, the General Assembly, the Human Rights Council, the Office of the High Commissioner for Human Rights, the International Court of Justice, the International Criminal Court, International Criminal Tri- bunals, treaty bodies and various committees on specific areas of concern such as the rights of the child, torture and discrimination against women. g g An assessment of the overall impact of the UN’s human rights work reveals that it is particularly effective at promoting and developing human rights, but is failing to adequately protect those rights when countries opt not to respect their obligations. This stems from a reliance on political and diplomatic pro- cesses as the means to achieving its objectives. Such an approach works for offering resources and technical assistance to countries, which in its very na- ture is non-confrontational, but has posed significant challenges when inter- vention is required to protect individuals from abuses (Freedman 2015: 54). Politicization is a defining feature across the human rights machinery; one that has hindered its success for decades. Selectivity and bias are widespread, and national interests consistently triumph over principled action. Diplomacy fa- vours dialogue and cooperation, which rarely produces results where grave vi- olations have occurred. An utter lack of enforcement mechanisms across UN human rights bodies has worsened the problem. Unlike domestic laws, inter- national laws are often not enforceable. The international community can apply pressure to uncooperative states through tools such as sanctions or military in- vasions, but aside from these extreme measures and simple diplomatic pres- sure, sovereign states cannot be obliged to do anything. The following section examines the complex system of human rights bodies at the UN, including their strengths and weaknesses and proposals for reform. It focuses on the bodies exclusively concerned with human rights and human- itarian affairs, though it is worth noting that much of the work of the General Assembly and Security Council centres on human rights issues. Through set- ting standards and passing resolutions to condemn or draw attention to human rights violations, the General Assembly’s role in advancing human rights is mostly an indirect one. The tremendous cost of violations Security and intelligence forces in Iran have arrested journalists, bloggers and social media acti- vists, who were given heavy sentences. The Chinese government notori- ously censors all forms of media, communication and education, inclu- ding the internet, television, film, literature and video games. Russia has also suffered a worsened reputation recently for intensifying its harass- ment and persecution of critics and activists. Use of torture, ill-treatment of detainees and enforced disappear- ances – The United States’ detention facilities at Guantánamo Bay in Cuba came under intense fire for human rights abuses including the use of torture. In its effort to combat organized crime, Mexican security forces have been involved in countless violations including extrajudicial killings, enforced disappearances and torture. Among numerous other a- buses, Egyptian officials have in recent years committed torture and en- forced disappearances, and mistreatment of detainees have led to many deaths. 104 From the Commission on Human Rights to the Human Rights Council From the Commission on Human Rights to the Human Rights Council When the Human Rights Council replaced the Commission on Human Rights in 2006, observers hoped that it would not suffer from the same defects as did its futile predecessor. Established in 1946 as the UN’s principal mechanism and forum for the promotion and protection of human rights, the Commission had a 70-year run marked by a few early successes followed by countless chal- lenges. During its first 20 years it successfully established the international le- gal framework outlined earlier in this chapter. It was also responsible for the many conventions adopted over the years. Having succeeded in setting norms and standards for states to follow, it shifted its attention in the late 1960s to monitoring and implementing human rights. This meant going beyond simply elaborating treaties to investigating, reporting on, and condemning violators. This new focus quickly turned politi- cal and states viewed it as infringing on the principle of sovereignty. It soon exposed the inherent contradiction between the political nature of the Commis- sion (it was, after all, a body made up of member states that represented their own national interests) and its mandate to act for the principled protection of human rights. A main criticism of the Commission was that its membership included known human rights abusers. The complete absence of membership criteria beyond geographic composition meant that not only did the body not select members based on an exemplary human rights record, but nor did it exclude states with appalling records. At the time of the Commission’s collapse, Sudan enjoyed membership despite the genocide taking place in Darfur, as did Zim- babwe while the Mugabe government pursued forced evictions and demoli- tions that left 700,000 people homeless and created a “humanitarian crisis of immense proportions” (Hoge 2005). This had been an issue for years but wors- ened in the early 2000s. The straw that broke the camel’s back and ultimately led to the demise of the vexed human rights body was the election of Muammar Gaddafi, the Libyan leader whom many labelled as a dictator and tyrant, as chair of the Commission in 2003. Over time it became clear that human rights abusers were actually seeking membership on the Commission as a means of protecting themselves from closer examination. And since violators were less likely to condemn other violators, their presence further threatened the body’s credibility. The United Nations’ Record in Upholding Human Rights The Security Council plays a more central role, holding the authority to reach a binding “decision” that a situation is a threat to or breach of international peace and security. It has occasionally done so during particularly serious human rights abuses. We do not provide a detailed account of how these two UN organs have defended or failed to defend human rights. Suffice it to say, they have been plagued by some of the same challenges as the UN’s principal human rights bodies explored below, namely the primacy of power and politics. Meanwhile, innovations in the human rights field such 105 as the Responsibility to Protect norm and the International Criminal Court have emerged largely to provide limits to sovereignty, which continues to dominate both the General Assembly and the Security Council. From the Commission on Human Rights to the Human Rights Council The political nature of the Commission led to marked regionalism and group-blocking action. Regional alliances that were particularly visible were 106 the G77, the Non-Aligned Movement, and the Organization of the Islamic Co- operation. Members were frequently encouraged by regional allies to vote against resolutions about grave violations in order to protect group members. In a flagrant show of contempt for the Bush administration, the Muslim-Afri- can bloc voted the United States off the Commission in 2001, while countries like Sudan and Pakistan were elected as members (Freedman 2011: 35). In many instances, regional alliances and friendships overrode misconduct be- cause membership is decided by votes from the world’s regions. Country-specific resolutions were the primary mechanism for addressing human rights abuses in countries. They became highly political weapons used by states to shield allies from scrutiny as well as to disproportionately lambast opponents. The United States would frequently focus on human rights viola- tions in Cuba but stay silent on more serious abuses in allied states like Guate- mala and El Salvador. During the Cold War, the Soviet Union similarly used human rights as a political tool when it criticized rights violations in Chile un- der Pinochet (an ally of the US), while ignoring the grave human rights situa- tion in communist states (Weiss et al. 2014: 211). Too often, states’ actions revealed political motivations and obvious double standards. The Commission was ultimately scrapped and replaced with the Human Rights Council following former Secretary General Kofi Annan’s recommen- dation in a 2005 report. It was believed that the challenges with politicization and selectivity were so great that a complete overhaul was the only option for reform. But the ensuing discussions around what a more effective human rights body would look like proved more difficult and divisive than expected. First, there was no consensus on the optimal size of the Council. Annan suggested limiting membership to 15, while others wanted it to be universal. Nor was there agreement over what, if any, membership criteria should be established. Many developing and non-democratic nations already saw human rights as a Western tool being used against them, so the idea of excluding violators and undemocratic states would have only acted to reinforce this belief. And creat- ing an exclusive club of likeminded, Western countries would have likely led to further polarization between the two groups. From the Commission on Human Rights to the Human Rights Council As well, given the variation in states’ understanding of human rights that was explored earlier in this chapter, it would have been near impossible to settle on any common definition of a ‘tolerable’ human rights record to begin with. As a way to promote the primacy of human rights over politics, the notion of having human rights experts rather than government representatives on the Council was discussed. However, many states were not willing to concede power and see the new body become an expert rather than political one. In the end, after a great deal of deliberation, the newly established Human Rights Council did not differ greatly in architecture, function or composition from the ineffective Commission: it has 47 rather than 53 members, no mem- bership criteria is used, and representatives are government officials concerned 107 with national interests. It was not identical to the Commission and introduced some new features explored below, but it was unable to tackle the heart of the issue. It is therefore little surprise that the Council has fallen short of expecta- tions. The Council did not get off to a good start. During its first year in 2006, China, Russia, Egypt, Cuba and others were elected as “champions”. The US decided not to even run, but rather to be an observer, as a means of protesting the UN’s inability to address the flaws of the Commission, especially the mem- bership of human rights violators. After two years it withdrew altogether (though under Obama the US reversed this decision of the Bush administration, running and getting a seat in 2009). Politics were omnipresent. The first session saw Israel condemned nine times, yet no other country. Over the Council’s first four years, six of the 12 special sessions that members held were focused on Israel. During the same period, the human rights situations in Myanmar, Dar- fur, the Democratic Republic of Congo and Sri Lanka were the subject of a single session each. There is little doubt that the Council has maintained the Commission’s strong bias against Israel. And while later sessions centred on Syria and other countries, it is still too early to proclaim a permanent shift. The 2016 Human Rights Council election saw several countries with less than stellar human rights records elected or re-elected as members, including China, Egypt, Cuba and Saudi Arabia. From the Commission on Human Rights to the Human Rights Council This points to the continued need for some minimal criteria for membership beyond the current voluntary pledge stating that the country will uphold international standards of human rights and listing their actions in advancing and protecting human rights. In the absence of strict guidelines, the international community may in some cases decide it- self that a country is not worthy of a seat on the council. We witnessed this in October 2016 when Russia was defeated in its bid for re-election. But this is only possible when a regional group puts forward more candidates than the number of seats available, as was the case in Eastern Europe where Russia, Croatia and Hungary competed for two seats. There was no such competition for Saudi Arabia, a country that had been indiscriminately bombing civilians in Yemen during the 18 months prior to the vote. Given that the Asia group had put forward just four candidates—Saudi Arabia, China, Iraq, and Japan— for four seats, it came as little surprise when they all won. Despite the prede- termined results, Abdullah bin Yahya Al-Moallami, the Saudi Ambassador to the UN, still claimed that “the re-election of Saudi Arabia reflects the interna- tional community’s trust in the pioneering and leading role of the Kingdom in the Human Rights Council.” (Saudi Press Agency 2016). This potential to by- pass a competitive process needs to be addressed for the Council to maintain its credibility. The Council has enjoyed some achievements, alongside criticisms. Not un- like the Commission, its greatest strengths lie in setting international standards and norms, raising awareness on human rights issues, and convening actors at 108 global forums. It has fared even better than its predecessor when it comes to considering the human rights situations in specific countries, particularly through the Universal Periodic Review mechanism that emerged from the re- form process. Each year the human rights situation in 42 countries is reviewed, meaning every country gets its turn every four and a half years. The process requires countries to submit a 20-page report on their efforts to improve human rights and overcome any challenges to the enjoyment of these rights. To reduce bias, two supplementary reports are submitted alongside the “national report”. From the Commission on Human Rights to the Human Rights Council The first contains the views of Special Procedures (i.e., independent human rights experts), human rights treaty bodies, and other UN entities, while the second includes information provided by NGOs and national human rights in- stitutions. Discussions are held and an outcome document is drafted with rec- ommendations for states. Once adopted, states are expected to confirm which recommendations they accept. It is fairly rare that states reject recommenda- tions, with the acceptance rate at 70-90 per cent (Ramcharan 2011). Whether countries implement the recommendations remains to be seen, but it is encour- aging that the Universal Periodic Review has been fairly well received by states. Its universal nature has meant that all countries are treated equally, while the emphasis on dialogue over the naming and shaming has reduced impres- sions of being targeted or singled out. Diagnosing a problem can be infinitely easier than developing a good solu- tion. Despite this, there is no shortage of proposals for improving the flawed human rights body. A number of workable ideas are outlined in the final chap- ter. But reforming the Human Rights Council will certainly not be straightfor- ward, especially if the reform process of the Human Rights Commission is any indication. The Secretary-General and the High Commissioner for Human Rights Not all parts of the UN human rights apparatus are as political as the Security Council, General Assembly and Human Rights Council. At the opposite ex- treme are the UN treaty-based bodies, which are the least politicized, biased and selective. These bodies are comprised of experts rather than government officials and do not have jurisdiction over states that are not party to the rele- vant treaties. They serve to monitor the implementation of key conventions and protocols. There are 10 such bodies, including the Human Rights Committee (monitoring civil and political rights), Committee on Economic, Social and Cultural Rights, Committee on the Elimination of Discrimination against Women and the Committee against Torture. Somewhere in the middle are the secretary-general and the High Commissioner for Human Rights. Both roles require a careful balance of impartial human rights advocacy and political sen- sitivity. Failure to advocate for principled human rights protection would result 109 in criticism from human rights NGOs and commentators, while neglecting pol- itics would lead to a loss of support of member states. There has been tremendous variation in the degree of outspokenness of past secretaries-general. From the Commission on Human Rights to the Human Rights Council Typically the world’s top diplomats have prioritized pro- gress on peace and security over human rights, presumably to reduce the risk of offending states by singling them out as violators. Kofi Annan, a vocal hu- man rights defender, was a notable exception. He used his position as a means to push states to behave better, albeit he did so cautiously. His successor Ban Ki-moon spoke out about some grave violations, but was fairly non-confron- tational and would usually defer public statements until major powers had taken stances (Weiss et al. 2014: 204). The job of the High Commissioner for Human Rights is particularly de- manding. The General Assembly created the post in 1993 following a success- ful lobbying campaign by several actors including many NGOs, the Carter Presidential Centre and the US government (ibid: 207). Earlier that year, the UN Vienna conference on human rights had also recommended it be created. Unlike with the secretary general who juggles multiple priorities and can choose not to make human rights a prime concern, the high commissioner en- joys no such luxury. Ruffling some feathers is effectively in the job description for this key human rights post. This sheds light on why so many who have assumed the position have been harshly criticized, even to the point of having to step down. This was the fate of the former president of Ireland Mary Rob- inson who was nominated as high commissioner for human rights by Secre- tary-General Annan. After Robinson drew much public attention to human rights violations in China and Israel, the US, which had close relations to these states and preferred quiet diplomacy to public condemnation, grew disillu- sioned with her and pressured her to resign in 2002. Louise Arbour, who as- sumed the post in 2004, also became disliked by Washington over time, while her successor Navanethem (Navi) Pillay upset many governments by high- lighting violations against LGBT people and by focusing on specific abuses in Rwanda, Sri Lanka and Syria (ibid: 208-9). Since accepting the role in 2014, Prince Zeid Ra’ad Al Hussein of Jordan denounced violations in many states. Prior to Donald Trump’s election in the US, Zeid called him “dangerous”—a rare move for his office and one that suggests further clashes with Washington are to come (Cumming-Bruce 2016). Dealing with the worst violations: the International Criminal Court and ad hoc tribunals Dealing with the worst violations: the International Criminal Court and ad hoc tribunals Crimes against humanity, war crimes and genocide represent the most heinous human rights abuses. What distinguishes these ‘international crimes’ from other violations of human rights is that they are widespread or systematic 110 attacks knowingly committed against a civilian population. Crimes against hu- manity can include murder, extermination, enslavement, deportation, torture, apartheid, imprisonment, enforced disappearances, and other offences if they are committed on a large scale against a population that is predominantly ci- vilian. Unlike war crimes, they can take place either during a time of peace or of war. As their name would suggest, war crimes can only occur during con- flict, and are violations of international humanitarian law or the law of armed conflict. Many of the offences are similar to those categorized as crimes against humanity, though it also covers areas like hostages and prisoners of war. Gen- ocide occurs when an intentional attack is carried out to destroy a national, ethnic, racial or religious group (United States Institute of Peace). International criminal courts and tribunals have been established to prose- cute individuals who, in committing these grave crimes, violate international criminal law. The Tokyo and Nuremberg trials were the first tribunals created to prosecute and punish those guilty of international crimes. After the Second World War the world was appalled by the atrocities that had taken place. Mil- itary commanders and political leaders in Nazi Germany were guilty of crimes against humanity, war crimes and genocide. Since there was no body of inter- national criminal law at the time, it was effectively ‘invented’ and ‘applied retroactively’ to ensure the crimes committed did not go unpunished (Freed- man 2015: 29). International criminal law later became codified and incorpo- rated in international, regional and national law. A number of international criminal tribunals and special courts have since been set up after wars and mass killings to bring justice to victims and deter others from committing such crimes. Criminals were prosecuted for genocide and crimes against humanity through tribunals in Rwanda and the former Yugoslavia, while special courts in Sierra Leone, Lebanon, Cambodia and East Timor dealt with serious viola- tions in those countries. The ad hoc nature of these tribunals and courts was the impetus for the even- tual creation of a permanent court, the International Criminal Court (ICC). Dealing with the worst violations: the International Criminal Court and ad hoc tribunals Based in the Hague, the Netherlands, the ICC is the first-ever permanent, treaty-based court of its kind that hears cases against individuals (the Interna- tional Court of Justice is also a permanent, treaty-based international court, but only deals with disputes between states). It cooperates with the United Nations while remaining formally independent from it. The ICC was born through the Rome Statute adopted in 1998, but only began work formally after the Statute had entered into force in 2002. It has 18 judges elected by states parties for a term limited to nine years, no two from the same country. The ICC does not have universal jurisdiction to investigate any situation it pleases; rather, states decide to be bound by the Court’s rules by signing and ratifying the Rome Statute. For countries that are party to the Rome Statute, situations may only be brought to its attention when national courts are unwill- ing or unable to prosecute individuals. As such, the Court was designed to 111 complement existing national judicial systems. Crimes in non-party states that remain outside of the Court’s jurisdiction can still be investigated if they are referred to the ICC by the Security Council. Similar to the courts and tribunals that came before it, a main objective of the ICC is deterrence—stopping leaders from believing they can get away with heinous crimes. g y g y Since its inaugural session in 2003, the ICC has opened ten investigations and publicly indicted 39 individuals, including Ugandan rebel leader Joseph Kony, Sudanese president Omar al-Bashir and Libyan leader Muammar Gad- dafi. Its first judgement was issued in 2012 when it found Thomas Lubanga, Congolese rebel leader, guilty of war crimes related to child soldiers. Several others have been found guilty since. Aside from the most recently opened in- vestigation in Georgia, every case investigated has been in Africa, and all 39 people indicted have been Africans. This prompted a perception among Afri- can countries in particular that the court is biased against them. In October 2016, three African countries—Burundi, Gambia, and South Africa—an- nounced that they would withdraw from the ICC, becoming the first countries to do so. In explaining his country’s decision to leave the Court, Gambia’s Information Minister Sheriff Bojang described the global judicial body as “an International Caucasian Court for the persecution and humiliation of people of color, especially Africans” (Sieff 2016). Dealing with the worst violations: the International Criminal Court and ad hoc tribunals Many observers fear the decisions to leave will spark a mass exodus of African countries. Already Kenya and Na- mibia’s parliaments have voted to withdraw, while Uganda’s president has harshly criticized the Court, even calling it “useless” (Gaffey 2016). Whether the ICC is biased against Africa has been the focus of much debate. The challenge with such a claim is that many of the cases that have been in- vestigated were referred by African governments themselves—34 of which are members of the ICC. The situations in Sudan and Libya, countries that are not party to the ICC, were referred by the Security Council. So when the Court is accused of discriminating against Africa for not investigating situations in other countries, it is a criticism that should in fact be directed toward the Se- curity Council. The ICC Prosecutor did cause an upset when on his own au- thority he decided to investigate post-election violence in Kenya, effectively challenging the Kenyan government which objected to the decision. However, this case was the exception rather than the rule. Electing an African Prosecutor, Fatou Bensouda of Gambia, in 2011 to replace the Luis Moreno Ocampo of Argentina helped combat anti-Africa perceptions to some degree. And looking at the preliminary examinations currently underway (in the pre-investigation phase), many are in countries outside Africa, including in Afghanistan, Colom- bia, Iraq/UK, Palestine and Ukraine. Since its inception, the much-needed human rights institution has struggled with the consequences of states being allowed to choose whether to opt into the ICC or not, as many have chosen not to. Before any country had announced it would withdraw, 124 states were parties to the ICC. If a state decides not to 112 be bound by the Court’s rules, its citizens can avoid arrest and prosecution simply by remaining in their country or not stepping into a country that is party to it. The US, which has been accused of committing war crimes at an Iraqi prison in 2003, has not sought membership in the Court (and with the US hold- ing veto power, the Security Council will not refer this case to the ICC). Nei- ther has Sudan, where the ICC has issued arrest warrants for those allegedly responsible for the genocide in Darfur, including the Sudanese president Omar al-Bashir. Responsibility to Protect and human security Responsibility to Protect and human security The Responsibility to Protect, or R2P, norm was a similarly promising inno- vation in human rights protection. As with the ICC, it was developed in the early 2000s to overcome the principles of non-intervention and sovereignty in the face of mass atrocities. The idea behind R2P was simple. Sovereignty is not a one-way street that leaders can use with impunity. Alongside a state’s right to sovereign independence comes the duty and responsibility to protect its citizens from gross violations. If a state proves unable or unwilling to do so, or if the state itself is the perpetrator, the principle of non-intervention yields to the international responsibility to protect. It came about as an alternative to the hotly-debated “humanitarian intervention”. In the 1990s, two devastating events—the genocide in Rwanda and war crimes committed during the Kosovo War—had demonstrated the deadly consequences of the UN Security Coun- cil’s failure to authorize interventions in order to protect civilians. States rec- ognized that the controversial “right to intervene” centred on coercive military intervention needed to be replaced by a more nuanced “responsibility to pro- tect” that framed military action as a last-resort measure and favoured preven- tion over intervention. The R2P norm was first articulated in the 2001 report of the International Commission on Intervention and State Sovereignty (ICISS), The Responsibil- ity to Protect, financed by Canada for the UN. The report outlined three spe- cific responsibilities of the international community: to prevent, to react and to rebuild. State sovereignty was framed as contingent on meeting certain obliga- tions rather than an absolute right, and the notion of ‘human security’ was pri- oritized over state security. Fortunately the report’s fate differed from that of many other international committee reports that largely go unread. Instead, the norm was widely acknowledged and quickly gained traction. It was endorsed by the 2005 UN World Summit and included in Kofi Annan’s report In Larger Freedom. The issue of ensuring ‘right intention’ was recognized from the outset. The ICISS report noted it as a precautionary principle, stating, “The primary pur- pose of the intervention, whatever other motives intervening states may have, must be to halt or avert human suffering.” The report went on to propose spe- cific criteria which must be met to justify intervention but these have never been adopted by the Security Council. Dealing with the worst violations: the International Criminal Court and ad hoc tribunals Since the country is not party to the ICC, the individuals can only be arrested if they enter a country that is. Even then, the ICC must rely on that country to make arrests as it cannot arrest individuals itself. However when Bashir flew to South Africa in 2015 for a summit, the South African govern- ment declined to arrest him. The dispute escalated politically, ultimately lead- ing to South Africa’s decision to leave the ICC, a surprise move given it had been a vocal supporter of the Court in its early days. This series of events served as a reminder that even if states join an international organization and agree to be bound by its rules, they can only be encouraged, not forced, to fulfil their commitments. In addition to the US, several other influential countries have decided not to join including great powers Russia and China. Russia had signed but not rati- fied the ICC’s statute, but in November 2016, a day after the Court classified the Russian annexation of Crimea as an occupation, it formally withdrew its signature. Though significant, the move was purely symbolic, given Russia was not subject to the Court’s jurisdiction to begin with. The combination of powerful countries deciding not to join and perhaps more importantly the series of recent departures from the Court have caused real harm to the institution as a body that derives its legitimacy from members’ willingness to be bound by its jurisdiction. j When the ICC was established, there was a strong sense of hope among NGOs and UN officials that it would effectively challenge state sovereignty and international politics and put human rights first. Human Rights Watch ex- claimed at the time, "The International Criminal Court is potentially the most important human rights institution created in 50 years. It will be the court where the Saddams, Pol Pots and Pinochets of the future are held to account.” (Human Rights Watch 2002). Sadly, what we have witnessed instead is that state power and politics have prevailed over attempts to prosecute the worst human rights violators, even where mechanisms to seek justice exist under in- ternational law. Until this is addressed, the ICC will remain a flawed institu- tion. 113 113 Responsibility to Protect and human security Thus R2P critics often note the uneven application of the principle, pointing to cases where great powers have used R2P as an excuse to intervene when it has been in their interest to do so, while ignoring grave situations where they stood to gain little or nothing. Commonly cited examples of abuses of the norm include: the US and UK in Iraq in 2003 where R2P was a post-hoc justification for the war after original claims of Al- Qaeda links and weapons of mass destruction fizzled out; Russia in Georgia in 2008 which was inspired by imperial aims more than protecting human rights; 114 and France’s intervention in Burma in 2008 after a hurricane hit and the local government was slow to react (Weiss et al. 2014: 29). Recent events in the Middle East have reaffirmed that the decision to protect and assist vulnerable individuals ultimately rests on political will and military capacity. In 2011, the Security Council was swift to authorize military inter- vention in Libya to protect the population from the ongoing atrocities. Mean- while it has utterly failed to do the same in Syria, where the moral justification for intervention has been at least as strong as it was in Libya at the time (many would argue it has in fact been stronger). A host of factors explain the paralysis over action in Syria: the military challenge was tougher, potential costs of co- ercion were believed to exceed the benefits, and the geopolitics differed greatly with both Russia and China exercising veto power to prevent action and ex- press their general opposition to foreign intervention (Weiss 2014b). The intervention in Libya was initially lauded as a successful instance of R2P application. As explained in Chapter 1, a problem arose when the US, UK and France went beyond the Security Council’s agreed-upon mandate of lim- ited civilian protection to helping usher in a regime change. This move upset the BRICS states (Brazil, Russia, India, China and South Africa) in particular and contributed to states’ unwillingness to respond to the Syrian crisis soon after. It also prompted Brazil to table the “Responsibility while Protecting” (RwP) proposal, which stresses that military action must be pursued as an ab- solute last resort, and that implementation should be subject to close monitor- ing and regular review (Evans 2015). Responsibility to Protect and human security Edward Luck, the UN’s first-ever Special Advisor on the Responsibility to Protect (2008-12), reiterated these concerns while reflecting on R2P’s first ten years. Despite the wide acceptance of the prevention and protection principles, he stated that questions remain for states on implementation. He noted unfin- ished business with regard to operational practice, accepting individual respon- sibility, and developing a more nuanced understanding of sovereignty. Echo- ing Brazil’s RwP proposal, he stressed the need for exercising greater respon- sibility before and after protecting. According to Luck, a key challenge stems from the 2005 Summit’s failure to specify who is responsible for protecting, as individual responsibility can get lost in collective responsibility. “The chal- lenge now is highly political and highly practical. We need to convince na- tional policymakers: (1) that curbing atrocities serves their broader foreign pol- icy and security objectives; and (2) that we know how to do prevention and protection in a cost-effective and sustainable manner.” (2015: 503). He makes a useful distinction between territorial sovereignty and “decision-making sov- ereignty”. Few countries have used territorial sovereignty claims to protest against actions under the R2P principle, yet many insist on retaining the right to decide whether and how to act—something he sees as a real stumbling block for the implementation of R2P. 115 More than a decade after the 2005 World Summit, R2P undoubtedly re- mains a crucial norm. Evidence of its growing normative traction lies in the general acceptance and support for R2P in both the General Assembly and the Security Council. Between 2005 and early 2011 the Security Council had only endorsed four resolutions mentioning R2P, but between March 2011 and Oc- tober 2015 it had adopted 31 resolutions that referenced this principle—figures that may surprise some given the strong disagreements over the Libyan inter- vention in March 2011 and ensuing claims of R2P’s demise (ibid). Still, the pillars related to preventing and rebuilding have proven less controversial than that which calls on states to react, thus going against the core norm of non- intervention. And as we have seen, R2P is by no means a guarantee that those in danger will receive assistance; geopolitics continues to dictate who is helped and who is left behind. Box 15: Key terms related to the movement of people Refugees – People fleeing armed conflict or persecution by crossing their national border to seek safety in nearby countries. Refugees are defined and protected in international law and the status guarantees access to as- sistance from states, UNHCR and other organizations. Governments have specific responsibilities towards anyone seeking asylum on their territories, as defined in national legislation and international law. Refugees – People fleeing armed conflict or persecution by crossing their national border to seek safety in nearby countries. Refugees are defined and protected in international law and the status guarantees access to as- sistance from states, UNHCR and other organizations. Governments have specific responsibilities towards anyone seeking asylum on their territories, as defined in national legislation and international law. Internally Displaced Persons – Those who are forced to flee their homes, often for the very same reasons as refugees—war, civil conflict, political strife, and gross human rights abuse—but who remain within their own country and do not cross an international border. Migrants – Unlike refugees, migrants do not relocate due to a direct threat of persecution or death but rather to improve their lives. Typically they are searching for better job opportunities, but they may also relocate for education, reuniting with family or other reasons. Countries accept or reject migrants based on their own immigration laws and processes. Migrants – Unlike refugees, migrants do not relocate due to a direct threat of persecution or death but rather to improve their lives. Typically they are searching for better job opportunities, but they may also relocate for education, reuniting with family or other reasons. Countries accept or reject migrants based on their own immigration laws and processes. Stateless Persons – Those who are not recognized as citizens by any state. The conflict in Syria undoubtedly accounts for a large portion of those dis- placed. Since the civil war began in March 2011, over 11 million Syrians have fled their homes—more than half the population of Syria. Despite a focus on the refugees pouring into Europe, most of those displaced remain in the region. In early 2017, the UN estimated that over 5 million were refugees in neigh- bouring countries (Turkey, Lebanon, Jordan, Egypt and Iraq) and 6.3 million were internally displaced within Syria (UNHCR 2017). Migration, refugees and the humanitarian response Migrants – Unlike refugees, migrants do not relocate due to a direct threat of persecution or death but rather to improve their lives. Typically they are searching for better job opportunities, but they may also relocate for education, reuniting with family or other reasons. Countries accept or reject migrants based on their own immigration laws and processes. Stateless Persons – Those who are not recognized as citizens by any state. Migration, refugees and the humanitarian response In 2016, the UN reported that the number of forcibly displaced people world- wide—a staggering 65.3 million—was the highest in human history. The pre- vious record had been set in the aftermath of the Second World War. While displaced people refers to refugees, internally displaced persons (IDPs) and migrants, only those in the first two categories are considered to be ‘forcibly displaced’ (see Box 15). The conflict in Syria undoubtedly accounts for a large portion of those dis- placed. Since the civil war began in March 2011, over 11 million Syrians have fled their homes—more than half the population of Syria. Despite a focus on the refugees pouring into Europe, most of those displaced remain in the region. In early 2017, the UN estimated that over 5 million were refugees in neigh- bouring countries (Turkey, Lebanon, Jordan, Egypt and Iraq) and 6.3 million were internally displaced within Syria (UNHCR 2017). While the world spot- light has been on Syrian refugees, conflict and unrest in Iraq, Libya, Somalia, Afghanistan, Yemen and elsewhere have equally led to an exodus of people from these countries. Under international law, refugees have the right to safe asylum. This in- cludes physical safety but also freedom of thought, speech, religion and move- ment, freedom from discrimination and torture, and economic and social rights such as access to health care, education and work. The key legal document pertaining to refugee protection is the 1951 Refugee Convention, which has been signed by 144 states. It defines ‘refugee’ and spells out their rights, as well as the legal obligations of states to protect them. 116 Box 15: Key terms related to the movement of people Refugees – People fleeing armed conflict or persecution by crossing their national border to seek safety in nearby countries. Refugees are defined and protected in international law and the status guarantees access to as- sistance from states, UNHCR and other organizations. Governments have specific responsibilities towards anyone seeking asylum on their territories, as defined in national legislation and international law. Internally Displaced Persons – Those who are forced to flee their homes, often for the very same reasons as refugees—war, civil conflict, political strife, and gross human rights abuse—but who remain within their own country and do not cross an international border. Box 15: Key terms related to the movement of people While the world spot- light has been on Syrian refugees, conflict and unrest in Iraq, Libya, Somalia, Afghanistan, Yemen and elsewhere have equally led to an exodus of people from these countries. Under international law, refugees have the right to safe asylum. This in- cludes physical safety but also freedom of thought, speech, religion and move- ment, freedom from discrimination and torture, and economic and social rights such as access to health care, education and work. The key legal document pertaining to refugee protection is the 1951 Refugee Convention, which has been signed by 144 states. It defines ‘refugee’ and spells out their rights, as well as the legal obligations of states to protect them. The United Nations High Commissioner for Refugees (UNHCR) is the UN’s refugee agency and the guardian of the Refugee Convention. It was es- tablished in 1950 to help the millions of Europeans displaced after the Second World War. UNHCR was only meant to exist for three years, and was to con- clude its work once it had successfully dealt with the refugee problem in Eu- rope. No one could have predicted that this would not be the last refugee crisis 117 to face the international community. Not only has it been dealt new challenges every few years, but the number of people that fall under its mandate has risen steadily each decade (see Figure 7). Since 2012, the increase has been drastic. The scenes of mass population movements have shifted over time—from Eu- rope to Asia (and to a lesser extent Africa and Latin America) beginning in the 1960s, and then to the Middle East at the turn of the century. Figure 7: Populations of concern (refugees, asylum-seekers, IDPs, return- ees, stateless persons), 1951-2015 Figure 7: Populations of concern (refugees, asylum-seekers, IDPs, return- ees, stateless persons), 1951-2015 Source: UNHCR data. Available at: http://popstats.unhcr.org/en/overview The UN is generally well regarded for its humanitarian work and support for refugees. UNHCR has helped more than 50 million refugees since inception and received two Nobel Peace Prizes for its worldwide assistance to refugees. Yet the magnitude of the current emergency has left it overwhelmed and under- resourced. In 2014 the UN’s World Food Programme (WFP), which relies en- tirely on voluntary contributions, ran out of money and food supplies and was forced to suspend food assistance to 1.7 million Syrian refugees. Box 15: Key terms related to the movement of people Without a reliable source of funding, it had to resort to online crowdfunding to raise the $64 million needed to reinstate the food vouchers program. The 72-hour social media campaign with the hashtag #AdollarAlifeline was a desperate plea, ask- ing members of the public to donate $1 to the cause (WFP 2014). Source: UNHCR data. Available at: http://popstats.unhcr.org/en/overview The UN is generally well regarded for its humanitarian work and support for Source: UNHCR data. Available at: http://popstats.unhcr.org/en/overview Source: UNHCR data. Available at: http://popstats.unhcr.org/en/overview The UN is generally well regarded for its humanitarian work and support for refugees. UNHCR has helped more than 50 million refugees since inception and received two Nobel Peace Prizes for its worldwide assistance to refugees. Yet the magnitude of the current emergency has left it overwhelmed and under- resourced. In 2014 the UN’s World Food Programme (WFP), which relies en- tirely on voluntary contributions, ran out of money and food supplies and was forced to suspend food assistance to 1.7 million Syrian refugees. Without a reliable source of funding, it had to resort to online crowdfunding to raise the $64 million needed to reinstate the food vouchers program. The 72-hour social media campaign with the hashtag #AdollarAlifeline was a desperate plea, ask- ing members of the public to donate $1 to the cause (WFP 2014). The UN is generally well regarded for its humanitarian work and support for refugees. UNHCR has helped more than 50 million refugees since inception and received two Nobel Peace Prizes for its worldwide assistance to refugees. Yet the magnitude of the current emergency has left it overwhelmed and under- resourced. In 2014 the UN’s World Food Programme (WFP), which relies en- tirely on voluntary contributions, ran out of money and food supplies and was forced to suspend food assistance to 1.7 million Syrian refugees. Without a reliable source of funding, it had to resort to online crowdfunding to raise the $64 million needed to reinstate the food vouchers program. The 72-hour social media campaign with the hashtag #AdollarAlifeline was a desperate plea, ask- ing members of the public to donate $1 to the cause (WFP 2014). 118 It is little surprise that the escalation of the current crisis has led to a renewed discussion around addressing the root causes of refugee movements and mass migration. Box 15: Key terms related to the movement of people In his former role as head of UNHCR, UN Secretary General Anto- nio Guterres was forthright about the need to address this, stating “To those who trigger and prolong conflicts, leaving humanitarians to clean up the mess, it is time to say that this must stop. We as humanitarians can no longer pick up the pieces.” (UNHCR 2014). Unfortunately, the UN has had far less success in ending conflict than it has in providing humanitarian assistance and aiding ref- ugees. g The UN has also struggled to get states to do their part in accepting refugees and respecting their obligations outlined in the Refugee Convention. Fears of Islamic radicalization and terrorism have driven nationalism and anti-immi- grant public narratives in many Western countries. Some states were reluctant to welcome refugees from Syria and elsewhere in the Middle East, whom they perceived as a greater threat than refugees of past eras. While the UNHCR has been praised for its work with refugees, the General Assembly has been criticized for failing to get states to make the commitments needed to end the suffering of millions worldwide. The first-ever Summit for Refugees and Migrants was held at the opening of the 71st UN General As- sembly in September 2016. Member states adopted an agreement aimed at im- proving educational opportunities for refugee children and working conditions for displaced adults, while fighting to counter xenophobia and fear. Meanwhile the US convened a Leaders’ Summit on Refugees the following day to get in- dividual states to agree to accept more refugees and to encourage states and companies to contribute material support (both funding and in-kind contribu- tions) for refugee assistance programs. NGOs were largely disappointed by the outcome of the summits and human rights critics held that the international community has not done enough. Salil Shetty, the Secretary-General of Am- nesty International, stated that, “Faced with the worst refugee crisis in 70 years, world leaders have shown a shocking disregard for the human rights of people who have been forced to leave their homes due to conflict or persecution.” (Amnesty International 2016). In March 2017, Secretary-General António Guterres appointed Louise Ar- bour of Canada as his Special Representative for International Migration. What next? Reforms: big and small Reforms: big and small This chapter examined the major failings of the United Nations in protecting human rights globally. Despite having a solid reputation for promoting human rights, specifically in the areas of education, capacity building and technical assistance, the global body has struggled to reconcile its political nature with the principled protection of human rights. A lack of judicial authority and en- forcement mechanisms have limited its success. Scholars and experts have put forth many reform proposals—both big and small—to improve the UN’s role in advancing human rights worldwide. A particularly ambitious idea is to establish a World Court of Human Rights that would address the lack of judicial and enforcement mechanisms at the in- ternational level to protect individuals from abuses and punish violators. The scholars behind the proposal, Nowak and Scheinin, argue that there is a gap in the judicial system and many violations of international human rights law raised by individuals (rather than by states) are outside the jurisdiction of existing forums. Indeed, the ICC was created to deal with breaches of interna- tional criminal and humanitarian law, but not cases that fall exclusively under human rights law, while the International Court of Justice only hears cases be- tween states. Meanwhile decisions made by UN committees monitoring core international human rights treaties do not carry enough weight to fill the re- maining void. An early vision for a World Court of Human Rights was developed in the 1970s but it took years for the idea to be fully fleshed out. In 2014 a Statute for the World Court of Human Rights (the Treaty of Lucknow) was drafted through an international collaboration of judges, lawyers and scholars. Propo- nents are currently raising awareness of the project and encouraging states to adopt the Treaty of Lucknow to actualize the Court. They hope to see India and the US agree to champion the new institution (World Court of Human Rights website). Not all human rights commentators are as enthusiastic though. Many express concern that the Statute in its current form does not adequately address the problems that other international legal bodies suffer from, namely whether countries could be convinced to accept the Court’s jurisdiction as binding and how it would enforce its judgements. A range of less radical reform proposals have been put forward over the years. Box 15: Key terms related to the movement of people Hav- ing served as UN High Commissioner for Human Rights and Chief Prosecutor of the International Criminal Tribunals for the Former Yugoslavia and Rwanda, Arbour is well placed to lead the follow-up to the migration-related aspects of the 2016 Summit, and to work with member states and other stake- holders to develop a global compact on safe, orderly and regular migration. 119 What next? Freedman has suggested that a more practical solution than a World Court would be to strengthen and utilize regional human rights courts. Three of the world’s regions have human rights mechanisms: Europe, the Americas and Africa. Each of these mechanisms relies on a regional rather than universal understanding of human rights, which helps explain why compliance has been strong. A similarity in norms, culture, practices and governance has made 120 countries more willing to engage with regional mechanisms than those at the international level. Equally crucial has been the geographic, political and eco- nomic linkages that connect countries within a region. These ties often lead to tacit pressure on states to cooperate on human rights, which in turn understand the potential ramifications of noncompliance. To start, Freedman proposes that human rights mechanisms be created in Asia and Eastern Europe, and that pres- sure be exerted on all countries to join a regional system. Financial aid and technical assistance would then need to be provided to ensure that regional courts could afford acceptable human rights procedures. The pitfall of this ap- proach is that it appears to abandon the aim of truly universal human rights protection. In an effort not to forgo the notion of universality, observers have offered diverse options for improving the UN human rights machinery. The Human Rights Council, not surprisingly, has been the primary target. The concluding chapter outlines reforms suggested by Bertrand Ramcharan, former acting High Commissioner for Human Rights, which are particularly sensitive to real- world constraints. Schwartzberg (2013) also provides useful ideas for strength- ening the essential human rights body. Weiss (2009) blames bureaucracy and lack of leadership for the UN’s inaction in situations like the Rwandan geno- cide, while Hanhimä ki (2008) maintains that there are simply too many human rights bodies and that reform efforts should emphasize consolidation. This pri- mer does not intend to explore proposals in detail, but rather to inspire further reading on the topic. g p There is clearly no shortage of good ideas, but how likely are we to see reform of the UN human rights machinery in the near future? If the process of replacing the flawed Commission on Human Rights with the Human Rights Council taught us anything, it is that achieving substantial change is no easy task. That colossal effort wrapped up just over a decade ago. Can change happen? That the United Nations is merely an organization of sovereign states acting on their own cannot be overstated. As is the case with maintaining peace and security and achieving sustainable development, progress on human rights de- pends entirely on member states. It would be as unrealistic to expect states to concede power to the UN (or any other global institution) to protect and pro- mote human rights as it would be to hope for a radical shift in attitudes with regards to state sovereignty. Still, states that do not currently prioritize human rights can be swayed over time. A crucial factor in getting states to act is pres- sure from domestic private groups, foreign NGOs, and international organiza- tions. Whether it is in a liberal democracy or in a repressive government, non- state actors often succeed over time in exerting influence on states to respect human rights. International human rights NGOs like Amnesty International and Human Rights Watch have been particularly instrumental in supporting the UN over the past decades to protect and promote human rights. Through their work doc- umenting violations, engaging in fact finding and advocating for action, they play a crucial role in exposing abuses and pressuring states to address them. Without their tireless efforts, it is unlikely that the ICC would have been cre- ated, or that states would have adopted the International Ban on Landmines when they did. Sadly, many countries—Russia, Turkey and China are particu- larly prominent examples, but there are countless others—have been increas- ingly cracking down on NGOs, journalists and the media. When an authoritar- ian regime suppresses freedom of speech, limits the activity of NGOs and har- asses human rights defenders, it makes it more difficult to identify what Freed- man refers to as ‘hidden abuses’. These unreported or under-reported abuses are taking place around the world, in countries like North Korea, Equatorial Guinea, Gambia, Qatar, Saudi Arabia, and Turkmenistan. Yet even authoritarian regimes are finding that technology and social media are disrupting their ability to exert absolute control within their borders and dominate the public discourse. In an already complex world of human rights protection, ‘citizen journalism’ has emerged as a promising mechanism to ex- pose human rights violations, particularly in countries where freedom of press, speech or expression is not guaranteed. What next? Despite under- whelming results, it is doubtful that another ambitious reform will be on the table any time soon. For one thing, doing so would be to admit defeat. As well, overhauling institutions or entire systems is costly in terms of time and re- sources which could otherwise be allocated elsewhere. More plausible would be a series of smaller reforms or tweaks that would lead to incremental change in the right direction. Human Rights Up Front, a Secretary-General initiative launched in 2013, is an example of how the UN has sought to improve how it delivers on its human rights mandate through means within its control—in other words, non-Charter, internal reforms. The initiative strives to realize a culture change within the UN system, by increas- ing awareness among UN staff of human rights and their responsibilities, while encouraging them to take action to prevent violations. 121 121 Can change happen? Can change happen? Chapter 5 – Workable Global Institutions: How to Get from Here to There? Chapter 5 – Workable Global Institutions: “The Charter of the United Nations frequently maps out a chasm between its aspi- rations and the means to achieve them. War is to be renounced, human rights are to be advanced, and development to be a priority. Yet peace is beholden to the five permanent members of the Security Council, human rights obligations remain lim- ited to voluntary commitments taken on by states, and development is the paradigm example of an unfunded mandate.” Simon Chesterman (2015: 505) This book has attempted to show, first, what the United Nations is and does and the astounding contributions it makes to the world. Second, we have tried to explain why the UN is so forcefully criticized for being unable to deal with urgent world problems. Third, we have wanted to indicate the sort of analysis that is required to understand this world institution. In this concluding chapter we consider possible reforms to the United Na- tions, building on the analytical foundations that have been laid throughout the book. We begin by looking at what others have been saying about how to ap- proach global reform. Then we list some of the types of reforms that are ur- gently required. Finally, we analyze how we can bring about global reform. Specifically, how do we get citizens to want to transfer authority to world in- stitutions so that institutional transformation is on the political agenda? Can change happen? During the Arab Spring when foreign reporters were frequently denied access and state-run media served only to spread government propaganda, individual citizens captured abuses with cell phone cameras and shared them on social media websites and blogs such as Facebook, Twitter, YouTube and WordPress. Some observers believe that human rights protection is shifting outside the realm of the state, as governments find it harder to hold onto their accustomed control. There may be an element of truth to this, but it would be premature to predict the demise of the state. 122 The subjective nature of human rights complicates matters further. States adopt their own understandings of human rights and choose to prioritize certain rights over others, as this chapter has explored. That they are party to the same treaties and declarations gives a false sense of consensus. Even among other- wise similar states (not to mention within states), disagreements often occur. We have witnessed this in Western states over issues like abortion, banning religious clothing or symbols, and whether health care should be considered a public good. Human rights are matters of moral judgement after all. Norms do change and adapt in the long run. But until there is actual consen- sus among states, the UN will find it difficult to provide systematic and author- itative protection. Progress on some fronts suggests there is reason to be hope- ful, yet backtracking on others justifiably raises concern. Whereas the overall trend is positive in the areas of peace and security and development, it is diffi- cult to discern with human rights. Although the UN has succeeded in placing the concept of ‘universal’ human rights on the international agenda, actually protecting human rights still remains as much of a challenge for the UN today as it was in 1945. 123 123 What we have learnt about understanding world institutions Our goal is to find a possible path toward more workable 21st century global institutions—that is to say, authoritative institutions that the world will obey. What did the history of international organizations and the analysis of their present operations tell us about transforming the UN into an effective global institution? The complex picture is not always clear. Nevertheless, let us draw the best conclusions we can. If the founding of the League and the UN taught us one thing it is that it took great leadership, diplomacy, and public communication. It took a Wood- row Wilson or a Franklin Roosevelt. They contributed ideas and deep thought, but they were also skilled at selling the concept to the public. Roosevelt dreamt of an organization that could overcome unilateralism, alliances, spheres of in- fluence, and balance of power politics. This did not materialize but the ideals led the way to what was possible. The leaders were backed by solid secretari- ats, and the impetus and ideas of private individuals and civil society also played a role. So, can we conclude that it takes a great power to lead the way? Perhaps. Certainly, the major powers cannot be opposed. Does it take a specific 124 personality? This is not certain either. Wilson failed to implement the League in the US and Roosevelt passed the baton to his successor Harry Truman, a sound but not a notable leader. There was a definite need for diplomacy and persistence to deal with contradictory goals. Member states wanted both coop- eration and independence; internationalism compatible with their national in- terests; and hierarchy alongside equality and participation. This is exactly the United Nations we got. Aside from the actors of the moment, the international organizations were also the result of their historical context. The League and the UN would not have come about without the devastation of war. Nor would they have come about without the cumulative historical precedents and models. They were, in part, the result of years of international thinkers, ideas, and conferencing— given birth to by cumulative ‘internationalism’. So, we may say that new in- ternational organizations resulted from what we have called the ‘big bang the- ory’ as well as ‘evolutionary historicism’. They were also children of their epoch. They were spawned by the post-war economic, social and technological conditions. What we have learnt about understanding world institutions Now the question is: are we finding ourselves in a ‘global era’ cor- responding to new ‘global’ challenges and requiring ‘global’ institutions? p g g g q g g Our analysis of the United Nations—including its significance, the need for its transformation, proposals for reform, and barriers to achieving them—also provides useful insights when considering a path forward. To begin with, the importance of the UN is enormous. This book has discussed many of its nu- merous achievements at length. As coordination managers, conveners, and the setter of standards, the UN’s contributions to peace are staggering. In 2016 there were 16 ongoing multidimensional UN peace operations with 120,000 military, police and civilian personnel trying to limit conflict in the world. At the same time, the UN is the world’s largest coordinator and supplier of hu- manitarian aid and is responsible for managing the gigantic 2030 Agenda for Sustainable Development (the SDGs). The organization has also been respon- sible for introducing and mainstreaming new concepts such as human rights, R2P, and gender equality, as explored in The Power of UN Ideas. Under Ban Ki-moon’s leadership, the UN made great strides in ensuring that climate change would be on the world’s agenda, including through convening the suc- cessful 2015 Climate Change Conference in Paris. Just as the need for the UN is evident, so is need for its transformation into an effective global institution. Critics point to the UN’s lack of legitimacy as a decision-making body, and the absence of democratic participation and fair representation. The Security Council’s two-tier system that accords a veto to the powerful is seen as unequal treatment. Many do not consider its decisions to be moral or justified, not to mention the issue of impasses, when vetoes are used. Meanwhile, the General Assembly that relies on ‘one member, one vote’ rather than a form of weighted voting is also charged with being unrepresenta- tive. The Human Rights Council struggles to protect human rights globally as 125 it struggles with a fundamental contradiction: it is a political body expected to take principled action. The Economic and Social Council deals with its own challenges, namely being a slow and bureaucratic organization that cannot make binding decisions and whose mandate greatly overlaps with that of the General Assembly. What we have learnt about understanding world institutions The result has been countless calls for reform of the UN system and the need for a global movement to bring them about. Beyond simply exposing the weaknesses of our present institutions, our analysis also revealed that there is no shortage of proposed improvements. Many aim to improve representation, fairness and effectiveness of the major organs, particularly the General Assembly, Security Council, ECOSOC and the Human Rights Council. Others focus on issues such as securing adequate financing, autonomous emergency peace services and increased engagement and consultation with stakeholders. Later in this chapter we will go into nine key reform ideas in greater detail. y g Despite all the good ideas that have surfaced over the years, we have also seen why the UN’s hands are tied and why we are not moving ahead. There is indeed a considerable gap between the ideas for reform and the political will to implement them. Most states do not want a more powerful and effective UN. And although the significant steps taken toward the international responsibility to protect have limited sovereign impunity, we are still living in an era of na- tion-state predominance. The primacy of national power and politics is hardly disputable. Meanwhile, there has been no sustained campaign to communicate the UN’s achievements or to speak of its need for additional authority to deal with global challenges. These are a couple of reasons we have yet to see gen- uine change. Reviewing the literature on revamping the UN “To respond to today’s and tomorrow's threats to peace and security, the United Nations must become more relevant, more credible, more legitimate and more capa- ble.” Mogens Lykketoft, President of the General Assembly (UN 2016) Before outlining reform proposals and discussing how to get citizens to want to transfer authority to global institutions, we should take a peek at what vari- ous experts have been saying on the topic. In this section we examine three major, recent examples of thinking about revamping the UN. In UN 2030: Rebuilding Order in a Fragmenting World, a 2016 report by Kevin Rudd, former Australian Prime Minister and Chair of the Independent Commission on Multilateralism, 10 principles are put forth for reforming the United Nations in response to the “globalization of everything”. To ensure a more relevant and responsive UN, Rudd highlights the need for agenda setting that is future-oriented (dealing with, for example, cybersecurity and lethal au- tonomous weapons systems) and proposes strengthening policy-planning 126 capability. He argues for breaking institutional silos through a ‘Team UN’ ap- proach of multi-disciplinary teams dealing with specific challenges on the ground, as well through the structural integration of the UN’s peace and secu- rity, sustainable development, humanitarian and human rights agendas. To im- prove member state participation and cooperation, he calls for a formal com- mitment to multilateralism by member states and for the UN to build bridges between the great powers to reduce tensions. Rudd also suggests a greater fo- cus on prevention, better results management, and enhanced efficiency to deal with the reality of budgetary constraints. Finally, he makes the case that women should be at the centre of the UN agenda and that youth need to have their voices heard in UN councils. While all this looks like a reasonable agenda for change it should be noted that it is very light on structural modifications. In fact, it looks more like ad- ministrative adaptation to the new demands of globalization, most of which is going on to some degree within the UN as it is. Although good principles are announced, there are no institutional changes for bringing them about. The 2016 report Reinventing Development: Reforming the UN for People and Planet by Barbara Adams and Karen Judd specifically warns that institu- tional transformations that would demand shifts in power structures within the United Nations may take a long, long time. Reviewing the literature on revamping the UN Thus, they claim, efforts aimed at major structural changes may stand in the way of the kind of urgent reforms that can make the UN a more capable force for people and the planet. There are deeply entrenched interests that support the status quo, so it may be more effective to ignore difficult institutional reforms and instead concentrate on the critical issue of development. According to Adams and Judd, there is already a framework in place for member states to transform the UN. It is composed of the three new global ‘agendas’ hammered out in 2015 by states and civil society: the 2030 Agenda for Sustainable Development (SDGs); the Addis Ababa Action Agenda on fi- nancing for development; and the Paris Agreement on climate change. The agendas reflect a political effort to come to terms with new global realities. They can potentially forge international cooperation under the auspices of the UN. Unlike past development agreements, the 2030 Agenda is universal, thus requiring all countries to measure and report on progress. The authors point to rising corporate influence and the emerging gap in ac- countable governance as obstacles to realizing development goals and serving public interests. They explain that universal norms and standards are not ap- plied consistently across the UN nor are they necessarily accepted by other institutions such as the WTO or IMF. Since trade and investment largely by- pass the UN, public interest is not always guaranteed. Another challenge they outline is that the UN’s assessed contributions have become increasingly inad- equate for covering the organization’s increased responsibilities. 127 In order to overcome these obstacles, Adams and Judd list four priorities for member states, which are necessary for achieving the three new development agendas: greater integration of the three pillars of the United Nations (peace and security, human rights and sustainable development); implementing a new funding formula to finance goals and commitments; adopting a values-based framework for intergovernmental processes; and creating a culture of account- ability. Taken together, these imperatives constitute an agenda for reform. All this seems very plausible and reasonable. The question is, of course, will the three agendas be respected without any authority to ensure compliance? Is a bunch of NGOs snapping at the heels of recalcitrant states enough to make them keep their promises? It never has been in the past. Do the three agendas have enough clout to ensure change at the UN? Reviewing the literature on revamping the UN The authors offer good sugges- tions for achieving these, and the three agendas include timelines and measur- able goals. And experts and civil society are working hard to try to ensure states fulfil their obligations. But will ‘should’ and ‘must’ become ‘shall’ and ‘will’? Is it just another case of planned reforms without teeth? Will improvements in development spill over into power politics? The fundamental question we have to ask ourselves is whether ‘process agendas’ can transform international pol- itics without any substantive change to institutional structures and their under- lying power imbalance and entrenched interests. y g p In June 2015, the Report of the Commission on Global Security, Justice & Governance was published. It is one of the most significant, recent studies pro- posing the renaissance of the UN. In presenting their report, Ibrahim Gambari, co-chair of the Commission and former Nigerian Foreign Minister and UN Under-Secretary-General of Political Affairs, noted that “the UN and global governance institutions are ill-suited to address many modern evolving threats and must reform or risk prolonging and deepening global crises.” According to the other co-chair, former U.S. Secretary of State Madeleine Albright, the world requires “more capable tools of global governance, with different kinds of public, private, and mixed institutions designed for twenty-first-century challenges.” The report, which is over 100 pages in length, presents a comprehensive agenda for reform. It focuses on the priority objectives of security (specifically state fragility and violent conflict), climate governance, governing the global economy, reforming global institutions, and engaging partners. Some of its key recommendations are summarized below:  Security: The Commission advocates focusing on conflict prevention, strengthening the role of women in peace and security, and building consensus on criteria for determining when to apply R2P. To deal with conflict situations, it calls for enhanced UN capacity in military, police and civilian response, through rapid deployment and military planning, designated military units, state-formed police units, and a standing and reserve capacity of civilian specialists.  Security: The Commission advocates focusing on conflict prevention, strengthening the role of women in peace and security, and building consensus on criteria for determining when to apply R2P. To deal with conflict situations, it calls for enhanced UN capacity in military, police and civilian response, through rapid deployment and military planning, designated military units, state-formed police units, and a standing and reserve capacity of civilian specialists. Reviewing the literature on revamping the UN 128  Climate change: The creation of a number of new climate change en- tities is suggested to support the implementation of the COP21 climate commitments. These include: an International Carbon Monitoring branch, a Climate Research Registry, a Climate Action Clearinghouse, and a Climate Engineering Advisory Board. It is also proposes that ad- ditional investments be made in climate adaptation and that private en- terprise be engaged on market-based incentives to reducing emissions.  Climate change: The creation of a number of new climate change en- tities is suggested to support the implementation of the COP21 climate commitments. These include: an International Carbon Monitoring branch, a Climate Research Registry, a Climate Action Clearinghouse, and a Climate Engineering Advisory Board. It is also proposes that ad- ditional investments be made in climate adaptation and that private en- terprise be engaged on market-based incentives to reducing emissions. p g g g  Governing the global economy: To foster a renewed framework for global economic cooperation and crisis response, the Commission turns to what it calls the G20+, where the plus signifies enhanced coordina- tion with the UN and Bretton Woods institutions. Interestingly, this pro- posal comes despite acknowledging a lack of evidence that G20 agree- ments led countries to reshape national actions in a significant way. Other priorities outlined include: combating illicit financial flows and extremist financing, instituting effective governance of natural re- sources, and securing the digital economy while promoting internet ac- cess in the Global South.  Reforming global institutions: For the General Assembly, its recom- mendations include streamlining its agenda, leading the post-2015 de- velopment agenda and developing a consultative UN Parliamentary Network that would have a formal relationship to the UN as an advisor to the GA. The purpose of the network would be to bring together elected parliamentarians from their national legislatures to discuss and advise on issues of UN governance. It would also function as an addi- tional channel to civil society, and to expand public knowledge and par- ticipation in UN work. Regarding the Security Council, the Commis- sion proposes that membership be expanded and immediate re-election of non-permanent members be allowed. It adds that the SC should con- sult more with civil society and business. On modernizing the Secretar- iat, it is recommended that the secretary-general selection procedure be improved and that the SG be given more discretion to manage the Sec- retariat. Reviewing the literature on revamping the UN Other ideas are put forth related to the Peacebuilding Commis- sion, international courts and human rights bodies.  Reforming global institutions: For the General Assembly, its recom- mendations include streamlining its agenda, leading the post-2015 de- velopment agenda and developing a consultative UN Parliamentary Network that would have a formal relationship to the UN as an advisor to the GA. The purpose of the network would be to bring together elected parliamentarians from their national legislatures to discuss and advise on issues of UN governance. It would also function as an addi- tional channel to civil society, and to expand public knowledge and par- ticipation in UN work. Regarding the Security Council, the Commis- sion proposes that membership be expanded and immediate re-election of non-permanent members be allowed. It adds that the SC should con- sult more with civil society and business. On modernizing the Secretar- iat, it is recommended that the secretary-general selection procedure be improved and that the SG be given more discretion to manage the Sec- retariat. Other ideas are put forth related to the Peacebuilding Commis- sion, international courts and human rights bodies.  Engagement: The Commission stresses the importance of the UN en- gaging with critical regional, local, civil society and business actors in global governance.  Engagement: The Commission stresses the importance of the UN en- gaging with critical regional, local, civil society and business actors in global governance. To realize these changes, the Commission recommends a transitional strategy for reform—a longer-term approach to modernizing global governance struc- tures. Specifically, it calls for the building of ‘smart coalitions’ to mobilize support to sustain reforms, the convening of a Conference on Global Institu- tions in 2020, and establishing a mechanism to monitor and coordinate reform. g We can see that there is certainly no lack of proposals for reform in this report. In fact, the Commission’s work is an excellent example of combining 129 high-minded analysis of the need for better global governance with its mem- bers’ judgement of what is actually possible. It is pragmatic, but not at the expense of good ideas and necessary changes. The only caveat is that it could go further in terms of offering ideas for institutional transformation. For in- stance, it stops short of suggesting an autonomous, emergency peacekeeping force or independent funding sources for the UN. Reviewing the literature on revamping the UN Finally, in devoting the last chapter to “Getting from Here to There”, the report goes further than most in setting us on the path to thinking about how we can achieve reform. Nine popular proposals to transform the UN To conclude this book, we present in greater detail a list of widely-known pro- posals for strengthening the United Nations before terminating with a set of ideas about how the renaissance of the UN can be brought about. To guide the process of understanding and contextualizing these proposals, we begin by of- fering some reflections on the various types of change (with their correspond- ing level of ambition) along with contrasting approaches to achieving change. It is helpful to think about change in terms of different gradations. It has been said that reform falls into two categories: a more up-to-date alignment of status and authority and better management of the system (Fasulo 2015: 237). But, in fact, it is a little more complicated. First, there are simple adjustments to administrative, financial, personnel and budget considerations—often called ‘reform’. These may be purposeful, managed improvements to administration. The UN has been quite effective at this sort of continuing reform. The terms ‘reform’ and ‘change’ can cause confusion as they are often used as catchall terms to include the whole spectrum of change at the UN. Second, we have ‘adaptation’. This refers to modifications made on an ad hoc basis to deal with short-term problems, without any overall plan or purpose. Third, a more fun- damental form of change is what is called ‘transformation’. Transformation entails structural modifications to the composition, organization, norms and rules of an institution to allow it to better cope with the effects of socio-eco- nomic, technical and political change. It may question the goals, purposes, val- ues and functions of organizations. This is sometimes referred to as changing the ‘architecture’ of the international system. Far more than a merely academic question of definitions, the terms ‘reform’, ‘adaptation’ and ‘transformation’ are critical to our understanding of what we are trying to achieve (for an elab- oration, see Trent 2007: 24-30). Once the desired change is determined, there are multiple strategies that can then be considered for how to achieve such change. Reformers (and we hope this will include you) struggle over whether to aim for radical and immediate change, or for incremental steps that may amount to a transformation over time. In historical terms the debate has been over revolution or evolution—what is 130 theoretically necessary or what is politically attainable. Nine popular proposals to transform the UN In the first chapter we introduced this debate, concluding that the founding of the League and the UN were the result of both ‘big bangs’ (the world wars) and ‘historical evolution’ (a cumulation of progressive steps). On the one hand, a gradual approach to change that considers political con- straints can be seen as practical and realistic. The hope is that smaller, achiev- able changes will eventually lead to a larger shift. We recall this recommenda- tion from leading scholars when it comes to improving the Human Rights Council. The challenge is that these minimalist arguments may focus on what is prob- able at the expense of what is desirable and what may, at a future time, be possible. Ambitious dreams for an alternative world order are often driven down to the lowest common denominator by those counselling practicality and prudence. Reformers must be inspired by the vision of a cooperative world, as were the founders of the League and the UN. This means being challenged by the end target of workable institutions that can respond to the challenges of tomorrow. It means being animated by the goals at the tip of our grasp while accepting all practical steps to move ahead. So perhaps the two are not irreconcilable after all. As Ban Ki-moon wisely put it: “Keep your head above the clouds, and your feet firmly on the ground. And move up, step by step. That means: Dream big—but be practical.” (UNSG 2016). Finally, a brief note on how we selected these nine ideas. We must stress that all the following proposals have their critics. They are also not intended as an exclusive plan for revamping the United Nations, but rather examples of reforms that could allow the United Nations to gain legitimacy and develop the institutional capacity to make respected, authoritative decisions. Each of the proposals is what we call “workable”, meaning it is practical, necessary and can be implemented. However, unlike the lists prepared by various interna- tional commissions and high level panels, our choices are not the proposals that might be considered the most politically acceptable. They are instead based on what is necessary to make the UN a credible global institution that can give leadership to the world. They range from grandiose to minute. 1. A more legitimate Security Council One fairly radical but ‘workable’ proposal for restructuring the Security Coun- cil is made by Joseph Schwartzberg in his powerful book, Transforming the United Nations System: Designs for a Workable World. He recommends a three-pronged approach. First, to surmount national interests and help smaller countries to work through larger groupings, he suggests a system of represen- tation in the Council by 12 world regions rather than individual nation-states. 131 Each multinational region would nominate slates of candidates from which one would be elected to the Security Council by the General Assembly. Second, he proposes a mathematically determined weighted vote for each region. Third, in exchange for phasing out the veto, the P5 would be rewarded with a larger weighted vote in a more empowered General Assembly. Additional new proposals for reforming the Security Council have been made in the 2015 Report of the Commission on Global Security, Justice and Governance. To achieve the twin goals of effectiveness and acceptability, it is suggested that the Council’s membership be expanded to reflect the tremen- dous increase in UN membership (as was done in 1965) and that immediate re- election of non-permanent members be allowed. The Commission then pro- poses ideas for resolving the question of the veto and improving peace opera- tions through improved worked methods (see Box 16). Box 16: Improving the working methods of the Security Council  Where there are reasonable grounds to justify it, members should be requested to defend their ‘no’ votes publicly in the Security Council.  Permanent members should be given the option of casting a ‘dissen- ting vote’ that does not rise to the level of a veto and therefore does not block passage of a resolution.  The Council should ensure sufficient resources and political support to new peace operations where there are potential risks of war cri- mes.  For each peace operation a ‘Group of Friends’ should be constituted, States that can help bring political and diplomatic pressure to bear on the situation.  The Council must consult troop and police contributing countries whenever there are problems and address their concerns.  It should undertake a rolling analysis of those terrorist, criminal and extremist elements that are capable of influencing the context of peace operations. 2. A more balanced and focused General Assembly 2. A more balanced and focused General Assembly Joseph Schwartzberg (2013) tells us that the General Assembly owes its legit- imacy to being an almost universal body with 99.6 per cent of the world’s peo- ple. This population has tripled since 1945, but for all this time the voting power in the GA has steadily been skewed to favour relatively minor states including a proliferation of microstates. No fewer than 39 current members have less than a million inhabitants each, and 13 have fewer than 100,000 in- habitants each. In financial terms, 128 members collectively pay less than 1.3 per cent of the total UN budget. If one can be opposed to an ‘undemocratic’ veto then one should be equally opposed to GA coalitions of the very weak. It has been called the ‘immoral egalitarianism’ of the ‘one-member, one-vote’ rule. At present, the three most populous countries (China, India and the United States) have 42 per cent of the world’s population but only 1.6 per cent of votes in the General Assembly. The major powers resort to bribery or threats to bend the weak to their demands. Because of these faults, the biggest states have opted to create the G20 where they can discuss economic matters without being hobbled by what they consider to be the noisy, ‘marginal’ states. A well-de- signed system of weighted voting would mitigate these defects. The aim, as at the beginning of the UN, should be to combine power with principle. The three basic principles for the weighted vote of each member would be: 1) the democratic principle in which population is the determining factor; 2) economic capacity represented by contributions to the UN budget; and 3) the sovereign equality principle whereby each state is treated equally. The three components of the vote would be combined in a simple formula as if they were of equal relevance (Schwartzberg 2013: 21-26). Over time, say, every ten years, a neutral agency would adjust these weights according to changed con- ditions. The aim is that states might start listening to each other. Rather than having weak and poor states pass a plethora of meaningless resolutions that destroy the reputation of the GA and are largely ignored by the rich and pow- erful, debates could become more consequential. Once the voting power within the General Assembly is more balanced, one could proceed to making its operations more focused. Box 16: Improving the working methods of the Security Council  Where there are reasonable grounds to justify it, members should be requested to defend their ‘no’ votes publicly in the Security Council.  Permanent members should be given the option of casting a ‘dissen- ting vote’ that does not rise to the level of a veto and therefore does not block passage of a resolution.  The Council should ensure sufficient resources and political support to new peace operations where there are potential risks of war cri- mes.  For each peace operation a ‘Group of Friends’ should be constituted, States that can help bring political and diplomatic pressure to bear on the situation.  The Council must consult troop and police contributing countries whenever there are problems and address their concerns.  It should undertake a rolling analysis of those terrorist, criminal and extremist elements that are capable of influencing the context of peace operations. Finally, in assessing the current requirements for social, economic and legal supports for peace and security, the Commission favours instituting a formal mechanism of consultation for regular, structured discussions between the Se- curity Council and representatives of civil society, business and municipalities. After all, these stakeholders have a demonstrated interest in and make specific contributions to the new dimensions of security (health, protection of civilians, women and children, climate change, natural disasters). 132 3. An Economic, Social and Environmental Council 3. An Economic, Social and Environmental Council This book has discussed some of the reasons why analysts have been decrying ECOSOC as the weak sister among the UN’s principal organs since at least the 1980s. The UN’s founders intended for it to coordinate the economic and social work of the UN system with its specialized agencies and other bodies. Yet they gave the agencies their own governing arrangements; set the Bretton Woods financial institutions on an autonomous path; provided ECOSOC and the Gen- eral Assembly with overlapping mandates; and failed to give ECOSOC a dis- tinctive profile. From there, everything went downhill. Critics say it is too large for high-level consultations and flexible decision-making, but not large enough to perform credibly as a plenary body. Heads of state, foreign secretaries and finance and trade ministers prefer the smaller, shorter and more focused meet- ings of the World Bank and IMF. They find ECOSOC to be incapable of deal- ing with crises, unable to exercise any significant influence over the special- ized agencies, and thus only attracting low level delegates to endless debates and little apparent action. Over the years, there have been a plethora of proposed reforms from differ- ent groups. Fearing hang-ups with structural reforms, some observers have limited themselves to suggesting short-term process changes. However, show- ing the weight of discontent with ECOSOC, structural transformations have been proposed by many groups such as: UBUNTU (World Forum of Civil So- ciety Networks), the Commission on Global Governance, Reimagining the Fu- ture, the United Nations Association of the USA, the South Commission and the World Commission on Environment and Development. p The major suggestion coming from these groups is that ECOSOC be trans- formed to a Social and Economic Security Council or an Economic, Social and Environmental Council (ESEC), with powers and methods parallel to those of the Security Council (without P5 vetoes). Its policy proposals would be trans- ferred back to the GA for debate and majority approval. As with the original intentions of the Charter, the new ESEC would have effective control and co- ordination over all agencies, financial institutions and multilateral groups in the UN system in the spheres of economics, social development and the envi- ronment. 2. A more balanced and focused General Assembly Because global problems require global solutions, Schwartzberg argues that the time has come to give the GA limited capacity to pass legally binding resolutions (in areas not being considered by the Security Council). In matters of a worldwide nature that can- not be addressed at the regional or national levels, the GA should be authorized to legislate binding international law when there is a two-thirds majority that includes 50 per cent of the total world population. Some issues might require a supermajority of 75 per cent of the weighted votes. 133 4. A reconfigured Human Rights Council In Chapter 4 we discussed how the UN has achieved far greater success in developing and promoting human rights than it has in protecting them, and how the politicization of UN human rights bodies, particularly the Human Rights Council, is to blame for this. We also looked at why it may be unlikely that we will see a complete overhaul of the flawed Human Rights Council any time soon, for a host of reasons including the time and resources involved and the optics of another major reform so soon after the 2006 effort. Box 17: Ideas for reforming the Human Rights Council  Representation: Governments could remain members but the repre- sentatives they select should be judges or academics with human rights expertise. Membership could also be expanded to include in- dependent experts in addition to government representatives.  Status within UN: The Council could eventually be made a principal UN organ, rather than a subsidiary organ of the General Assembly, though doing so at this time may only elevate its politicization.  Prevention: Strategies that emphasize preventing conflict and vio- lence that lead to human rights abuses should be pursued.  Protection: A provision on implementing the Responsibility to Pro- tect could be added to the Council’s mandate.  Universal Periodic Review: The UPR process could integrate a pro- cedure for dealing with violators, to ensure that dialogue and coop- eration leads to action.  NGOs: A more formal institutional framework around the relation- ship of NGOs to the Council would be beneficial. The consultative status that NGOs enjoy with ECOSOC would be a model to follow. 3. An Economic, Social and Environmental Council The Second and Third Committees of the General Assembly (dealing with economic and financial issues; and social, humanitarian and cultural is- sues, respectively) would be wound up and their activities transferred to the new ESEC. The UN Conference on Trade and Development (UNCTAD) and the UN Industrial Development Organization (UNIDO) would be closed down. The votes in the World Bank and the IMF would be more equitably distributed. To govern the new council, a ministerial board of some 25 governments could be created along with an advisory commission. Alternatively, the General Committee of the General Assembly could be the leader of ECOSOC. The 134 governing bodies of the major development agencies would be combined into one executive committee. The Commission on Peace, Justice and Governance outlined an alternative idea to strengthen the United Nation’s role in the field of economics without completely redesigning ECOSOC. It proposed transforming the G20 into the G20+. The term ‘co-optation’ is not used, but this is the essence of the proposal to institutionalize the G20 and strengthen its coordination with the UN, the World Bank, IMF, WTO, ILO and regional organizations. The proposed G20+ would meet every two years at the UN. The UN would furnish a secretariat, a liaison mechanism, a UN deputy secretary-general, and a technical body of experts to frame its activities. The details are shrewd and complex, but this appears to be a subtle manner to bring the G20 within the UN—which is indeed a necessity. Box 17: Ideas for reforming the Human Rights Council  Representation: Governments could remain members but the repre- sentatives they select should be judges or academics with human rights expertise. Membership could also be expanded to include in- dependent experts in addition to government representatives.  Representation: Governments could remain members but the repre- sentatives they select should be judges or academics with human rights expertise. Membership could also be expanded to include in- dependent experts in addition to government representatives. g  Status within UN: The Council could eventually be made a principal UN organ, rather than a subsidiary organ of the General Assembly, though doing so at this time may only elevate its politicization.  Status within UN: The Council could eventually be made a principal UN organ, rather than a subsidiary organ of the General Assembly, though doing so at this time may only elevate its politicization.  Prevention: Strategies that emphasize preventing conflict and vio- lence that lead to human rights abuses should be pursued.  Protection: A provision on implementing the Responsibility to Pro- tect could be added to the Council’s mandate.  Universal Periodic Review: The UPR process could integrate a pro- cedure for dealing with violators, to ensure that dialogue and coop- eration leads to action.  NGOs: A more formal institutional framework around the relation- ship of NGOs to the Council would be beneficial. The consultative status that NGOs enjoy with ECOSOC would be a model to follow. 135 But that does not mean we cannot make certain changes that would have a measurable impact on the Human Rights Council’s work. In doing so, we should focus on what is realistic and achievable. Ramcharan (2011) outlines a number of actionable ideas that could help make the body more fair, impartial and effective (see Box 17). These vary in level of ambition. Reforming the Human Rights Council will not be straightforward—espe- cially if the reform process of the Commission on Human Rights is any indi- cation—but in the long run, failing to even try will have devastating conse- quences. 5. Improved staffing and management practices There is a general consensus among experts that the UN’s approach to hiring and managing the international civil service needs fixing. One of the chief criticisms of the Secretariat relates to the politicized ap- proach to hiring, particularly when it comes to top appointments. Since it was launched in 2014, the 1 for 7 Billion campaign has made appeals for greater transparency regarding top UN appointments, and an end to the current mo- nopoly over the decisions. It explains how permanent Security Council mem- bers have dominated the selection process for secretaries-general, often sup- porting candidates in exchange for promises to reserve senior posts for their own nationals. Multiple examples are provided, for instance how since 1997 the Department of Peacekeeping Operations has been led by four successive nationals of France, and how five successive US nationals headed the Depart- ment of Management from 1992-2007 (since 2007, Americans have led the Department of Political Affairs). There are similar cases of posts held by UK, Russian and Chinese nationals for multiple terms. This practice thus prevents nationals from other countries from holding these positions. 1 for 7 Billion argues that the selection process should be based exclusively on merit, as stip- ulated in the UN Charter, so that the secretary-general can recruit a strong lead- ership team from all the world’s regions (1 for 7 Billion 2016). The Commission on Global Security, Justice and Governance made related calls, stating that, through the General Assembly, member states politicize hir- ing at all levels by micromanaging the budget. As well, despite having some influence on the selection process, the Secretary-General can neither appoint nor let go the heads of UN agencies, programs, and funds. The Commission highlighted a need for the Secretary-General to have greater discretion to man- age the Secretariat. For the appointment of under-secretaries-general and as- sistant secretaries-general, it proposes that member states continue to approve the selections, but that the Secretary-General be presented with an array of candidates from which to choose (2015: 93). 136 A second issue increasingly in the spotlight relates to management prac- tices. In former Australian Prime Minister Kevin Rudd’s recent report, he calls for “deep reforms of the UN management system”, arguing that the organiza- tion relies on “rigid staffing” and that its “excessively hierarchical structure is a legacy from an earlier age” (2016: 21,35). 5. Improved staffing and management practices He draws attention to some of the main challenges, from the inability of managers to move employees either within or between agencies, to the length of time involved in hiring people (up to 12 months) and the difficulty of firing people. He also disapproves of what he refers to as the “demise of a permanent, professional, international public service”, whereby the ability of long-standing and highly-experienced UN staff to rise to senior management positions is being weakened by an increase in external, political appointments. He makes a strong case for a flatter, flexible, effective, and cross-disciplinary structure that can better respond to complex challenges. Finally, Rudd claims that more of the Secretariat’s staff should be in the field, rather than at headquarters (at present they are roughly equally divided). This would help shift the UN’s work from report writing to executing its man- date on the ground. At the same time he states that the opportunities for ad- vancement are disproportionately at headquarters, and that deserving field staff should be encouraged, rewarded and promoted (ibid: 33). Weiss (2009) has echoed these sentiments, calling for an end to the tendency for promotions to result from work and contacts in pleasant headquarter settings. 6. Autonomous emergency services for the UN It is unlikely that reformed structures alone will be enough to help the United Nations to fulfil its mandate for global protection and development. The or- ganization will also require additional resources to give it the autonomy to carry out its work. This proposal will look at a suggestion that has come from multiple experts for giving the UN its own emergency, rapid reaction peace force, while the next will cover ideas for independent sources of income. One of the leading proponents of a United Nations Emergency Peace Ser- vice (UNEPS), Peter Langille (2015), has pointed out that UN peacekeepers, over the years, have helped improve conditions in 69 armed conflicts world- wide. The chief problem is that it currently takes 6-12 months for the UN to mount a peace operation—as opposed to the seven days it took to deploy forces to the first UN peacekeeping in the Suez in 1956. In addition, Western coun- tries with their advanced militaries are now only contributing a minimal num- ber of the 120,000 troops the UN has in the field. In part because of this slow reaction, the UN is now spending $8.2 billion annually on peacekeeping, but only after conflicts have spread, thousands have died and countries have been destroyed. 137 Creating a UNEPS would help prevent the spread of conflict along with the ensuing mass atrocities and huge costs. Its principal characteristics would be:  a permanent standing, integrated UN formation;  a permanent standing, integrated UN formation;  highly trained and well-equipped;  ready for immediate deployment by the Security Council;  composed of soldiers, police and civilian experts (‘multidimensional’);  capable of diverse assignments (e.g., security, environmental and health crises);  13,000 to 15,000 professional volunteers;  equitable regional and gender representation; and  a first responder to cover the initial six months until member states can deploy.  ready for immediate deployment by the Security Council; y p y y y  composed of soldiers, police and civilian experts (‘multidimensional’);  capable of diverse assignments (e.g., security, environmental and health crises); )  13,000 to 15,000 professional volunteers;  equitable regional and gender representation; and q g g p  a first responder to cover the initial six months until member states can deploy. The central aim of UNEPS would be to deter aggression and its spread. 6. Autonomous emergency services for the UN There would be sufficient military forces and police to restore and maintain order and civilian teams to provide essential services. Its approximate start-up costs of $3 billion and recurring costs of $1.5 billion annually, while significant, are trifling in comparison to the $13.6 trillion in annual military expenditures glob- ally (Global Peace Index). A very similar proposal has been presented in great detail by Robert Johansen in “A United Nations Emergency Peace Service: To Prevent Genocide and Crimes Against Humanity”. 7. Financing the UN Over the years, UN proponents have put forth a whole series of ideas for find- ing innovative, alternative sources of funding for the organization. The basic problem is that the regular assessments of member states (even when they are paid) have never provided a sufficient budget to finance the UN’s multitude of operations. When these are supplemented by voluntary contributions from states, they are still insufficient and tend to reflect the priorities of the particular donor. They have been called the ‘UN à la carte’. So, there has been an ongoing search for additional and independent financial resources for the UN. There is no lack of ingenuity in proposals, which have included imposing levies (a sort of international tax) on air and sea travel, arms sales, transnational movements of currencies, international trade, the production of polluting materials, and production from the global commons such as mining of sea nodules—even an annual UN lottery has been suggested. The trick is to keep it international, find huge areas of untaxed transactions, and place the levy low so that it will not cause a backlash. A good example is the air travel tax that France introduced in 2005 to finance Unitaid, which was implemented by nine countries. Yet none of these relatively simple and ingenious ideas has ever been im- plemented on a global scale. Many critics believe that member states have not 138 given the UN permission to impose such levies because they do not want the organization to have its own independent sources of revenue. Rather, they wish to retain full control. It is this attitude on the part of governments that must be one of the major targets of a reform movement. This latter consideration caused Joseph Schwartzberg to propose that the easiest path would simply be for the UN to return to its original financial well in a more astute manner. He proposes scrapping the present complicated and contentious system used to calculate member state assessments, and replacing it by one wherein all states are assessed at a “very small, affordable and equal percentage” (say 0.1 per cent initially) of their respective gross national in- comes (GNI). He calculates that if such a GNI assessment had been in place in 2010, it would have generated $58.65 billion—more than twice the total cur- rent spending for the entire UN system (2013: 201-24). 7. Financing the UN Tax evasion is another issue that harms national budgets, and thus the UN’s budget. In his book Options for Strengthening Global Tax Governance, Wolf- gang Obenland calls on the UN to create new institutions for a platform for tax collaboration. He claims the world is losing hundreds of billions in tax reve- nues annually because of a lack of international cooperation on tax issues which permits rich individuals and corporations to evade or avoid taxes in off- shore tax havens or to demand preferential treatment in their host countries. 8. Principles and criteria for the Responsibility to Protect 8. Principles and criteria for the Responsibility to Protect It is time to complete what the United Nations neglected when it adopted the resolution on the Responsibility to Protect (R2P) at the UN Summit in 2005. In itself, it was one of the most significant steps to place some limits on the abuse of national sovereignty. But the resolution omitted two of the most im- portant elements of the report of the International Commission on Intervention and State Sovereignty, which had formulated the concept of R2P. These were the ‘Principles for Military Intervention’ that formed the criteria for when the Security Council needed to act, and, even more alarmingly, the whole chapter on ‘the Responsibility to Prevent’ which dealt with early warning, analysis of root causes, and the techniques of prevention. Without clear principles for intervention, the UN lacked the key instruments for deciding when to put R2P into action. Some wicked tongues claimed this was exactly the intention of some of the P5. If this was the case, it backfired. When the Security Council authorized the NATO intervention in Libya, the principles of ‘right intention’ and ‘proportional means’ and the operational ‘principle of limitations’ were not on hand to guide the resolution. So, NATO went on to overthrow the government of Libya rather than just halting or avert- ing human suffering. As we have discussed, this has been the basis of Russia and China vetoing UN intervention in Syria with the results we know. 139 The Commission put forth six principles that should be respected before the UN would decide on military intervention in the name of protecting civilian populations:  The ‘just cause threshold’ stated that there had to be actual or appre- hended large scale loss of life or ethnic cleansing. Human rights viola- tions, the overthrow of democracy, or the desire to rescue one’s nation- als were not sufficient cause.  The ‘right intention’ principle said the intervention must be to avert human suffering which could best be assured by ‘multilateral opera- tions’ supported by regional opinion and the victims concerned.  The ‘last resort’ principle stipulated that every form of negotiation and non-military forms of arm-twisting had to have been exhausted.  The principle of ‘proportional means’ asserted that the scale, duration and intensity of the intervention has to be the minimum necessary to achieve the human rights objectives. 8. Principles and criteria for the Responsibility to Protect  The principle of ‘proportional means’ asserted that the scale, duration and intensity of the intervention has to be the minimum necessary to achieve the human rights objectives.  The principle of ‘reasonable prospects’ insisted there has to be reason- able chances of success and that action will not be worse than inaction.  The principle of ‘right authority’ stipulates that the Security Council is the appropriate body for authorizing interventions, but it should act promptly. In addition, the Permanent Five should not apply their veto power except when their vital state interests are involved. If the Secu- rity Council fails to act in a reasonable time, an alternative option would be for the General Assembly to consider the matter in an Emergency Special Session under the ‘Uniting for Peace’ procedure, or a regional organization might take action. In addition to these R2P principles for military intervention, the Commission also proposed ‘operational principles’ including clear objectives, unity of com- mand, limitations in the application of force (“for the protection of the popula- tion, not for defeat of the state”), adherence to international law, and coordina- tion with humanitarian organizations. It is high time the UN gave due consideration to these criteria and principles for the use of R2P. 9. The dispersion and control of global power Most experts agree that providing the United Nations with greater authority is a necessity. Thomas Weiss has courageously taken the bull by the horns in his recent publications. In his 2009 book, What’s Wrong with the United Nations and How to Fix It, he closes with the statement, “global government rather than global governance is a necessary part of future analytical perspectives.” (232). Latterly, in Governing the World, Weiss concludes, “We cannot continue to 140 ignore and rationalize the absence of overarching authority… Humanity col- lectively is capable of better and more fairly governing the world.” (2014a: 101). Similarly, French thinker Jacques Attali summed this view up well, stating “Neither an empire nor the marketplace will be able to master the immense problems which await the world. For that it will require a world government. This government will take a form fairly close to today’s federal regimes. ... Leaving to the national governments the responsibility for assuring the specific rights of each people and the protection of each culture, the global government will be in charge of the general interest of the planet and verifying that each nation respects the rights of each citizen of humanity.” (2011: 10). However, there is a widespread fear of a more authoritative global institu- tion. Since the time of Immanuel Kant, the fear of a global leviathan has been one of the main arguments against giving enhanced authority to the United Nations or any other international institution. Unfortunately, even cosmopoli- tans share this fear of big government—even if, aside from this, they are some of the strongest proponents of a reformed international system. It is reflected too in science fiction where we have the forces of the ‘Federation’ confronting those who want to install an evil and all-powerful oppressor. One of the most serious warnings came from Michael Barnett and Martha Finnemore in their book Rules for the World. Their research focused on inter- national organizations as bureaucracies. They point out that we have known ever since the work of the German sociologist, Max Weber, that bureaucracy is a distinctive social form with its own internal logic that generates specific behavioural tendencies which might be good or bad. According to the authors, bureaucracies can govern complex situations be- cause of their rules-based, hierarchical, continuous, impersonal, and expert na- ture. They control access to knowledge and their rational process makes them both proficient and efficient. 9. The dispersion and control of global power Moreover, rules shape their activities, under- standings, identity, and practices, along with how bureaucrats see the world and perceive social problems. Bureaucrats often make rules that prescribe the behaviour of others. When we think about the activities of the UN and the European Commission in this light, we can see why there has been growing reaction against often inappropriate rules applied by distant, impersonal, so- called experts who are unaware of local conditions. This is, in large part, driv- ing the rise of populism. The analysis by Barnett and Finnemore shows that there is a danger lurking in the nearly 300 international organizations that manage much of the world’s relationships. These organizations are lauded for advancing liberal goals such as human rights and economic growth, but behind this liberal façade is the reality that they do so using undemocratic procedures. They note how Weber recognized that “a bureaucratic world had its own perils, producing increas- ingly powerful and autonomous bureaucrats who could be ‘spiritless’ and 141 driven by rules, who could apply those rules in ways that harmed the people whom they were expected to serve.” (2004: 172-73). We must take these warnings to heart as we contemplate ever-more power- ful international organizations. There will be bureaucracies. Yet the well- founded concerns some have of a ‘world government’ can be addressed by designing transformed global institutions based on the diffusion and control of power. As Trent began to elaborate in his Modernizing the United Nations Sys- tem (2007: 237-40, 256-8), the best way to do this is by applying what democ- racies have learnt about diffusing and controlling power during the past two hundred years. The techniques of federalism, subsidiarity, checks and bal- ances, the division of powers, constitutionally guaranteed freedoms, rights and equality, liberalism, the rule of law, transparency and participation all help to decentralize power. To these can be added the more specific freedoms of speech, thought, association and the freedom of the press. While at first glance it may not appear so, the rising inequality between and within countries also has a tendency to reinforce established power. The wealthy often use their money to influence the political elite to develop policies that reinforce their wealth—creating a vicious circle. The control and dispersion of power must include the struggle against inequality. Democracy itself is at the core of the dispersal of power. 9. The dispersion and control of global power Many associate democracy with electoral techniques, but it is far more complex than that. There is a long list of attributes often considered as the defining properties of democracy: free, fair and frequent elections, competition, participation, con- testation, civil liberties, rights, freedom of expression, information and associ- ation, rule of law, an effective state, accountability, equality, inclusive citizen- ship, individual and minority rights, checks and balances, constitutional pro- tection, and an active civil society (Geissel et al, 2016). Taken together, these properties ensure the circulation, distribution, division and control of power, a key objective of democracy. Two centuries ago, few gave the nascent democ- racies much chance of persisting but because of the control of power we have come a long way. These techniques must be replicated in any proposals for the reform of global institutions. Another concept we may briefly expand upon is world federalism. Federal- ism is a mechanism for creating rules and norms for the distribution of juris- dictions and for a continuing, principled struggle for power. Under federalism, each political unit maintains its own ‘sovereign’ responsibilities and authori- ties. In functioning federations with a constitutional enforcement and multiple political parties, there is no concentration of power. It is a fundamental tech- nique for ensuring that the ‘centre’ does not accumulate too much ‘authority’. It is also what has allowed vast territories such as Canada and the United States to have one central government along with a great dispersal of power in the provinces/territories or states. Thus, world federalism would promote a contin- uing balancing of power between states and a UN with more authority. So, we 142 can assume that if the UN had small but autonomous emergency peace forces and sources of funding, the member states would still have their national mili- tary and police forces along with their own legislatures, judiciaries, public ser- vices and everything else that goes to make sovereign nation-states. It should be added that so-called federalism is also the governing authority in countries such as Russia which are not exactly shining models of democracy and freedom. So, we must understand that federalism is a necessary technique for the dispersion of power but by no means a sufficient one for ensuring the decentralization and control of authority. 9. The dispersion and control of global power Worrying about the techniques of global democracy and world federalism may not appear to be of immediate concern, but considering the multiple tech- niques for the control and distribution of power in international organizations with more decision-making authority needs to be a priority. Sequencing reform proposals: where to start This list of possible transformations of the UN leads immediately to the ques- tion: where to start? The next section, which will conclude this book, explores a possible path to mobilizing public and political support for reform so it is placed on the international agenda. Before getting to this, it is necessary to consider how to begin to sequence reform proposals. As we have said, it is not our intention to present a comprehensive program or step-by-step plan for reform. Rather, we wanted to demonstrate to the reader the extent of thinking that has already gone into UN reform and offer a panoply of proposals to work from. There are many more that have come from scholars, civil society and other UN experts. To sort or rank these issues is no small task. Developing a methodological approach is helpful. Some of the questions we need to ask when considering how to prioritize are: How urgently is the reform needed? How large is it in scale and scope? How easily could it be achieved? What would the cost be? How much time might it take? Is there/would there be political will? Is there/would there be public support? How developed are the proposals? Is there consensus among experts on what needs to be done? Is there a strong champion behind it? Joseph Schwartzberg (2013) gives considerable thought to the sequencing of UN reforms. He suggests that some changes are easier to get accepted, and that starting with these may open the door to further reforms. His preference would be to begin with: improving UN funding via a new assessment system; regaining legitimacy through weighted voting in all the UN organs (i.e., in- cluding population and the economic contributions of states in their voting power rather than just one-state-one-vote); creating a Security Council that is universally representative with weighted regional voting and no veto; and es- tablishing a standing, all volunteer UN peace force. 143 Few disagree that the UN must have its own sources of revenue, although many would prefer this to come from some form of taxation. Many also accept the need for some form of emergency peace force. Still, to get these two re- sources, the organization must have legitimacy. This is why weighted voting is such an important concept. The European Union has used weighted voting and it appears to work quite well. Sequencing reform proposals: where to start In the context of global institutions, it could help address the issue of large countries fleeing the cacophony of the UN to- wards the G7 and G20, in part to have their voices heard for their just value. Four steps toward the renaissance of the United Nations We have come to the conclusion that significant groundwork needs to be done before reforms will get under way. The whole issue of renewing the UN must be placed on the international agenda. This will take considerable leadership, education and communication. At the same time, overcoming established in- terests and natural tendencies for maintaining the status quo will require im- mense mobilization of popular support to create political will. In all likelihood, neither of these will be achieved without the efforts of a committed and sus- tained movement pushing for change in this direction. This final section ex- plores how we can bring about a civil society-led UN renaissance movement, and how strategic campaigning and advocacy can garner the public support and political will required for transformative change. 1. Mapping actors and interests 1. Mapping actors and interests A useful first step in building a successful movement for reform, one that gar- ners enough popular support to lead to political action, is having a deep under- standing of the players involved. This is as true for allies as it is for opponents. Let’s begin by identifying potential allies to help spot opportunities for co- alition building. A first step is seeking out civil society organizations that ad- vocate for more effective global governance and share a common vision for a fairer and more legitimate UN. There are also a number of groupings of states that have voiced their concerns about the status quo and have called for reforms at the UN. Some of these governments may be willing to join efforts to collec- tively push for action. There are also allies within the UN and other interna- tional organizations who would like to see a more effective global institution. The core of change will come from a partnership of NGOs and like-minded states plus reformers in the UN. Not all those identified as allies will be focused on the same issue areas, nor will they necessarily agree on how to fix some of the problems. Issue-specific groups or committees can be formed to deal with this, as was done with the NGO Coalition for an International Criminal Court, discussed in Box 18. 144 Of course, not everyone favours a rational and progressive path to more workable global institutions. The most obvious opposition has come from member states themselves, particularly the most powerful ones. The recent wave of right-wing populist nationalism spreading across the West poses an additional threat to attempts at reform, as anti-globalism sentiment is paired with isolationist policies, trade barriers, and restrictions on the movement of people. Such politicians are responding to the understandable frustration that many have felt as a result of the gains from globalization not being equitably shared, and from the challenges that have come with greater regional and global integration. Yet rather than working to improve global institutions to better manage the harmful consequences of living in a globalized world, they are proposing to reverse the positive achievements of the past decades, includ- ing gains made on climate change policies, trade, regionalism and multilater- alism, diplomacy, efforts to improve taxation and environmental regulation, humanitarianism, multiculturalism, arms reductions and peace and security. 2. Form smart coalitions of allies For many of the more substantial reforms ideas to materialize, a coalition, or perhaps multiple coalitions, will likely need to be formed to drive a wider movement. Among the attributes of these ‘smart coalitions’ comprising NGOs and some governments are: ideas, leadership, expertise, skilful negotiations, mobilization of networks and resources, concrete agendas, targets and indica- tors, and tools to measure progress and respond to setbacks. In terms of personnel, they will require NGO experts, international practi- tioners, communicators, academics, retired politicians and ideally some repre- sentatives of willing governments. Often, complex reforms must be organized into manageable, issue-specific areas that can be overseen by actors with spe- cific expertise. It is useful to learn from past examples of successful coalitions, such as the Coalition for an International Criminal Court, the International Coalition for R2P, the International Campaign to Ban Landmines, and 1 for 7 Billion, all of which were mobilized by the World Federalist Movement. These campaigns came along when conditions in the General Assembly opened the door to cre- ating momentum for the specific reform. NGOs and governments made use of each other’s relative advantages. NGOs applied their expertise, their ability to inform the media and frame the discourse, and their means for embarrassing dissenting governments through naming-and-shaming campaigns. Friendly governments used their money and resources to manage negotiations and de- cision-making. Campaigns were based on a strong and simple normative mes- sage. Instead of meeting opposition governments head-on (even the P5), the coalitions moved around them through the support of a strong majority of states. The Coalition for an International Criminal Court is explored in greater detail in Box 18. This idea is not new. The report by the Commission on Security, Justice and Governance (which we have analyzed at length in this chapter) called for a World Conference on Global Institutions that could “serve as a rallying point for smart coalitions and simultaneously generate political momentum for mul- tiple, urgent global reforms” (109-110). The Commission proposes that the Conference take place in 2020 to mark the UN’s 75th anniversary, and that it be the culmination of a three-year multilateral negotiation process on global institutional reform. It highlighted the need to include diverse voices, including those at the most local level and those of under-represented groups. 1. Mapping actors and interests Potential opposition could also come from all those who benefit from inter- national conflict, inequality and general disarray. Among them are the wealth- iest one per cent (who collectively control over fifty per cent of the world’s capital), who may not support a more regulated international regime that might, for example, combat offshore tax havens. There are also those who benefit economically from war and world military spending, which in 2016 was esti- mated at $1.7 trillion. And of course there are the many perpetrators of conflict and war. Not all sets of actors will have clearly identifiable interests or roles. The media (which admittedly is a broad term that reflects a wide diversity of or- ganizations, from CNN to DemocracyNow! to Breitbart News) has played dif- ferent roles at different times. On the one hand, there was a great deal of bal- anced, high quality reporting in the mainstream media around the time of the UN’s 70th anniversary that recognized the organization’s many achievements while also pointing to a need for change. At the same time, the media has often painted the UN in a negative light, as was the case in late 2016 when it failed to report on a successful example of countries uniting against Security Council inaction in Syria, opting instead to lambaste it over appointing Wonder Woman as honorary ambassador. Given the fairly direct link between media coverage and public opinion, this example may help explain why we have a relatively ill-informed and ambivalent public. There are additional actors and interests that should be mapped out too, many of which may not be clear allies or opponents. These include various parts of the UN system itself, regional groupings, faith-based organizations, multinational corporations and others that have been explored throughout this book. 145 2. Form smart coalitions of allies A number of scholars have also called for the creation of NGO coalitions for UN reform, including Trent in his 2007 book Modernizing the United Nations System. And indeed, a small number of NGOs are already working toward a 2020 UN re- form congress. 146 Box 18: An NGO coalition that made history Box 18: An NGO coalition that made history Box 18: An NGO coalition that made history The Coalition for the International Criminal Court (CICC) started in 1995 when 25 civil society organizations joined together to advocate for a permanent international criminal court. By 1997, the Coalition had grown to 450 organizations. After three and a half years of intense advo- cacy and deep collaboration among NGOs and between NGOs, govern- ments and the UN Secretariat, governments held a diplomatic conference in Rome and 120 countries adopted the Rome Statute that established the ICC. The Coalition’s role was enormous. The information-sharing, legal analysis and advocacy that it undertook prior to and during the con- ference is widely seen as having shaped the outcome. The Coalition is credited with a number of achievements, namely the independent nature of the Court and its prosecutor, strong victim and witness protection, and the inclusion of gender crimes. g Coordinating the 200+ NGOs that attended the conference and achieving the results they did was a major feat. The Coalition included a steering committee of a dozen NGOs and regional caucuses that re- presented all the world’s regions. Thematic caucuses were formed to delve into specific issues. These were: Women’s Initiative for Gender Justice, Victims’ Rights Working Group, Faith-Based Caucus, Universal Jurisdiction Caucus, Children’s Caucus, and Peace Caucus. The World Federalist Movement–Institute for Global Policy (WFM–IGP) and its Executive Director, William R. Pace, served as Secretariat for the entire network. Over 20 years later, the Coalition remains as active as ever. Its recent efforts in the fight for international justice have focused on encouraging African countries to stay with the ICC, restraining the Security Council veto (which prevented the investigation of crimes in Syria) and trying to halt government attempts to undermine the Court. Source: Coalition for the ICC website 3. Marketing the UN and its achievements to build public support A main task of a smart coalition would be to generate public support for more authoritative global institutions through an effective marketing campaign. Those in other fields and industries seem to understand the importance of mar- keting far better than those desiring UN reform. Consider the entrepreneurs who often spend more time on marketing than they do on inventing. Or the athletes who work as hard on their game plans as they do on muscle building. Or the movie moguls who put as much effort into attracting the public as they do in producing films. Those seeking reforms need to convey the UN’s im- portance as well as its numerous achievements to an audience that remains 147 unconvinced that better global governance is even necessary. This will take considerable leadership, education and communication. It will also take out- side specialists from diverse disciplines—like strategists, communicators, ed- ucators, psychologists, behavioural economists, marketers, and mobilizers. Throughout this book we have observed our collective incapacity to develop policies and take actions to adequately deal with the challenges that go beyond the capacities of any single state. We explored how the UN needs new institu- tional capacities to fill this void, and how a first step in this direction is to continue efforts to harness sovereignty. The question is, how precisely do we lead citizens to look favourably on more UN authority? Some useful insights were provided by Matthias Ecker-Ehrhardt in his arti- cle “Why do citizens want the UN to decide?”. He studied the fifth wave of the World Values Survey and drew several conclusions about public attitudes to- ward giving more decisional authority to the UN. First, he found that, in gen- eral, public support for UN authority is strongly linked to individuals believing that many problems we face are of a global scope and that nation-states are unable to handle them on their own. A second finding is that citizens of pow- erful nations view UN authority more favourably because their power base al- lows them to establish international institutional arrangements that preserve their interests. Finally, he found that the social legitimacy of world institutions is already in short supply. Global governance ‘architects’ or ‘entrepreneurs’— those pushing for reform—should be aware that any attempt to simply ‘upload’ more authority to existing institutions is likely to lead to further backlash. 3. Marketing the UN and its achievements to build public support Ecker-Ehrhardt concluded that institutional inequality and skewed distribution of power must be convincingly addressed before effective global institutions will be widely accepted. The takeaway from this research is that the more people think global chal- lenges surpass the capacities of states, the more they will support global au- thorities—except when they think present world institutions are skewed in the favour of major powers or special interests, in which case they will demand institutional reform that spreads power more equitably. Marketing and com- munications efforts must address these lessons. Of course, engaging the public should not be the sole responsibility of a civil society-led coalition for reform. The UN itself needs to invest more time and energy in its public relations. This observation was made recently by Kevin Rudd, who recommended that the UN appoint a chief communications officer to “overhaul the UN’s communications structure and strategy in order to effec- tively communicate its message to member states, the general public, the me- dia, and the rest of the UN system” (2016: 62). His rationale is precisely what we have argued, “The UN has a good story to tell, but it is not telling it effec- tively.” He proposed that the UN develop a new communications strategy that makes use of all media platforms in multiple languages, and uses plain lan- guage rather than “UN dialect, which is incomprehensible to the rest of the 148 world.” While it is made clear that this would not be a substitute for the indi- vidual communications efforts of UN agencies, Rudd stressed the need for a UN-wide approach to enhancing the integrity of its brand. 4. Transforming public support into political action Can we assume that an effective social movement and a corresponding shift in public opinion will lead to political will for change? The short answer is yes. A wealth of research has been produced over the past decades on the impact of public opinion on public policy, and the evidence is compelling. In his book How Change Happens, Duncan Green, author and Head of Research at Oxfam, explores how activists have succeeded in achieving norm change (2016: 48- 56). While acknowledging that on rare occasions governments have been the source of new norms, he explains how many core features of the state, such as social protection, education and healthcare, were formulated by activists before being taken up by the state. 3. Marketing the UN and its achievements to build public support This was similarly the case with securing various group rights, including women’s rights, children’s rights, disabled people’s rights and gay rights. The typical trajectory is that activists gain public support, and politicians respond to voters’ desires by implementing change. p p y p g g In their piece “When the pillars fall: how social movements can win more victories like same-sex marriage”, Mark and Paul Engler (2014) describe how the fight for marriage equality in the US was one where the consensus view held by the public changed, for a host of reasons, and politicians and legal rulings responded. “The change has come about through a mass withdrawal of cooperation from a past order based on prejudice. It could be felt well before it was written into law, and well before it was acknowledged by those leaders now struggling to show that they have ‘evolved’.” As recently as 1990, 75 per cent of Americans saw homosexuality as immoral. Politicians knew that ex- pressing support for same-sex marriage could end their career. In 1996, the U.S. Senate passed the Defense of Marriage Act, which narrowly defined mar- riage and denied federal benefits to same-sex couples, by an overwhelming majority. But the movement slowly chipped away at the “pillars of support”, the institutions of society that legitimized the status quo. In this case, denying equal rights to same-sex couples was a system propped up by the churches, the media, the business community, the military, the educational system, the courts, and so on. Gains were made on a range of fronts and public support for same-sex mar- riage rose steadily until it reached a tipping point—in 2011, polls showed it to be more than 50 per cent for the first time in history. The tide of public opinion had turned, and legal and political victories came in rapid succession. In 2012, President Obama stated that he had changed his position on the issue, as did Bill Clinton who had led the Defense of Marriage Act just a decade and a half earlier. In a single week in April 2013 six U.S. Senators came out in support 149 for marriage equality. This example presents an important lesson for other so- cial movements: “Rather than being based on calculating realism—a shrewd assessment of what was attainable in the current political climate—the drive for marriage equality drew on a transformational vision.” (Engler and Engler, 2014). 3. Marketing the UN and its achievements to build public support Of course, this case is from the US, which has strong democratic institu- tions. To what extent does the assumption that public opinion leads to political change hold in places with undemocratic regimes? After all, demands are fil- tered through domestic political institutions and the sensitivity of political elites to the demands of voters varies greatly. Yet the “pillars of support” no- tion is every bit as applicable. If civil servants, the military, merchants and all others effectively propping up a regime were to suddenly refuse to cooperate, they could bring down even the most authoritarian dictatorship. By removing enough pillars, the temple will topple and the tyrant will tumble (ibid). It should be clear from this example and others explored in this book that social movements matter. In many cases, they are the single most important factor leading to change. Take for example the research conducted by Laurel Weldon and Mala Htun on what factors drive change in government policies on violence against women around the globe, which Green presents in his book (2016: 55-56). Gathering data from 70 countries over four decades on various forms of state action (legal and administrative reforms, prevention measures, training, etc.) and other factors such as women legislators, level of economic development, political regime. They concluded that, when all else is equal, it was the presence of strong feminist movements that made the single largest impact on whether a country had comprehensive policies addressing violence against women or not. Public opinion and political pressure can and do lead to political action. To summarize, we have now seen from domestic and international politics that creating significant political change has a number of components. In- formed citizens need to recognize a massive public problem, in this case that existing international organizations do not have the capacity to surmount global problems. Activists need to rally around a transformational vision, likely in partnership with politicians and other allies. A movement for reform needs to be launched—ideally one that seeks to undermine the pillars of sup- port for maintaining the status quo. Broad public support needs to be gained, in order to influence the decision-making of elites. “You may never know what results come of your actions but if you do nothing there will be no results” Mahatma Gandhi Concluding remarks History suggests that a reform movement will most likely start when a small number of like-minded states and numerous civil society organizations with strong leadership and communications skills can mobilize widespread and sus- tained support for a UN with adequate authority. This should be an immediate 150 focus of activists concerned with our current collective inability to govern the world. It is worth reminding ourselves that if nationalist and isolationist politics continue to gain traction, all these potential reforms may be cast aside—for the moment. If they are, they will come back into their own as ever more citizens recognize that the renaissance of world institutions is required to deal with global issues. 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Bibliography Citizen journalism 122 Commonwealth 26f., 39 Commonwealth 26f., 39 Communism 29, 72 Canada 37, 64, 69, 72, 114, 119, 142 Concert of Europe 23f., 27, 54 Controlling power 142 Controlling power 142 Coordination of the UN 18, 51, 79, 129, 135, 140 China 17, 33, 35, 37, 40, 48, 59f., 62, 68, 77f., 100, 102f., 108, 110, 113, 115, 122, 133, 139 Churchill, Winston 32f. Citizen journalism 122 Corporate social responsibility (CSR) 84f. Crimes against humanity 18, 44, 63, 110f., 138 161 Freedom of religion 104 Freedom of speech and press 104 Future United Nations Development System (FUNDS) 92-94 FUNDS survey 97 G20 15, 41f., 54, 61, 129, 133, 135, 144 G20+ 129, 135 G7 15, 41f., 54, 144 G77 39, 75, 107 Gaddafi, Muammar 37, 106, 112 Gender equality 16, 48, 71, 86, 92, 125 Darfur 18, 59, 62f., 106, 108, 113 Darfur 18, 59, 62f., 106, 108, 113 Freedom of religion 104 Democracy 38, 60, 67, 122, 140, 142f. Freedom of speech and press 104 Department of Peacekeeping Operations 65, 136 Development First World, Second World, Third World 39, 72 G20+ 129, 135 Development finance 78, 81 Addis Ababa Action Agenda 48, 127 Blended finance 81 Innovative financing 81 Official Development Assistance (ODA) 78 Remittances 78f. G7 15, 41f., 54, 144 G77 39, 75, 107 General Agreement on Tariffs and Trade (GATT) (see also World Trade Organization) 53, 74 General Agreement on Tariffs and d ( ) ( l ld Human Rights 16, 99-101 Human Rights International Bill of Human Rights 100 World court of Human Rights 120 International crimes 44, 110f. International Criminal Court (ICC) 18, 31, 44, 54, 60, 86, 105f., 110f., 113, 144, 146f. International criminal law 111 International criminal tribunals 37, 45, 105, 111, 119 International Criminal Court (ICC) 18, 31, 44, 54, 60, 86, 105f., 110f., 113, 144, 146f. 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Indonesia 69, 104 Inequality 48, 67, 79, 82, 142, 145, 148 Human Rights Watch 113, 122 Human security 56, 75, 114 Humanitarian assistance 74, 119 Hussein, Zeid Ra’ah Al 103 Independent Commission on Multilateralism 126 India 60, 62, 68f., 78, 90, 103, 115 120 133 Human Rights Watch 113, 122 Human security 56, 75, 114 Humanitarian assistance 74, 119 Hussein, Zeid Ra’ah Al 103 Independent Commission on Multilateralism 126 India 60, 62, 68f., 78, 90, 103, 115, 120, 133 Indigenous peoples 16, 85, 100f. Indonesia 69, 104 Inequality 48, 67, 79, 82, 142, 145, 148 Information and communication technology 90 Innovation 18, 25, 49, 84, 91, 114 Institute for Intellectual Cooperation 31 Institutions Authoritative 124 Workable 43, 124, 131, 145 Human Rights Watch 113, 122 Human security 56, 75, 114 Humanitarian assistance 74, 119 Hussein, Zeid Ra’ah Al 103 Independent Commission on Multilateralism 126 India 60, 62, 68f., 78, 90, 103, 115, 120, 133 Indigenous peoples 16, 85, 100f. General Agreement on Tariffs and Trade (GATT) (see also World Trade Organization) 53, 74 Development system, UN 18, 82, 90-92, 94, 97 Delivering as One 93 Drummond, Eric 28 East Timor 59, 66, 111 General Assembly Committees 40, 134 General Committee 40, 134 East Timor 59, 66, 111 Genocide 18, 44, 63, 106, 110f., 113f., 121 Economic and Social Council Economic and Social Council (ECOSOC) Functional commissions 42 Regional commissions 42 George, David Lloyd 27 Germany 30, 52, 68f., 76, 111 Global Compact 54, 84, 91, 119 Global partnerships 82, 87 Global partnerships 82, 87 Economic Community of West Africa (ECOWAS) 59 y Africa (ECOWAS) 59 Economic, Social and Environmental Council (ESEC 73, 134 El Salvador 59, 66, 107 European Recovery Program (see Marshall Plan) 72 European Union 16, 29, 36, 66, 144 Evolutionary historicism 125 Federation 141f. First World War 26, 28-31, 33 Food and Agriculture Organization (FAO) 32, 73 France 23, 25f., 28, 30, 35, 52, 62f., 68, 98, 115, 136, 138 Franco-Prussian war 23 Global power, dispersion and control of 140 Economic, Social and Environmental Council (ESEC) 73, 134 Global South 75, 78, 82, 87, 101, 129 Global South 75, 78, 82, 87, 101, 129 Globalization (see also Anti- globalism) 15, 45, 75, 85, 126f., 145 Global challenges 18, 56, 82, 126, 148 Global institutions 13f., 124, 126, 128f., 142, 144-148 G d 53 71 79 Global South 75, 78, 82, 87, 101, 129 Globalization (see also Anti- globalism) 15, 45, 75, 85, 126f., 145 Global challenges 18, 56, 82, 126, 148 Global institutions 13f., 124, 126, 128f., 142, 144-148 Good governance 53, 71, 79 Great Depression 29, 35 Great power politics (see also Permanent Five) 15 Groups of Friends 59 Guterres, Antonio 47f., 119 El Salvador 59, 66, 107 El Salvador 59, 66, 107 European Recovery Program (see Marshall Plan) 72 European Union 16, 29, 36, 66, 144 Evolutionary historicism 125 France 23, 25f., 28, 30, 35, 52, 62f., 68, 98, 115, 136, 138 Franco-Prussian war 23 162 Internally displaced persons 116 International Atomic Energy Agency 51, 62 International Ban on Landmines 122 International Bank for Reconstruction and Development 52 International Commission on Intervention and State Sovereignty 24, 114, 139 International Court of Justice (ICJ) 34, 40, 43f., 60f., 105, 111, 120 International Law Commission 44 Hammarskjöld, Dag 46f. Internally displaced persons 116 International Atomic Energy Hammarskjöld, Dag 46f. Hammarskjöld, Dag 46f. High Commissioner for Human Rights 103, 105, 109f., 119, 121 Hitler, Adolf 30 Hull, Cordell 33 Human rights Collective rights 99, 101 Negative vs. positive rigths 101 Traditional values 102 Universal Declaration of Human Rights 16, 99-101 i h High Commissioner for Human Rights 103, 105, 109f., 119, 121 Hitler, Adolf 30 Hull, Cordell 33 Human rights Collective rights 99, 101 Negative vs. positive rigths 101 Traditional values 102 Universal Declaration of Human Rights 16, 99-101 Human Rights High Commissioner for Human Rights 103, 105, 109f., 119, 121 International Ban on Landmines 122 International Bank for 122 International Bank for Hitler, Adolf 30 Hull, Cordell 33 Reconstruction and Reconstruction and Development 52 International Commission on Intervention and State Sovereignty 24, 114, 139 International Court of Justice (ICJ) 34, 40, 43f., 60f., 105, 111, 120 International Law Commission 44 Universal Declaration of Human Rights 16, 99-101 Indonesia 69, 104 Inequality 48, 67, 79, 82, 142, 145, 148 Information and communication technology 90 Innovation 18, 25, 49, 84, 91, 114 Institute for Intellectual Cooperation 31 Institutions Authoritative 124 Workable 43, 124, 131, 145 37, 45, 105, 111, 119 International Development Association 52 International humanitarian law 45, 111 International Monetary Fund 32, India 60, 62, 68f., 78, 90, 103, y 42, 47, 51f., 74 Internationalism 24-26, 28, 30, 125 Iran 17, 60, 62-64, 103f. Israel 60, 62, 108, 110 Italy 14, 26, 28, 30, 69 Japan 28, 30, 52, 54, 68f., 75, 108 Latin America 26, 39, 42, 69, 118 Law of armed conflict 111 League of Nations League Secretariat 33 Permanent Court of International Justice 28, 35 Innovation 18, 25, 49, 84, 91, Latin America 26, 39, 42, 69, 118 Authoritative 124 Workable 43, 124, 131, 145 163 Legitimacy 16, 18, 38, 58, 61, 67f., 85, 87, 113, 125, 131, 133, 143f., 148 Non-Aligned Movement (NAM) 39, 61, 75, 107 Non-Aligned Movement (NAM) 39, 61, 75, 107 Legitimacy 16, 18, 38, 58, 61, 67f., 85, 87, 113, 125, 131, 133, 143f., 148 Non-intervention 24, 114, 116 Lesbian, Gay, Bisexual and Transgender (LGBT) rights 104 Non-permanent members 38, 69, 129, 132 Lesbian, Gay, Bisexual and Transgender (LGBT) rights 104 Leviathan 141 Libya 14, 37, 102, 112, 115-117, 139 North Atlantic Treaty Organization (NATO) 36f., 58, 66, 139 Leviathan 141 Libya 14, 37, 102, 112, 115-117, 139 North Korea 57, 60, 62f., 122 North Korea 57, 60, 62f., 122 North-South divide 75, 101 North-South divide 75, 101 Major powers 13, 33, 35, 46, 62, 110, 124, 133, 148 Nuclear weapons 17, 61f. Comprehensive Test Ban Treaty 62 Nonproliferation Treaty 16 Marketing 147f. Marshall Plan 72 Member state assessments 139 One-state-one-vote 143 Middle East 14, 20, 28f., 37, 58, 115, 118f. Organization of the Islamic Cooperation 107 Orlando, Vittorio 27 Pakistan 62, 69, 93, 104, 107 Pandemics 14, 18, 60, 94f. Black Plague 29 Ebola 18, 95 Spanish influenca 29 Zika 14 Migration 14, 15, 57, 116, 119 Migrants 14, 63, 116f. p Organization (UNIDO) 134 Second World War 25f., 29, 32, 34f., 52, 58, 61f., 72, 98, 111, 116f. Second World War 25f., 29, 32, 34f., 52, 58, 61f., 72, 98, 111, 116f. UN Information Office 32 UN Parliamentary Network 129 Secretary-General 1 for 7 Billion campaign 48, 136 Self determination Declaration on the Granting of Independence to Colonial Countries and Peoples 72 Smuts, Jan 26, 27 Social enterprise 84 Social media 14, 79, 88, 90, 104, 118, 122 Somalia 66, 116f. South Africa 26f., 60, 68f., 78, 83, 112f., 115 Soviet Union 30, 32f., 46, 57, 62, 75, 107 Secretary-General 1 for 7 Billion campaign 48, 136 Self determination Declaration on the Granting of Independence to Colonial Countries and Peoples 72 Smuts, Jan 26, 27 Social enterprise 84 Social media 14, 79, 88, 90, 104, 118, 122 Somalia 66, 116f. South Africa 26f., 60, 68f., 78, 83, 112f., 115 Soviet Union 30, 32f., 46, 57, 62, 75, 107 UN Relief and Rehabilitation Human Rights 16, 99-101 Talleyrand, Prince 24 Terrorism 13, 59 Terrorism 13, 59 State-sponsored terrorism 63 Thirty Years War 23 Torture 100, 103ff., 111, 116f. T f L k 120 State-sponsored terrorism 63 Thirty Years War 23 Torture 100, 103ff., 111, 116f. Treaty of Lucknow 120 Regionalism 106, 145 Regionalism 106, 145 Treaty of Vienna (see Concert of Europe) 23, 27 Treaty of Vienna (see Concert of Europe) 23, 27 Truman, Harry 125 Trusteeship Council 43 Responsibility to prevent 139 Truman, Harry 125 Robinson, Mary 110 Rockefeller Foundation 29, 32 Trusteeship Council 43 UBUNTU (World Forum of Civil Roosevelt, Eleanor 25 UBUNTU (World Forum of Civil Society Networks) 134 Uganda 102, 104, 112 UN Children’s Fund (UNICEF) 41, 49, 73, 93f. UBUNTU (World Forum of Civil Society Networks) 134 Uganda 102, 104, 112 Roosevelt, Franklin D. 26, 32f., 35, 124f. UN Children’s Fund (UNICEF) 41, 49, 73, 93f. Rudd, Kevin 126f., 137, 148f. Rwanda 18, 45, 60, 62f., 66, 93, 110f., 114, 119 UN Conference on Trade and Development (UNCTAD) 134 UN Industrial Development , , San Francisco Conference 32, 34 Sanctions 28, 35f., 58, 60f., 105 Second World War 25f., 29, 32, 34f., 52, 58, 61f., 72, 98, 111, 116f. San Francisco Conference 32, 34 Sanctions 28, 35f., 58, 60f., 105 San Francisco Conference 32, 34 Development (UNCTAD) 134 Development (UNCTAD) 134 UN Industrial Development p ( ) UN Industrial Development Sanctions 28, 35f., 58, 60f., 105 Administration 32 UN Summit 2005 114-116, 139 UN War Crimes Commission 32 UN War Crimes Commission 32 UN Women 16, 92 Unilateralism 124 United Kingdom 16, 57, 81 i d i UN Women 16, 92 Unilateralism 124 Countries and Peoples 72 Human Rights 16, 99-101 Military intervention 17, 114f., 140 Millenium Development Goals (MDGs) 76-81, 83, 96 Mobilization 88, 144, 146 Monnet, Jean 29 Monroe Doctrine 26 Mozambique 66, 93, 103 Millenium Development Goals (MDGs) 76-81, 83, 96 Mobilization 88, 144, 146 (MDGs) 76-81, 83, 96 Mobilization 88, 144, 146 Mobilization 88, 144, 146 Monnet, Jean 29 Monnet, Jean 29 Pasvolsky, Leo 33 Monroe Doctrine 26 Peace and Security Agenda for Peace 64 Brahimi Report 66 Conflict prevention 47, 128 disarmament 17, 26, 40, 65f. Peace enforcement 36, 64f. Peacebuilding 36f., 64-66 Peacemaking 36, 64-66 Peace Conference (Paris 1919) Mozambique 66, 93, 103 Multilateralism 16, 32, 38, 127, 145, 151 Multinational corporations (see also Business and Private sector) 13, 15, 54, 145 disarmament 17, 26, 40, 65f. disarmament 17, 26, 40, 65f. Peace enforcement 36, 64f. Multi-sector partnerships 83, 97 Climate Resilience Initiative 83 Every Woman Every Child 83 Global Eduction First Initiative 83 Global Pulse 83 Mussolini, Benito 30 Namibia 66, 112 Napoleonic Wars 23 New Deal 32, 35 Peacebuilding 36f., 64-66 Peace of Westphalia 23f. Peace of Westphalia 23f. Peacebuilding Commission 129 Permanent Five or P5 35, 37, 48, 56, 68, 140 Pilla, Navanethem (Navi) 110 Populist nationalism 145 164 Specialized agencies 25, 42, 49, 51, 73f., 92, 134 Specialized agencies 25, 42, 49, 51, 73f., 92, 134 Stalin, Joseph 32 Suez 36, 65, 137 Private sector (see also Business and Multinational corporations) 48f., 52, 54, 78, 83-85, 97 Private sector (see also Business and Multinational corporations) 48f., 52, 54, 78, 83-85, 97 Queen Elizabeth I 25 Refugees Refugee Convention 116f., 119 Refugee crisis 18, 29, 117, 119 Refugee Settlement Commission 29 Regional human rights courts 120 Regionalism 106, 145 Responsibility to prevent 139 Robinson, Mary 110 Rockefeller Foundation 29, 32 Roosevelt Eleanor 25 51, 73f., 92, 134 Stalin, Joseph 32 Suez 36, 65, 137 48f., 52, 54, 78, 83-85, 97 Queen Elizabeth I 25 Refugees Refugee Convention 116f., 119 Refugee crisis 18, 29, 117, 119 Refugee Settlement Commission 29 Regional human rights courts 120 Regionalism 106, 145 Responsibility to prevent 139 Robinson, Mary 110 Rockefeller Foundation 29, 32 R l El 25 Queen Elizabeth I 25 Refugees Refugee Convention 116f., 119 Refugee crisis 18, 29, 117, 119 Refugee Settlement Commission 29 Regional human rights courts Sustainable Development Goals (SDGs) 16, 71, 77f. (SDGs) 16, 71, 77f. United Kingdom 16, 57, 81 United Kingdom 16, 57, 81 Smuts, Jan 26, 27 United Nations United Nations Achievements and failures 16 International civil service 28, 46, 136 Social enterprise 84 United Nations Development Programme (UNDP) 49, 81, 93f. South Africa 26f., 60, 68f., 78, 83, 112f., 115 South Africa 26f., 60, 68f., 78, Soviet Union 30, 32f., 46, 57, 62, 75, 107 United Nations Educational, Scientific and Cultural United Nations Educational, Scientific and Cultural 165 Organization (UNESCO) 41, 49, 73, 83 West African States 36, 66 Wilson, Woodrow 26-29, 124f. United Nations Emergency Peace Service (UNEPS) 137f. Woolf, Leonard 27 World Commission on Environment and Development 71, 134 United Nations High Commissioner for Refugees (UNHCR) 49, 117-119 World Federalists Movement 48 Uniting for Peace Resolution 57, 64 World Food Programme (WFP) 93f., 118 World Trade Organization (WTO) 51, 53, 75, 96, 127, 135 War crimes 18, 36f., 44, 63, 100, 110-114, 132 Washington Consensus 53 Washington Consensus 53 World Values Survey 148 Wright, Qunicy 30 Weighted voting 41, 125, 133, 143f. 166 166
https://openalex.org/W4246871614	https://hal.archives-ouvertes.fr/hal-02471294/file/Publi.pdf	English	null	Caryophyllene as a Precursor of Cross-Linked Materials	null	2,019	cc-by-sa	7,720	Keywords: terpene; sesquiterpene; ROMP; elastomer; vulcanization; click-chemistry; solvent- free. Caryophyllene as a precursor of cross-linked materials Anderson M. M. S. Medeiros,† Cédric Le Coz,† Etienne Grau†* Anderson M. M. S. Medeiros,† Cédric Le Coz,† Etienne Grau†* † Univ. Bordeaux, CNRS, Bordeaux INP, LCPO, UMR 5629, F-33600, 16, Avenue Pey Berland, 33607, Pessac, France. * etienne.grau@enscbp.fr Abstract: This paper aims at the synthesis of a new type of elastomers from caryophyllene. The adopted strategy was to cross-link the polycaryophyllene, which was synthesized by ring-opening metathesis polymerization (ROMP). The polycaryophyllene obtained showed Mn = 2 x 104 g.mol-1 (Đ = 1.5) with a glass transition temperature (Tg) of -35 °C. On the first hand, thermal cross-linking was performed in the presence of organic peroxides or sulfur system. On the second hand, thiol-ene coupling initiated by UV-light at room temperature was also investigated as an alternative pathway to cross-link the polycaryophyllene. The materials obtained were analyzed by TGA, DSC, and DMA. The Tg of cross-linked polycaryophyllene could be easily modulated from -35 °C to a range between -25 and 10 °C by changing the type of cross-linking agent. The curing process led to the improvement of thermal stability ranging from 200 °C to around 340 °C. Finally, the network storage modulus varied from 1 to 100 MPa at room temperature. Keywords: terpene; sesquiterpene; ROMP; elastomer; vulcanization; click-chemistry; solvent- free. 1 Introduction New strategies to develop materials from renewable feedstock have been continuously sought as greener alternatives to commercial oil-based materials. There is a significant interest in polymeric materials, taking into consideration their wide range of applications, especially for elastomers. The elastomers market has witnessed significant growth in the global production in recent years, with an estimated increase in revenue from current US$ 80.73 billion to close to US$ 104.17 billion by 2026.1,2 The adoption of vulcanized thermoplastics (TPV) in various application sectors has led to this development. Among several categories of thermoplastics, the natural rubbers (NR) emerge as a major players. The NR segment plays an important role in the TPV global market since the NR is one of the most used thermoplastic thanks to their versatility.2 Due to their properties such as high thermal stability, good chemical resistance, high tensile strength, low shrinkage, and greater design flexibility, they can be employed as devices and/or tools for automotive, medical, consumer goods, industrial, and other applications.3,4 In the framework of looking for an alternative to oil-based consumer goods, an important number of research groups have concentrated their studies and described the synthesis of vegetable-oil-based thermoplastics, such as polyester, polyurethanes, polyamides, and many other bio-based polymers.4–10 Terpenes and terpenoides have drawn considerable attention as potential starting precursors in the synthesis of elastomers.11–18 Studies on the synthesis of elastomers from pinene, myrcene, and limonene19–24 can be readily found in the scientific literature and some commercially available resins and adhesives (e.g. Piccolyte®) derived from polypinenes are already reported. However, there is little exploitation of sesquiterpenes by the scientific community.25,26 Among molecules defined as sesquiterpenes, caryophyllene and humulene stand out, since these molecules are made of interesting structural elements. These molecules are found in many essential oils such 2 2 as rosemary, cannabis sativa, hops, and mostly clove oil.27–29 With a worldwide production of 190 million metric tons per years, clove oil is the main source of caryophyllene. Indeed, clove oil primary use is to extract the eugenol from the oil by distillation. Caryophyllene, which represents around 10% of the clove oil, is a waste in this process and is usually burned. All these led to the fact that caryophyllene being one of the cheapest and most abundant sesquiterpene. Introduction Moreover, from a chemical point of view, caryophyllene is a versatile and significant molecule due to a bicyclic and a cyclobutane rings as well as unsaturated bonds present in its chemical structure.28 Thanks to them, the caryophyllene can be easily modified by attaching different moieties to its backbone chain. Moreover caryophyllene is, to the best of our knowledge, the only readily available bio-based molecule that can be polymerized by ring-opening metathesis polymerization (ROMP) leading to polycaryophyllene (PCar).30 PCar exhibits two carbon-carbon double bonds in its structure which can be further used to cross-link it. Keeping in mind this context, the present research has aimed to the cross-linking of polycaryophyllene by using different routes and investigating the thermo-mechanical properties of the newly produced cross-linked bio-based elastomer. Two main pathways have been considered to reach three dimensional cross-linked polycaryophyllene: i) thermal pathways: in the presence of organic peroxides [benzoyl peroxide (BPO) or dicumyl peroxide (DCP)], or by classical vulcanization based on sulfur system (SS) and ii) via UV light initiated thiol-click reactions at room temperature (see diagram in Figure 1) using different dithiols, such as 1,2-ethanedithiol (EDT), 1,4-butanedithiol (BDT), 1,6-hexanedithiol (HDT), and 1,10-decanedithiol (DDT). As far as we know, this is the first study about the cross-linking of polycaryophyllene. 3 3 Figure 1 Scheme of caryophyllene ROMP and subsequent cross-linking of polycaryophyllene by different pathways. Credit: hop picture by Hagen Graebner CC BY-SA 2.5, rosemary picture by Margalob CC BY-SA 4.0 Figure 1 Scheme of caryophyllene ROMP and subsequent cross-linking of polycaryophyllene by different pathways. Credit: hop picture by Hagen Graebner CC BY-SA 2.5, rosemary picture by Margalob CC BY-SA 4.0 Margalob CC BY-SA 4.0 Results & Discussion ROMP of caryophyllene was performed by dissolving Grubbs’ 2nd Generation catalyst ((tricyclohexylposphine) (1.3-dimesitylimidazolidine-2-ylidene) benzylideneruthenium dichloride) (0.2 mol % to monomer) in caryophyllene without any solvent at room temperature for 24 hours under N2 atmosphere. The obtained polycaryophyllene (Mn = 23 kg.mol-1, Đ = 1.5) was used without any further purification for thermal cross-linking or UV cross-linking (see Figure S1- S6). For thermal cross-linking, the polymer was homogenized with the selected cross-linker and 4 the mixture was kept at 160 °C under 30 bar for 1 hour in a machine press. For UV-light initiated cross-linking, the polycaryophyllene was mixed with dithiol and IRGARCUR 2959 (photo-cross- linker), placed into a Teflon mold and left in a UV-reactor (λ= 320-380 nm) for 4 hours. To confirm the cross-linking, the gel fractions were quantified by extraction in THF at 50 °C for 48 hours. The resulted gel fractions were 65 %, 90 %, and 72% for BPO, DCP, and SS respectively, as summarized in Table 1. The gel fraction appears to be limited for BPO cross-linking at 65% since even an increase of the curing temperature to 180 °C does not improve the gel fraction (see Table S1). Obtaining a higher amount of gel fraction with DCP compared to with BPO was already identified in other studies.31,32 Indeed, DCP is the most frequently used cross-linking agent since it is an organic peroxide which is more selective to vinyl moieties than benzoyl peroxide.33,34 The ability of cumyloxy radical from DCP to react with non-activated vinyl moieties in a free-radical cross-linking pathway via H abstraction may be a possible explanation of its higher reactivity compared to BPO.35 Additionally, the cumyloxy radical can undergo β-scission resulting in radical fragments (methyl radical and acetophenone) with high mobility.36 Furthermore, the unsaturated bonds are not easily accessible by benzoyloxy radicals due to the steric hindrance around double bonds as the increase of formation of cross-linked networks.29 The SEC analysis of the BPO soluble fraction testified to the presence of oligomers (see Figure S7, Mn=320 g.mol-1, Đ=1.5) which indicated that some polymer backbone cleavage also takes place during the cross-linking. In the case of the sulfur system, the soluble fraction is mainly constituted of the accelerator and the catalyst used. Finally, the IR analysis of the materials obtained (see Figure S8) shows that whatever the cross-linker, the vibration characteristics of the carbon-carbon double bond (i.e. Results & Discussion 5 =CH2 bending 887 cm-1, C=C stretching at 1645 cm-1, C-H stretching at 3115 cm-1) are still present indicating a crosslinking by hydrogen abstraction mechanisms. =CH2 bending 887 cm-1, C=C stretching at 1645 cm-1, C-H stretching at 3115 cm-1) are still present indicating a crosslinking by hydrogen abstraction mechanisms. In order to confirm the occurrence of cross-linking reactions, on-line rheology measurements were carried out, they are shown in Figure 2. The visco-elastic analysis also provided an estimation of the initial temperature of the cross-linking process. Figure 2. Rheology curves of polycaryophyllene with (black) or without (red) DCP, and temperature (blue). The solid line represents the loss modulus (G’’) and dashed line corresponds to storage modulus (G’). 0 20 40 60 80 100 120 140 160 180 1E-04 1E-02 1E+00 1E+02 1E+04 1E+06 0 5 10 15 20 25 30 35 40 Time (min) Storage and Loss Moduli (Pa) Temperature (°C) G’ > G’’ 160 °C Temperature Storage Modulus Loss Modulus 0 20 40 60 80 100 120 140 160 180 1E-04 1E-02 1E+00 1E+02 1E+04 1E+06 0 5 10 15 20 25 30 35 40 Time (min) Storage and Loss Moduli (Pa) Temperature (°C) G’ > G’’ 160 °C Temperature Storage Modulus Loss Modulus Figure 2. Rheology curves of polycaryophyllene with (black) or without (red) DCP, and temperature (blue). The solid line represents the loss modulus (G’’) and dashed line corresponds to storage modulus (G’). As expected, polycaryophyllene in the absence of a crosslinker behaves as a liquid for temperatures up to 160°C with the loss modulus (G’’) higher than the storage modulus (G’). Classically, as the temperature increases, both moduli continuously decrease and no cross-linking reaction takes place at 160°C since no change in moduli was observed over time. 6 6 In the presence of DCP, a shoot up in moduli is clearly observed with a gel point (intersection point) at 160°C. Similar curves were obtained for the curing of polycaryophyllene with BPO and SS (see Figure S9-10) with gel points occurring at 118°C and 160°C respectively and are in agreement with DSC online curing curves (Figure S11-12). After the gel point, both loss and storage moduli reached constant plateaus and no more significant changes are observed. Results & Discussion The magnitude of storage modulus increased up to 1.2 MPa, 40, and 60 kPa and the loss modulus to 0.6, 0.5, and 0.5 kPa respectively for polycaryophyllenes cross- linked by DCP, BPO, and sulfur system. Concerning the UV cross-linking by thiol-ene coupling, the soluble fraction remains between 0% and 11% depending on the dithiol used (see Table 1). Interestingly, when the cross-linking is performed with 0.5 eq. of thiol per carbon-carbon double bond (db), a slight improvement of the gel fraction is observed compared to the materials synthesized with 1 eq of thiol per db (see Table S2). This may be due to the presence of remaining free dithiol in the soluble fraction. IR analysis of the polycaryophyllene obtained with 0.5 eq. of thiol per db shows no regioselectivity of the thiol-ene addition since the bands which are characteristic of the two carbon-carbon double bonds decrease (see Figure S13). For 1 eq. of thiol per db, double bonds appear to have totally disappeared whatever the dithiols used (see Figure S14). However, in all cases with 1 eq of thiol per db, IR spectra show a broad peak around 3400 cm-1 which confirmed the presence of remaining thiols in the materials. To obtain further information about cross-linked polycaryophyllenes, TGA and DSC measurements were performed in order to investigate their thermal properties, as shown in Figure 3-4 and Table 1. 7 7 Figure 3. TGA and DTG curves of polycaryophyllene cross-linked by using different routes Figure 3. TGA and DTG curves of polycaryophyllene cross-linked by using different routes Table 1. Thermo-mechanical properties of cross-linked polycaryophyllenes. a Extraction in THF at 50 °C for 48 h (IF=md/m0 with md the mass of the dried sample and m0 the mass of the sample before extraction by THF) ; b Obtained by DSC ; c Obtained by TGA ; e Obtained by DMA; f Determined using the rubber-like elasticity theory. Entry Insoluble Fraction a (%) Tg b (°C) T10% c (°C) Tan e (°C) E’ e (MPa) Cross-linking degree f (103 mol.m-3 of elastomer) Polycaryophyllene 0 -35 200 NA NA 0 Organic Peroxide Pcar/DCP 90 12 335 4 28 11 Pcar/BPO 65 -6 245 -12 0.7 0.4 Vulcanization Pcar/SS 72 -9 315 -5 0.9 0.3 Dithiol (0.5 equiv. Results & Discussion SH to db) Pcar/EDT 100 -22 338 -16 0.9 1.1 Pcar/BDT 100 -16 325 -17 8.2 3.1 Pcar/HDT 95 -15 332 -15 2.0 0.8 Pcar/DDT 98 -6 336 -2 4.6 1.9 a Extraction in THF at 50 °C for 48 h (IF=md/m0 with md the mass of the dried sample and m0 the mass of the sample before extraction by THF) ; b Obtained by DSC ; c Obtained by TGA ; e Obtained by DMA; f Determined using the rubber-like elasticity theory. a Extraction in THF at 50 °C for 48 h (IF=md/m0 with md the mass of the dried sample and m0 the mass of the sample before extraction by THF) ; b Obtained by DSC ; c Obtained by TGA ; e Obtained by DMA; f Determined using the rubber-like elasticity theory. 8 According to TGA (see Figure 3 and Figure S15-S16), the crosslinking of polycaryophyllene increases its thermal stability. Indeed, the temperature of 10% of degradation (T10%) for polycaryophyllene is around 200 °C. After cross-linking, the T10% take place at temperature ranging from 310 to 340 °C. DSC measurements were performed to evaluate the effect of newly formed cross-linked structures on the glass transition temperatures (Tg) of polycaryophyllene (See Figure 4 and Figure S17). The Tg of cross-linked polycaryophyllene increased significantly compared to non-cross-linked polycaryophyllene (Tg = -35 °C). The curing by DCP provided more rigidity to polycaryophyllene (Tg = 12 °C) than the one by sulfur system (Tg = -9 °C) or DDT (Tg = -6 °C). Moreover the Tg of the network gradually increases with the chain length of the dithiol from -22°C for EDT to -6°C for DDT. Figure 4. DSC Curves of cross-linked polycaryophyllene. DCP SS DDT Polycaryophyllene - 35 °C - 9 °C - 6 °C 12 °C Figure 4. DSC Curves of cross-linked polycaryophyllene. Figure 4. DSC Curves of cross-linked polycaryophyllene. The mechanical properties of cross-linked polycaryophyllene were evaluated by dynamic mechanical analyses (DMA), displayed in Figure 5 and Figure S19. 9 9 The DMA curves show a typical behavior for cross-linked materials with a glassy plateau before the alpha transition temperature (T) followed by a rubbery zone after this point. The DMA curves show a typical behavior for cross-linked materials with a glassy plateau before the alpha transition temperature (T) followed by a rubbery zone after this point. Figure 5. Results & Discussion DMA analysis of cross-linked polycaryophyllene by DCP, DDT and SS. Storage modulus in solid line, tan δ in dashed line. Figure 5. DMA analysis of cross-linked polycaryophyllene by DCP, DDT and SS. Storage modulus in solid line, tan δ in dashed line. Polycaryophyllene crosslinked by DCP exhibits the highest glassy storage modulus (over 1 GPa). The T values were 4 °C, -2 °C and -5 °C for Pcar/DCP, Pcar/DDT and Pcar/SS respectively (see Figure 5), and can be considered in agreement with the one obtained by DSC (12 °C, -6 °C and -9 °C respectively). Finally, the rubbery plateau confirms the cross-linking of the materials and its value is directly proportional to the cross-linking density. Polycaryophyllene crosslinked by DCP exhibits significantly higher storage modulus above glass transition temperature (28 MPa) compared to SS or BPO systems (0.7 and 0.9 MPa respectively) (see Table 1) or even DDT cross- linking (4.6 MPa). Tensile tests were performed in order to obtain the values of tensile strength and elongation at break of cross-linked polycaryophyllene (see Figure S20). On the first hand, the cross-linked materials produced by BPO curing and vulcanization show elastomeric behaviors with a high value 10 of elongation at break (360 % and 187 %, respectively) and low Young moduli (1.7 and 2 MPa, respectively). On the other hand, in agreement with the DMA results, polycaryophyllene cross- linked by DDT exhibited much lower elongation at break (25 %) and higher Young modulus (40 MPa) compared to SS and BPO crosslinked materials. Conclusion In conclusion, caryophyllene was polymerized by ROMP and the polymer obtained was cross- linked using different routes: organic peroxides, sulfur system or dithiol. Rheological analyses confirmed the occurrence of cross-linking reactions in agreement with the extractions by THF results. Swelling experiments showed that the polycaryophyllene was entirely cross-linked by DCP or dithiols with an insoluble fraction over 90%. Cross-linking by sulfur system resulted in a gel content of 72%. The cross-linked materials were in-depth analyzed by TGA, DSC and DMA. Materials were obtained with Tg between -22 °C and 12 °C with improved thermal stability compared to non- cross-linked polycaryophyllene (T10% > 300 °C). At room temperature (20 °C) storage modulus of the cross-linked polycaryophyllene were between 1 MPa to 100 MPa with elongation at break up to 360%. Polycaryophyllene is a precursor of elastomers which can be cross-linked by various pathways. Its Tg of -35°C is close to the Tg of synthetic rubber such as EPDM, CR or NBR. To the best of our knowledge, it is the only polyterpenes in this range of temperature (see Table S3 in supporting information). Indeed, poly(-myrcene),20 poly(-farsene)25 exhibit Tg close to -70°C while poly(allocimene)18 have a Tg of -17°C. Moreover with a production potential of 20 million metric 11 tons per year, caryophyllene is widely available and in quantity compatible with the synthetic rubber market which is estimated to 15 million metric tons per year. ACKNOWLEDGMENT E.G. thanks Emergence@INC2019 for the research funding. The authors thank Equipex Xyloforest ANR-10-EQPX-16. 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Dicumyl Peroxide Thermal Decomposition in Cumene: Development of a Kinetic Model. Ind. Eng. Chem. Res. 2012, 51, 7493–7499. DOI 10.1021/ie201659a. 17 For Table of Contents Use Only Synopsis Caryophyllene was polymerized by ROMP, the polycaryophyllene obtained was then cross- linked leading to material exhibiting elastomeric-like properties Synopsis Caryophyllene was polymerized by ROMP, the polycaryophyllene obtained was then cross- linked leading to material exhibiting elastomeric-like properties Caryophyllene was polymerized by ROMP, the polycaryophyllene obtained was then cross- linked leading to material exhibiting elastomeric-like properties Caryophyllene was polymerized by ROMP, the polycaryophyllene obtained was then cross- linked leading to material exhibiting elastomeric-like properties 18 Supporting information Anderson M. M. S. Medeiros,† Cédric Le Coz,† Etienne Grau†* The number of pages: 17 The number of Figures: 20 The number of Tables: 3 The number of pages: 17 The number of Figures: 20 The number of Tables: 3 Figure S5 1H and 13C NMR spectrum of polycaryophyllene in CDCl3. gure S6. Three different samples of Polycaryophyllene and Caryophyllene SEC profile. Table S1. Gel fractions of cross-linked polycaryophyllene by BPO at varied temperatur Figure S7. SEC profile of the soluble fraction of polycaryophyllene crosslinked by BPO. Figure S8. FTIR Analysis of (a) polycaryophyllene and cross-linked polycaryophyllene by (b) DCP, (c) BPO, (d) SS. Figure S9. Online curing of polycaryophyllene by BPO followed by rheology. Figure S10. Online curing of polycaryophyllene by SS followed by rheology. Figure S11. DSC online curing measurement of polycaryophyllene by DCP. Figure S12. DSC online curing measurement of polycaryophyllene by BPO. Table S2. Gel fractions of cross-linked polycaryophyllene by different thiols. Table S2. Gel fractions of cross-linked polycaryophyllene by different thiols. Figure S13. FTIR Analysis of (a) polycaryophyllene and cross-linked polycaryophyllene by HDT with 0.5eq and 1 eq. of SH per double bond. Figure S13. FTIR Analysis of (a) polycaryophyllene and cross-linked polycaryophyllene by HDT with 0.5eq and 1 eq. of SH per double bond. Figure S14. FTIR Analysis of (a) polycaryophyllene and cross-linked polycaryophyllene by dithiols with 1 eq. of SH per double bond. Figure S14. FTIR Analysis of (a) polycaryophyllene and cross-linked polycaryophyllene by dithiols with 1 eq. of SH per double bond. Figure S14. FTIR Analysis of (a) polycaryophyllene and cross-linked polycaryophyllene by dithiols with 1 eq. of SH per double bond. 1 Figure S15. TGA and DTG curves of polycaryophyllene, and cross-linked polycaryophyllene by EDT, BDT, HDT, DDT. (0.5 equiv SH to DB.) Figure S16. TGA and DTG curves of cross-linked polycaryophyllene BPO. Figure S17. DSC curves of polycaryophyllene pure, and cross-linked polycaryophyllene by EDT, BDT, HDT, DDT with 0.5 of thiol per double bond. Figure S18. DSC curves of polycaryophyllene pure, and cross-linked polycaryophyllene by EDT, BDT, HDT, DDT with 1 equiv. of thiol per double bond. Figure S18. DSC curves of polycaryophyllene pure, and cross-linked polycaryophyllene by EDT, BDT, HDT, DDT with 1 equiv. of thiol per double bond. Figure S19. DMA analysis of cross-linked polycaryophyllene by EDT, BDT, HDT, DDT (0.5 eq. of SH per db). Storage modulus (E’) in solid line, tan δ in dashed line. Figure S19. S1. General experimental procedure Caryophyllene (≥80%), Grubbs’ 2nd Generation catalyst ((tricyclohexylposphine) (1.3- dimesitylimidazolidine-2-ylidene) benzylideneruthenium dichloride), dicumyl peroxide (DCP, 98%), IRGARCUR 2959, 1,2-ethanedithiol (EDT), 1,4-butanedithiol (BDT), 1,6-hexanedithiol (HDT), and 1,10-decanedithiol (DDT), dichloromethane and tetrahydrofuran were purchased from Sigma-Aldrich. Benzoyl peroxide (BPO, 75%) was purchased from Fischer Scientific. Sulfur curing system i.e. sulfur, zinc oxide, stearic acid and N-tert-butyl-2-benzothiazyl sulfenamide (TBBS) accelerator were supplied by EMAC company. All products and solvents (reagent grade) were used as received. Anderson M. M. S. Medeiros,† Cédric Le Coz,† Etienne Grau†* DMA analysis of cross-linked polycaryophyllene by EDT, BDT, HDT, DDT (0.5 eq. of SH per db). Storage modulus (E’) in solid line, tan δ in dashed line. Figure S20. Tensile strength and elongation at break of cross-linked polycaryophyllene by SS, BPO, and DDT 0.5 equiv. Figure S20. Tensile strength and elongation at break of cross-linked polycaryophyllene by SS, BPO, and DDT 0.5 equiv. Table S3. General properties of different polyterpenes. Table S3. General properties of different polyterpenes. 2 2 i) Synthesis of polycaryophyllene The synthesis of polycaryophyllene was based on Grau’s procedure. The caryophyllene was placed into a Schlenk flask in which 0.2 wt. % of Grubbs catalyst 2nd generation (C46H65Cl2N2PRu) was added under nitrogen atmosphere. The reaction was conducted for 24 h at room temperature. The scheme of the reaction is depicted in the figure below. Figure S1 Schematic reaction of the polymerization of caryophyllene. Caryophyllene Polycaryophyllene Catalyst / N2 Atmosphere 25 °C Polycaryophyllene Figure S1 Schematic reaction of the polymerization of caryophyllene. A sticky polymer was obtained at the end and used without any further purification. The product was characterized by 1H NMR, SEC and DSC, which are shown in Figs S4, S5, S6. ii) Cross-linking of polycaryophyllene using thermosensitive cross-linkers 1) Organic peroxide: The polycaryophyllene and cross-linking agent (15 wt. %) were homogenized and placed in a machine press. The mixture was kept in the machine press for 1 h at 160°C under 30 bar. 3 3 2) Vulcanization by sulfur system: The polycaryophyllene and sulfur system (2 phr of ZnO, 2 phr of stearic acid, 3 phr of sulfur, and 15 phr of TBBS accelerator) were homogenized and placed in a machine press. The mixture was kept in the machine press for 1 h at 160°C under 30 bar. At the end, a rubber-like cross-linked polymer was obtained. iii) Photocross-linking of polycaryophyllene under UV light radiation The cross-linking reactions under UV radiation were carried out in a reactor equipped with a UV lamp ( = 320-380 nm). The polycaryophyllene, IRGARCUR 2959 (0.02 equiv.), selected dithiol (0.25 or 0.5 equiv. to double bonds) and dichloromethane (3 mL) were homogenized and placed in a Teflon mould. The teflon mould was left overnight to evaporate the solvent. Then, the mixture was dried under vacuum for 4 hours to make sure that all solvent was removed. The Teflon mold was thus enclosed into the UV-reactor for 5h at room temperature. At the end, a film-like was obtained. In this study, thiols with different carbon chains were used 1,2-ethanedithiol (EDT), 1,4-butanedithiol (BDT), 1,6-hexanedithiol (HDT), and 1,10-decanedithiol (DDT) (Figure 1). Figure S2 Chemical structures of different thiols. 1,2-ethanedithiol (EDT) 1,4-butanedithiol (BDT) 1,6-hexanedithiol (HDT) 1,10-decanedithiol (DDT) Figure S2 Chemical structures of different thiols. Figure S3 Thiol-ene cross-linking reaction of polycaryophyllene. Figure S3 Thiol-ene cross-linking reaction of polycaryophyllene. 4 4 iv) Characterizations NMR spectra were recorded using a Bruker AC-400 NMR, at room temperature, in deuterated chloroform (CDCl3). Fourier Transform Infrared (FT-IR) spectra were performed on a Bruker VERTEX 70 spectrometer equipped with diamond crystal (GladiATR PIKE technologies) for the attenuated total reflection (ATR) mode. The spectra were acquired from 400 to 4000 cm-1 at room temperature using 32 scans at a resolution of 4 cm-1. Polymer molar masses were determined by size exclusion chromatography (SEC) using tetrahydrofuran (THF with 250 ppm of BHT as an inhibitor) as an eluent and trichlorobenzene as a flow marker. Measurements in THF were performed on a ThermoFisher Scientific Ultimate 3000 system equipped with Diode Array Detector, Wyatt light scattering detector and RI detector. The separation was achieved on three Tosoh TSK gel columns: G4000HXL (particles of 5 mm, pore size of 200 Å, exclusion limit of 400 000 g mol-1), G3000HXL (particles of 5 mm, pore size of 75 Å, exclusion limit of 60 000 g mol-1), G2000HXL (particles of 5 mm, pore size of 20 Å, exclusion limit of 10 000 g mol-1), at flow rate of 1 mL min-1. The injected volume was 20 µL. Columns’ temperature was held at 40 °C. Data were recorded and processed by Astra software from Wyatt. SEC was calibrated using polystyrene standards. iii) Photocross-linking of polycaryophyllene under UV light radiation The rheology measurements were performed on an Anton Paar MCR302 controlled- stress rheometer and frequency was modulated at 6.28 rad.s-1 with a shear strain () variation of 1%. The measurements of G’ and G’’ were performed as a function of temperature from 30°C to 160 °C at a rate of 10°C.min-1. The polycaryophyllene and cross-linker were previously homogenized and then placed between two plates of 8 mm diameter. Finally, the temperature was maintained at 160 °C for 30 minutes. Thermogravimetric analysis (TGA) thermograms were obtained using a TGA Q500 apparatus from TA instruments. Samples (10 mg) were heated from room temperature to 700 °C at a rate of 10 °C min-1 under nitrogen atmosphere. Differential scanning calorimetry (DSC) measurements of samples (5 mg) were performed using a DSC Q 100 apparatus from TA Instruments over temperature range from -120 °C to 240 °C, in a heating-cooling mode of 10 °C min-1. The analyses were carried out in a helium atmosphere using aluminum pans. Glass transition temperatures and melting points were obtained from the second heating runs. 5 5 Dynamic mechanical analysis (DMA) measurements were carried out using a RSA3 apparatus from TA Instruments equipped with a liquid nitrogen cooling system. The thermomechanical properties of the samples (width  5 mm; thickness  2 mm and length of the fixed section  10 mm) were studied from around -50 °C to 60°C at a heating rate of 3 °C min−1. The measurements were performed in tensile test at a frequency of 1 Hz, a strain sweep of 0.03% and an initial static force of 0.1 N. Tensile tests were performed on extension mode test system using RSA3 apparatus from TA instruments. The measurements were carried out at room temperature for rectangular samples (width ≅ 5 mm; thickness ≅ 2 mm) using a cross-head speed of 2 mm.min-1. The gel fractions were calculated by using the equation 𝑮𝑭= 𝒘𝒅 𝒘𝟎. 𝟏𝟎𝟎%, where wd is the mass of the dried sample and w0 is the mass of the sample before swelling test. The cross-link densities () of networks defined as the number of moles of elastically effective network chains per cubic centimetre of sample which can be calculated with the following equation with R= 8,314 J.mol-1.K-1, 𝑇+30 and = 1. iii) Photocross-linking of polycaryophyllene under UV light radiation = 𝐸′(𝑇+) .𝑅.𝑇+ (2) (2) 6 6 7 Figure S4: 1H and 13C NMR spectrum of caryophyllene in CDCl3 1 2 3 4 5 5 6 7 8 9 10 11 8 8 11 5 5 1 3 6 4 2 2 9,10 7,10 9 1 2 3 4 5 5 6 7 8 9 10 11 11 8 4 7 1 5 5310 9 6 2 1* 5* 8* 8* 1* 5* 1 2 3 4 5 5 6 7 8 9 10 11 8 8 11 5 5 1 3 6 4 2 2 9,10 7,10 9 1 2 3 4 5 5 6 7 8 9 10 11 8 8 11 5 5 1 3 6 4 2 2 9,10 7,10 9 Figure S4: 1H and 13C NMR spectrum of caryophyllene in CDCl3 1 2 3 4 5 5 6 7 8 9 10 11 11 8 4 7 1 5 5310 9 6 2 1* 5* 8* 8* 1* 5* Figure S4: 1H and 13C NMR spectrum of caryophyllene in CDCl3 Figure S4: 1H and 13C NMR spectrum of caryophyllene in CDCl3 7 7 8 Figure S5 1H and 13C NMR spectrum of polycaryophyllene in CDCl3. 1 2 3 4 5 5 6 7 8 9 10 11 10 5 5 1-E 1-Z 7 9 2,4 6 3,6 8 11-E 11-Z 1 2 3 4 5 5 6 7 8 9 10 11 1* 5* 8* 8 8* 11-E 11-Z 1-E 1-Z 1-E 5 1-Z 10 3 5 7 4 6 2 9 5* 1 2 3 4 5 5 6 7 8 9 10 11 1 2 3 4 5 5 6 7 8 9 10 11 10 5 5 1-E 1-Z 7 9 2,4 6 3,6 8 11-E 11-Z 10 5 1-E 1-Z 7 9 2,4 6 3,6 8 11-E 11-Z 1 2 3 4 5 5 6 7 8 9 10 11 1* 5* 8* 8 8* 11-E 11-Z 1-E 1-Z 1-E 5 1-Z 10 3 5 7 4 6 2 9 5* Figure S5 1H and 13C NMR spectrum of polycaryophyllene in CDCl3. 8 8 Figure S6 Three different samples of Polycaryophyllene and Caryophyllene SEC profile. iii) Photocross-linking of polycaryophyllene under UV light radiation ROMP 01-03 were performed using 5, 50, and 50g of caryophyllene respectively ROMP 01 ROMP 02 ROMP 03 Caryophyllene Flow marker SAMPLE Mn Ð ROMP 01 20850 1.6 ROMP 02 19800 1.6 ROMP 03 23090 1.5 Caryophyllene 130 1 Figure S6 Three different samples of Polycaryophyllene and Caryophyllene SEC profile. ROMP 01-03 were performed using 5, 50, and 50g of caryophyllene respectively Table S1 Gel fractions of cross-linked polycaryophyllene by BPO at varied temperatures. Curing temperature by BPO Gel Fraction (%) 15 wt. % 100 °C 27 120 °C 36 140 °C 43 160 °C 64 170 °C 63 180 °C 60 Figure S7 SEC profile of the soluble fraction of polycaryophyllene crosslinked by BPO. Table S1 Gel fractions of cross-linked polycaryophyllene by BPO at varied temperatures. Curing temperature by BPO Gel Fraction (%) 15 wt. % 100 °C 27 120 °C 36 140 °C 43 160 °C 64 170 °C 63 180 °C 60 Figure S7 SEC profile of the soluble fraction of polycaryophyllene crosslinked by BPO. Table S1 Gel fractions of cross-linked polycaryophyllene by BPO at varied temperatures. Curing temperature by BPO Gel Fraction (%) 15 wt. % 100 °C 27 120 °C 36 140 °C 43 160 °C 64 170 °C 63 180 °C 60 ble S1 Gel fractions of cross-linked polycaryophyllene by BPO at varied temperatures. 160 C 64 170 °C 63 180 °C 60 Figure S7 SEC profile of the soluble fraction of polycaryophyllene crosslinked by BPO. Figure S7 SEC profile of the soluble fraction of polycaryophyllene crosslinked by BPO 9 Figure S8 FTIR Analysis of (a) polycaryophyllene and cross-linked polycaryophyllene by (b) DCP, (c) BPO, (d) SS. 0 0,2 0,4 0,6 0,8 1 1,2 1,4 500 1000 1500 2000 2500 3000 3500 4000 Wavenumber(cm-1) Intensity (a.u.) (a) 3115 1645 887 (b) (c) (d) Polycaryophyllene CRLPcar/DCP CRLPcar/BPO CRLPcar/SS 1714 Figure S8 FTIR Analysis of (a) polycaryophyllene and cross-linked polycaryophyllene by (b) DCP, (c) BPO, (d) SS. Figure S9 Online curing of polycaryophyllene by BPO followed by rheology. iii) Photocross-linking of polycaryophyllene under UV light radiation 0 0,2 0,4 0,6 0,8 1 1,2 1,4 500 1000 1500 2000 2500 3000 3500 4000 Wavenumber(cm-1) Intensity (a.u.) (a) 3115 1645 887 (b) (c) (d) Polycaryophyllene CRLPcar/DCP CRLPcar/BPO CRLPcar/SS 1714 Time (min) Storage and Loss Moduli (Pa) Temperature (°C) G’ > G’’ Pcar/BPO 0 30 60 90 120 150 180 1E+00 1E+01 1E+02 1E+03 1E+04 1E+05 0 5 10 15 118 °C Storage modulus Loss modulus Temperature Figure S8 FTIR Analysis of (a) polycaryophyllene and cross-linked polycaryophyllene by (b) DCP, (c) BPO, (d) SS. 0 0,2 0,4 0,6 500 1000 1500 2000 2500 3000 3500 4000 Wavenumber(cm-1) Intensity (a) (b) (c) Polycaryophyllene CRLPcar/DCP CRLPcar/BPO d Loss Moduli (Pa) perature (°C) G’ > G’’ 90 120 150 180 1E+02 1E+03 1E+04 1E+05 118 °C Figure S8 FTIR Analysis of (a) polycaryophyllene and cross-linked polycaryophyllene by (b) DCP, (c) BPO, (d) SS. ure S8 FTIR Analysis of (a) polycaryophyllene and cross-linked polycaryophyllene by (b) DCP, (c) BPO, (d) SS. gure S8 FTIR Analysis of (a) polycaryophyllene and cross linked polycaryophyllene by (b DCP, (c) BPO, (d) SS. Figure S9 Online curing of polycaryophyllene by BPO followed by rheology. Time (min) Storage and Loss Moduli (Pa) Temperature (°C) G’ > G’’ Pcar/BPO 0 30 60 90 120 150 180 1E+00 1E+01 1E+02 1E+03 1E+04 1E+05 0 5 10 15 118 °C Storage modulus Loss modulus Temperature Time (min) Storage and Loss Moduli (Pa) Temperature (°C) G’ > G’’ Pcar/BPO 0 30 60 90 120 150 180 1E+00 1E+01 1E+02 1E+03 1E+04 1E+05 0 5 10 15 118 °C Storage modulus Loss modulus Temperature Figure S9 Online curing of polycaryophyllene by BPO followed by rheology. 10 10 Figure S10 Online curing of polycaryophyllene by SS followed by rheology. Figure S11 DSC online curing measurement of polycaryophyllene by DCP. 0 60 120 180 1E-03 1E+00 1E+03 1E+06 0 6 12 18 24 Storage and Loss Moduli (Pa) Time (min) Pcar/SS G’ > G’’ 160 °C Storage modulus Loss modulus Temperature Temperature (°C) Heat flow (W/g) Exo up 185 °C DCP online curing Figure S10 Online curing of polycaryophyllene by SS followed by rheology. 0 60 120 180 1E-03 1E+00 1E+03 1E+06 0 6 12 18 24 Storage and Loss Moduli (Pa) Time (min) Pcar/SS G’ > G’’ 160 °C Storage modulus Loss modulus Temperature Temperature (°C) Figure S10 Online curing of polycaryophyllene by SS followed by rheology. iii) Photocross-linking of polycaryophyllene under UV light radiation 0 60 120 180 1E-03 1E+00 1E+03 1E+06 0 6 12 18 24 Storage and Loss Moduli (Pa) Time (min) Pcar/SS G’ > G’’ 160 °C Storage modulus Loss modulus Temperature Temperature (°C) Figure S10 Online curing of polycaryophyllene by SS followed by rheology. Figure S11 DSC online curing measurement of polycaryophyllene by DCP. Heat flow (W/g) Exo up 185 °C DCP online curing Figure S11 DSC online curing measurement of polycaryophyllene by DCP. Heat flow (W/g) Exo up 185 °C DCP online curing Heat flow (W/g) Figure S11 DSC online curing measurement of polycaryophyllene by DCP. 11 11 Figure S12 DSC online curing measurement of polycaryophyllene by BPO. Heat flow (W/g) Exo up 121 °C BPO online curing Figure S12 DSC online curing measurement of polycaryophyllene by BPO. Table S2 Gel fractions of cross-linked polycaryophyllene by different thiols. Gel content (%) Dithiol 0.5 eq. of SH per double bond 1 eq. of SH per double bond EDT 100 100 BDT 100 90 HDT 95 94 DDT 98 89 Figure S13 FTIR Analysis of (a) polycaryophyllene and cross-linked polycaryophyllene by HDT with 0.5eq and 1 eq. of SH per double bond. Table S2 Gel fractions of cross-linked polycaryophyllene by different thiols. Figure S13 FTIR Analysis of (a) polycaryophyllene and cross-linked polycaryophyllene by HDT with 0.5eq and 1 eq. of SH per double bond. 12 Figure S14 FTIR Analysis of (a) polycaryophyllene and cross-linked polycaryophyllene by dithiols with 1 eq. of SH per double bond. Figure S14 FTIR Analysis of (a) polycaryophyllene and cross-linked polycaryophyllene by dithiols with 1 eq. of SH per double bond. Figure S15 TGA and DTG curves of polycaryophyllene, and cross-linked polycaryophyllene by EDT, BDT, HDT, DDT. (0.5 equiv SH to DB.) -0,5 0 0,5 1 1,5 2 2,5 3 0 20 40 60 80 100 20 100 180 260 340 420 500 580 Weight (%) Temperature (°C) Derivative Weight (%/°C) Polycaryophyllene Pcar/EDT Pcar/BDT Pcar/HDT Pcar/DDT Figure S14 FTIR Analysis of (a) polycaryophyllene and cross-linked polycaryophyllene by dithiols with 1 eq. of SH per double bond. Figure S14 FTIR Analysis of (a) polycaryophyllene and cross linked polycaryophyllene by dithiols with 1 eq. of SH per double bond. Figure S15 TGA and DTG curves of polycaryophyllene, and cross-linked polycaryophyllene by EDT, BDT, HDT, DDT. iii) Photocross-linking of polycaryophyllene under UV light radiation (0.5 equiv SH to DB.) -0,5 0 0,5 1 1,5 2 2,5 3 0 20 40 60 80 100 20 100 180 260 340 420 500 580 Weight (%) Temperature (°C) Derivative Weight (%/°C) Polycaryophyllene Pcar/EDT Pcar/BDT Pcar/HDT Pcar/DDT -0,5 0 0,5 1 1,5 2 2,5 3 0 20 40 60 80 100 20 100 180 260 340 420 500 580 Weight (%) Temperature (°C) Derivative Weight (%/°C) Polycaryophyllene Pcar/EDT Pcar/BDT Pcar/HDT Pcar/DDT Figure S15 TGA and DTG curves of polycaryophyllene, and cross-linked polycaryophyllene by EDT, BDT, HDT, DDT. (0.5 equiv SH to DB.) 13 Figure S16 TGA and DTG curves of cross-linked polycaryophyllene BPO. Figure S17 DSC curves of polycaryophyllene pure, and cross-linked polycaryophyllene by EDT, BDT, HDT, DDT with 0.5 of thiol per double bond. -35 °C -22 °C -16 °C -6 °C -15 °C 0.5 eq. SH to DB Polycaryophyllene EDT BDT HDT DDT Figure S16 TGA and DTG curves of cross-linked polycaryophyllene BPO. Figure S17 DSC curves of polycaryophyllene pure, and cross-linked polycaryophyllene by EDT, BDT, HDT, DDT with 0.5 of thiol per double bond. -35 °C -22 °C -16 °C -6 °C -15 °C 0.5 eq. SH to DB Polycaryophyllene EDT BDT HDT DDT -35 °C -22 °C -16 °C -6 °C -15 °C 0.5 eq. SH to DB Polycaryophyllene EDT BDT HDT DDT Figure S17 DSC curves of polycaryophyllene pure, and cross-linked polycaryophyllene by EDT, BDT, HDT, DDT with 0.5 of thiol per double bond. 14 1 eq. SH to DB - 35° C - 9° C 0.5° C - 3.8° C - 4.5° C Polycaryophyllene EDT BDT HDT DDT Figure S18 DSC curves of polycaryophyllene pure, and cross-linked polycaryophyllene by EDT, BDT, HDT, DDT with 1 equiv. of thiol per double bond. 1 eq. SH to DB - 35° C - 9° C 0.5° C - 3.8° C - 4.5° C Polycaryophyllene EDT BDT HDT DDT Figure S18 DSC curves of polycaryophyllene pure, and cross-linked polycaryophyllene by EDT, BDT, HDT, DDT with 1 equiv. of thiol per double bond. Figure S19 DMA analysis of cross-linked polycaryophyllene by EDT, BDT, HDT, DDT (0.5 eq. of SH per db). Storage modulus (E’) in solid line, tan δ in dashed line. iii) Photocross-linking of polycaryophyllene under UV light radiation EDT BDT HDT DDT -17 °C -15 °C -16 °C -2 °C EDT BDT HDT DDT -17 °C -15 °C -16 °C -2 °C Figure S19 DMA analysis of cross-linked polycaryophyllene by EDT, BDT, HDT, DDT (0.5 eq. of SH per db). Storage modulus (E’) in solid line, tan δ in dashed line. 15 Figure S20 Tensile strength and elongation at break of cross-linked polycaryophyllene by SS, BPO, and DDT 0.5 equiv. Figure S20 Tensile strength and elongation at break of cross-linked polycaryophyllene by SS, BPO, and DDT 0.5 equiv. 16 16 Table S3. General properties of different polyterpenes. Table S3. General properties of different polyterpenes. Terpene Unit Polymerization method Tg (°C) Molar mass (g.mol-1) Reference Free Radical Poly(-myrcene) -73 9x104 19 Cationic Poly(-pinene) 90 4x104 23 Free Radical Poly-allocimene -17 1x104 18 Anionic Poly(-farsene) -70 1x104 25 Free Radical Polylimonene 116 4x104 24 Cationic Poly(-phellandrene) 130 1x104 24 ROMP Poly(caryophyllene) -35* 2x104 This work 17
W2412101621.txt	https://hipatiapress.com/hpjournals/index.php/redimat/article/download/1469/pdf	en		How can I help my students with learning disabilities in Mathematics?	REDIMAT	2,016	cc-by	6,289	Instructions for authors, subscriptions and further details: http://redimat.hipatiapress.com How Can I Help my Students with Learning Disabilities in Mathematics? Gracia Jiménez-Fernández1 1) University of Granada, Spain th Date of publication: February 24 , 2016 Edition period: February 2016-June 2016 To cite this article: Jiménez-Fernández, G. (2016). How can I help my students with learning disabilities in Mathematics? REDIMAT, 5(1), 56-73. doi: 10.4471/redimat.2016.1469 To link this article: http://dx.doi.org/10.4471/redimat.2016.1469 PLEASE SCROLL DOWN FOR ARTICLE The terms and conditions of use are related to the Open Journal System and to Creative Commons Attribution License (CC-BY). REDIMAT, Vol. 5 No. 1 February 2016 pp. 56-73 How Can I Help my Students with Learning Disabilities in Mathematics? Gracia Jiménez-Fernández University of Granada (Received: 10 March 2015; Accepted: 14 February 2016; Published: 24 February 2015) Abstract Learning Disabilities in Mathematics (LDM) or dyscalculia are a frequent and disruptive problem within schools. Nevertheless, this problem has received little attention from researchers and practitioners, if compared with the number of studies published on disabilities in reading. Therefore, teachers do not have enough guidance to help children overcome their difficulties. Consequently, educators, practitioners and teachers are in need of some guidelines which help them choose teaching methods that are adapted for children with LDM. This paper proposes an interventional framework for improving skills in children who show problems in learning basic mathematics. Concretely, it suggests some guidelines which are focused on number sense and problem solving, two of the most important mathematical areas. Keywords: Learning disabilities, mathematics, educational guidelines, number sense, problem solving 2016 Hipatia Press ISSN: 2014-3621 DOI: 10.4471/redimat.2016.1469 REDIMAT, Vol. 5 No. 1 February 2016 pp. 56-73 ¿Cómo Puedo Ayudar a Mis Estudiantes con Dificultades de Aprendizaje de las Matemáticas? Gracia Jiménez-Fernández Universidad de Granada (Recibido: 10 Marzo 2015; Aceptado: 14 Febrero 2016; Publicado: 24 Febrero 2016) Resumen Las dificultades en el aprendizaje de las Matemáticas (DAM) o discalculia son un problema frecuente y disruptivo en las aulas de Educación Primaria. Sin embargo, este problema ha recibido muy poca atención desde la investigación e intervención si lo comparamos con el gran número de estudios publicados sobre las dificultades de aprendizaje de la lectura. Por ello, los maestros no tienen a su disposición suficientes materiales que puedan guiarles para ayudar a estos niños a superar sus dificultades. Consecuentemente, los profesionales de la educación necesitan conocer directrices que les permitan elegir la metodología didáctica que se pueda adaptar a los niños con DAM. Este artículo propone un marco general de intervención para mejorar algunas habilidades en niños que manifiestan problemas en el aprendizaje de las matemáticas. Específicamente, se sugieren directrices y orientaciones centradas en el sentido del número y en la resolución de problemas, dos de las áreas con dificultades más importantes. Palabras clave: Dificultades de aprendizaje, matemáticas, orientaciones educativas, sentido del número, resolución de problemas 2016 Hipatia Press ISSN: 2014-3621 DOI: 10.4471/redimat.2016.1469 56 Jiménez – Overcoming Learning Difficulties in Mathematics M athematics is one of the basic instrumental tools of learning and, along with language, contributes to enhancing logical thinking as well as being a fundamental basis for which other fields of knowledge relies on. Therein lays the importance of teaching mathematics in Elementary Education, which should be regarded as an essential goal to be achieved at this schooling stage. However, learning mathematics is not easy and its rate of failure is usually one of the highest, particularly in the final stages of Elementary Education (Alsina i Pastells, 2007). In fact, a significant percentage of students consider mathematics is a complicated subject and consequently show a certain lack of interest in it. Nevertheless, some children find more difficulties in this field than expected. These children will be referred in this paper as children with Learning Disabilities in Mathematics (LDM). LDM are also called dyscalculia that is, difficulties in the production or understanding of quantities, numerical symbols or basic arithmetic operations. The prevalence is considered to be around 6% and its symptoms become evident in about the second grade of Elementary Education (Shalev, 2007). Current data indicate that this learning disability is a brain-based disorder with a familial-genetic predisposition (Shalev & Gross-Tsur, 2001). Although people with LDM have an average or above average intelligence and may even stand out in fields other than mathematics, they find it difficult to estimate distance, size or time and usually count on their fingers in order to work out simple operations (Landerl, Bevan, & Butterworth, 2004). These kinds of situations are frequent in the daily live, and it is because of this, the problems associated with LDM may have huge negative impacts on children’s social and, of course, educational areas. Children with LDM can learn mathematical concepts but the progression they do in two years of schooling corresponds to what other students achieve in a year’s time (Ortiz, 2009). Likewise, Blakemore & Frith (2007) affirm that these children have a difficulty to learn but it does not make learning impossible, even though its development may be slower. Consequently, the learning processes of these students require additional and specific teaching methodologies. Despite the high prevalence of the LDM and the negative impact it has for children who have them, until now there are very few articles that provide intervention guidelines for teachers and other educational practitioners. Therefore, the main goal of this paper is to present a brief review about some general strategies and guidelines research-based from REDIMAT, 5(1) 57 Cognitive Psychology (e.g.: Artigas, 2011; Cohen, Dowker, Heine, Kaufmann, & Kucian, 2013; Defior, 1996; Ortiz, 2009; Sans, 2008) and the Neuroscience (see interesting review Radford & André, 2009). However, as Butterworth, Varma, & Laurillard (2011 p. 1051) pointed out: "Although the neuroscience may suggest what should be taught, it does not specify how it should be taught". Similarly, Bruno, Noda, Aguilar, González, Moreno, & Muñoz (2006) highlighted there are not many papers that focus on explain specific strategies and materials for children with special needs. In this sense, the present review aims to contribute to cover the gap between research and practice and offer some methodological strategies to compensate the LDM. First, general intervention strategies will be presented, these strategies are general because they can be applied various areas of mathematics learning. Subsequently, this review will cover three of the main areas where children with LDM show more difficulties: number sense, basic arithmetic operations and problem solving. In each area, this paper will include suggestions evidence-based and concrete examples that can facilitate the use in the classroom. Characteristics of Children with LDM These characteristics will be presented in relation to the main fields of mathematical knowledge. These fields are the numerical sense, basic arithmetic operations and problem solving (Santiuste & González-Pérez, 2005). These fields are considered to follow a pyramidal structure, being the number sense the base where the learning process of basic arithmetic operations (addition, subtraction, multiplication and division) and problem solving stand. Moreover, these specific fields of knowledge within mathematics are influenced by various cognitive processes such as attention, memory, language and knowledge of basic concepts (e.g. size or shape). Number Sense The number sense or numerosity concept is the ability to understand the meaning of numbers. This ability is ancestral and it is prior to language and 58 Jiménez – Overcoming Learning Difficulties in Mathematics reading, obviously, and is developed without need of any formal education at a very early stage. The development of the number sense comprises different stages: 1. Development of a general number sense: innate ability with which we are able to differentiate between one and multiple elements. 2. Development of the verbal number sense: ability to associate a quantity with a specific word. 3. Development of the symbolic number system: ability to associate a quantity with a specific number. 4. Development of the representation of a numerical sequence, called mental number line: ability to represent numbers in a consecutive manner on an imaginary number line. However, these stages do not develop linearly in all case; in this sense, the knowledge in one stage can facilitate the acquisition of others (see Dehaene, 2011 for details). The number sense is primarily developed from informal and nonintentional learning, that is to say, through the child’s every day experiences, without the need of formal learning. The number sense is a fundamental aspect in order to acquire a proper number processing ability. It is essential, for example, to the learning of basic arithmetic operations. In addition, there are other factors involved in the development of the number processing ability. These can be personal, like the intellectual ability, or environmental, like teaching at the school. In the same way, the number sense is the basis of several mathematical abilities such as numbering, mental arithmetic and estimation. In this sense, some authors regard an inadequate development of the number sense as the core of the learning disabilities in mathematics, that is, children with LDM do not develop this knowledge or they develops more slowly than typically developing children (Berch, 2005). However, although the development of number sense in children with LDM does not occur spontaneously (incidental fashion), they can acquire it with explicit instruction (see this paper, section 4.1.). The deficit in the development of number sense makes children with LDM have problems to associate numbers and objects; to understand that each 10 units make up a ten; for counting; to understand the place value of figures in a quantity (e.g. 3 does not have the same place value in 328 as in REDIMAT, 5(1) 59 23); to sort numbers in decreasing or increasing order; to decompose and compose numbers (e.g. identify units, tens, hundreds, etc.); and/or to complete a sequence of related numbers (e.g. 147-140-133...). Moreover, these children fail at a high rate in recalling numerical information from the long-term memory, i.e., knowledge stored over a long period of time, often over the entire life span of the individual. The type of mathematical knowledge stored in this type of memory is, for example, the multiplication tables and the learning and storing of this kind of information is a hard task for children with LDM. In the same way, they usually make mistakes when reading and writing multi-digit numbers, especially those which have zeros. These problems remain throughout the school period, so these children’s’ progress is different to that of their partners. This is generally slower and lacking the necessary automation to do basic mathematical procedures. All in all, the learning of mathematics stands upon an innate ability called number sense, even though its learning context corresponds to that of the school. This skill is not taught but gained incidentally from a repetition of experiences with the environment. This innate ability would be the main explanation for learning disabilities in mathematics. However, more research is needed in order to set its limits and define the exact cause of the problem. Basic Arithmetic Operations Basic arithmetic operations (BAO) are addition, subtraction, multiplication and division. The learning of these operations is an essential goal in all stages of Elementary Education. Children with LDM get to know the different concepts related to basic arithmetic operations but do not proceed automatically. In this sense, they do not use the appropriate arithmetic strategies for their level of development (e.g. counting from the addend of greatest value), nor automate basic combinations of numbers (e.g. 2+2). Consequently, these children take more time to do an operation and usually need complementary materials, tangible or visual, for help (e.g. using sticks or counting on their fingers). Moreover, they often have problems with the spatial location of numbers when doing operations (e.g. 352+47= 822) and make mistakes in 60 Jiménez – Overcoming Learning Difficulties in Mathematics the direction of the operation (they start from right to left). In general, these students make many procedural errors, especially in operations with borrowings. Going into more into detail, they may find more difficulties with subtractions since it is a complex concept that requires having acquired the concept of reversibility. In this respect, one of the most common mistakes is to subtract the bigger number from the smaller without considering if it is the minuend or the subtrahend. Another frequent mistake is confusing mathematical signs, doing the wrong operation and sometimes mixing addition and subtraction. Furthermore, they usually omit or change some step of the algorithm; for example, they only do an operation with the first number in a division with a two-figure divisor. The division is the most difficult arithmetic operation for children with learning disabilities in mathematics. Some of the reasons are the following: it is essential to have fully acquired the other BAOs upon which the division stands, and also it is the only operation which has two results (quotient and remaining) and has to be reviewed because the remaining cannot be bigger than the quotient, for example. Concerning calculation, these children do not only have trouble doing BAOs but also with the content that is delivered during the last years of Elementary Education, like operations with fractions and decimal numbers. In most of the cases, these difficulties are due to an inappropriate acquisition of basic arithmetic operations. Therefore, it is crucial to ensure that children, especially with LDM, are taught new knowledge only when previous knowledge has been fully understood. The Importance of Error Analysis Error analysis is considered to be an exact way to detect and determine children’s difficulties. Research clearly shows that children with LDM do not make mistakes by chance or as a consequence of their lack of attention, but because of systematic wrong ways of procedure or knowledge (Defior, 1996). Teachers should regard errors as signs of deficit, being these ones the point of departure from which solutions for the student’s difficulties should start. Errors should be presented as normal things, as part of the whole process of improvement. Therefore, if a child makes a mistake in REDIMAT, 5(1) 61 computation the teacher has to analyze the error pattern and encourage the student to do the operation again, this time with the support that the error analysis has determined. The identification of the error pattern will let us put all complementary strategies into operation so that the child can overcome it. Seven error patterns, which frequently appear in arithmetic operations, have been identified (Enright, 1989). They are: 1. Borrowing from another operation. This mistake indicates that the child has not understood correctly the place value of numbers or the steps to follow in order to do an operation. For example, let’s assume the child has to do the subtraction 460-126 and the solution they propose is 340. The error is due to the 0 in the minuend because this number has different values in additions, subtractions, multiplications and divisions and they get confused. Another error of this kind would be 460-126= 346. 2. Replacement in the process. Errors are due to the substitution of one or several steps of the algorithm for another that has been made up or is wrong. In the following example, it is shown how the student only does the first multiplication and then copies the rest of the numbers: 232x3 = 236. 3. Omission. The error comes after the omission of any of the steps of the algorithm (the child does not invent it, only forgets it). In the following example, it is shown how the student only pays attention to the decimals and forgets about the integers: 2.75+0.44=1.19. 4. Direction. Errors in the order or direction of the proceedings (they start from left to right), even though the computation is correct. 5. Place. The calculation is right but the student changes the place of the elements when they are writing the operation. For example: 9+5=41. 6. Operational symbols. The error is due to a wrong interpretation of the operational symbol or that the sign is simply ignored. 7. Guessing. Sometimes, errors do not follow any logical explanation. This indicates that the basis of the operations has not been understood. Other types of error that appear frequently are: Mismatching of quantities when doing an addition or when organising intermediate products in multiplications or the omission of zero (e.g.: it is thought that 0+N=N, for example: 208-62=166). Error patterns of children with LDM have to be studied carefully in order to detect and correct them. Teachers have to learn to distinguish systematic errors which result from a lack of knowledge or the use of 62 Jiménez – Overcoming Learning Difficulties in Mathematics wrong strategies from those which are due to an isolated lack of attention. As Defior (1996) has pointed out, systematic errors are not solved by doing more practice and exercises but by a teaching method focused on every mistake considered individually. Problem Solving Problem solving is a complex area of mathematics, which requires knowledge from both of the above-mentioned areas (number sense and basic arithmetic operations) and language knowledge. Children with LDM difficulties in problem solving relate to an inadequate way of carrying out the different processes (representation and planning, carrying out of the operations and reviewing and evaluation tasks) that are implied in this activity (Romero & Lavigne, 2005). Difficulties in the representation and planning processes. In order to solve a problem, it is necessary to build a mental representation of the problem statement. That is to say, the student has to understand the terms in which the problem is expressed and relate them. Translating the narrative language to mathematical language is essential to understand it. Children with learning disabilities in mathematics can find this translation process difficult and, consequently, have more difficulties to build a representation of the statement. These problems are more visible when the semantic structure of the problem is complex, for example a comparison, or if the solution of the problem only results by doing multiple steps, like two or more arithmetic operations. Furthermore, it is important to highlight that some of the children that have difficulties in problem solving also have problems with reading comprehension. Consequently, the lack of reading comprehension explains the fact that these children usually do arithmetic operations and algorithms correctly but make mistakes translating the problem. One of the most frequent mistakes that children with LDM make is called "direct or literal translation". This incorrect strategy consists of focusing on looking for the numbers that appear in the problem statement as well as some key words to identify the operations they have to do. For example, the problem "Pablo had 16 Euros and he spent half of his money. His grandfather gives 12 Euros more. How many Euros does Pablo have? Children with LDM pay attention to the word "more" and some numbers REDIMAT, 5(1) 63 and they solve (incorrectly) the problem with the operation 16+12. Likewise, children with LDM have trouble identifying pertinent information and discarding that which is not necessary for the resolution of the problem. Difficulties carrying out the operations. It is possible for a child with LDM to have translated, embedded and planned a mathematical problem correctly but to make a mistake doing the operation due to any of the problems we commented previously about counting. Difficulties during the reviewing and evaluation processes. While the student is solving the problem, they should control all the process so that it results in a solution that suits the plan. In other words, the student have to review what has been done, along with the final result, in order to check if it fits the plan and to identify and correct, if necessary, any possible error. As a general rule, children should turn to their mathematical knowledge to review and control the efficiency of the methods they are using. However, children with LDM do not usually review the final solution or, if done, they have problems to differentiate when something is right or wrong, so they tend to consider their work correct even when it is wrong. Apart from these behavioural features concerning number sense, computation and problem solving, children with LDM usually show some cognitive deficits such as a lack of attention, memory problems, visualspatial problems and difficulties in processing audible information. It is important to point out that children with LDM do not necessarily present all characteristics that have been mentioned, since, as we have already said, this is not a uniform group. The evaluation and analysis of errors will help to determine the concrete characteristics that a child with LDM presents and use them to design an adequate educational treatment for the student. General Strategies of Intervention As in the case of other learning disabilities, the first step is to plan a specific educational intervention to identify the child’s particular deficits and abilities. This identification is comprised from both the assessment results and the error analysis. Once the attainment of the student has been defined, the teacher will be able to work with some clear and quantifiable objectives. As noted Alsina i Pastells (2007), the traditional methodology based on the repetition of mathematical activities makes no sense. Sometimes even the 64 Jiménez – Overcoming Learning Difficulties in Mathematics repetition of activities is applied as a punishment for their poor performance, which has emotional consequences beyond academic scope. In this respect, the solution lies on activating mental processes involved in the different math activities, specifically, the general strategies of intervention (Kaufmann, 2008; Martínez, 2010; Sans, 2008) should be based on:  The usual use of contexts that simulate problems and arithmetic operations relevant to their daily life.  Procedures that include guidance by direct demonstration along with verbal instructions and/or consecutive attempts by the child with feedback (e.g. making rules explicit in order for the child to achieve autonomy).  The assurance that all necessary previous knowledge is well developed before starting to learn new content.  Include scaffolding teaching practices of the content, using a multisensorial approach.  A learning process built on sequences which go from abstract to concrete things.  Include, from time to time, review exercises which represent varied topics and different situations so that the child can fully master the ability.  The suggestion of individual assignments to support specific difficulties.  Give extra time to solve mathematical tasks at home and at the school, since children with LDM have not yet automated the basic processes.  Treatment of possible associated disorders such as dyslexia (GrossTsur, Manor & Shalev, 1996) and/or attention deficit hyperactivity disorder (Monuteaux, Faraone, Herzig, Navsaria & Biederman, 2005). Strategies Specifically Adapted According to the Field of Difficulty It is very important to bear in mind that mathematical skills are heterogeneous and a mathematical learning demands a wide range of cognitive mechanisms. There are differences among learning multiplication tables, doing estimations of the number of elements or using the area of the REDIMAT, 5(1) 65 circle formula in a problem. Thus diverse kinds of abilities take part even in the same mathematical operation. The heterogeneity of mathematical knowledge makes it clear that children with LDM will not be a uniform group, as it is in other learning disabilities. Therefore, it is essential to determine their specific symptoms in order to create an educational intervention that is specific to their needs. Although many processes are involved in the learning of mathematics, this review focus on the three areas of significant importance in Elementary School and which seems to be the most problematic for children with LDM. Number Sense Prior research has shown that the core of the deficit presented by children with dyscalculia is an inappropriate development of the concept of number or number sense (e.g. Butterworth, Varma and Laurillard, 2011; Dehaene, 1997; Otálora Sevilla & Orozco Hormaza, 2006). Therefore, an assessment of this aspect may detect future difficulties in mathematics (see details of number sense development in Sowder, 1992). The first step to reinforcing the concept of number sense is to strengthen basic concepts which were taught in previous stages of education (e.g. in kindergarten). In this sense, it is sometimes necessary to teach the principles of quantity, order, size, distance and space. The teacher must begin teaching these basic concepts using concrete and familiar materials like seeds or pencils. Once children have fully acquired these concepts, it is advised that the teacher start working on activities which reinforce the meaning of numbers such as magnitude activities (see Figure 1) and/or simple numerical sequences. Figure 1. Activity to reinforce the number magnitude. 66 Jiménez – Overcoming Learning Difficulties in Mathematics In any event, the teaching process must move slowly from concrete to abstract concepts. Therefore, teachers should use tangible materials and other supportive measures to support and build upon the process of number abstraction (Chan, Au & Tang, 2014). The mental number line can be reinforced by using real number tables and lines available in the classroom or written in the students’ exercise books. Likewise, it would be useful for them to count objects or events connected to real life such as lists of attendance, items available in class, schedules, votes, heart beats or Parcheesi. In addition, it is necessary to reinforce the understanding of the place value of numbers. In order to understand this concept, it is important to repeat and make clear that multi-digit numbers are numerical expressions to be decoded in a process that follows certain rules. In other words, every single number has its own value depending on the place they are in. It is highly recommended that you use tangible materials such as sticks or an abacus to strength the composition/decomposition process of numbers. An example of an activity to improve writing numbers using templates is shown in Table 1. Table 1 Example thought to improve the writing of numbers by means of templates (Adapted from Martínez, 2010). Activity A. Write the number in figures Three thousand four hundred and sixty eight 3 4 Three thousand four hundred and sixty Three thousand four hundred and eight Three thousand four hundred Three thousand and sixty Three thousand and eight 6 8 Basic Arithmetic Operations Error analysis will provide us with concrete guidelines for the intervention in BAOs. However, it will be necessary to strengthen both the conceptual REDIMAT, 5(1) 67 knowledge (meaning of operations) and procedural knowledge (steps to follow in order to do an arithmetic operation). In this sense, and although the objective of the intervention is to automate procedural knowledge (so there are cognitive resources available), the importance of conceptual knowledge should be reminded. For example, it will be easier to eventually do a subtraction algorithm automatically if the principles of subtraction are understood. It is important to take into account that to achieve automatization, repetitive practice is required and that children with LDM will need a higher number of repetitions. There are some materials and software very advisable to encourage this practice in a motivational environment. Educational software is particularly useful, as computation exercises are generally very tiring for children with LDM. Moreover, there are some computation support activities we could do with daily materials (for more information, see Martínez, 2010). For example, two rules could serve to get used to doing subtractions. One of them will be the minuend and the other the subtrahend. In the subtraction operation 13-9, both rules would be overlapped in the mentioned numbers so that the zero in the second rule would be situated exactly above the solution of the subtraction, that is, 4 (see other interesting activities about mental calculation in Gálvez, Cosmelli, Cubillos, Leger, Mena, Tanter, et al., 2011). An explicit instruction on counting strategies is very advisable for children with LDM, since they do not usually develop them automatically. This teaching method starts by showing the strategy to the child, verbalizing all the steps. Next, the reasons as to why this strategy is better should be explained, and lastly, the child should be told to do some exercises to practice it. Some examples of strategies which are useful in teaching are the following:  Efficient computation: when doing an addition, it is better to start by the figure of higher value. For example, we will add three to the number 9 in the operation 3+9.  Understanding and using composition and decomposition out of context. For example, using tens to do an addition, e.g. 3+9=2+10=12. 68 Jiménez – Overcoming Learning Difficulties in Mathematics Problem Solving Improving problem solving is not a matter of doing more practical exercises but rather teaching solving strategies that will help a child develop specific competencies and make their performance better (Cabrera & Campistrous,1999). Those strategies must be trained explicitly and appropriately to be applied to a wide range of problems (Castela, 2005). In order to teach the problem solving process, teachers sometimes focus on the arithmetic operations which are implied in the problem. However, the complexity resides primarily in the semantic structure of the statement (Carey, 1991). In this sense, the teacher must take into account the importance of semantic structures of various problems on the pupils´ ability to identify the operation required to solve them. For example, combined semantic structures are easier than compared problems (see Figure 2) and teachers must make explicit the different semantic structure and high or low complexity of them. Example of combine problem (easy semantic structure): Mary has 3 pencils. Peter has 9 pencils. How many pencils do they have altogether? Example of compare problem (difficult semantic structure): In a group there are 5 men more than the women. There are 15 men in the group. How many women are there? Figure 2. Examples of different semantic structures. Additionally, it is essential to teach strategies which are aimed at improving the representation, planning and reviewing processes implied in problem solving (Romero & Lavigne, 2005). The supportive activities should be divided into steps (so that the representation process is reinforced and the statement understood) and end by asking the child to reread the statement and check the result (reviewing process). For instance, it is very effective to include a sentence at the end of each problem such as: Well REDIMAT, 5(1) 69 done! But before moving on to the next exercise, it is very important to reread the statement and check if the answer you have given is possible. Moreover, it should be highlighted that teaching problem solving processes should include different kinds of tasks and these tasks have to go beyond the classical view of a statement to be solved. In other words, the appropriate teaching strategy necessarily includes different kinds of problems, that is, problems with no exact solution, with unnecessary data, or with non-explicit data to be previously inferred by the student. Teachers could pose these questions to their students, which could be solved using the given data, or to discriminate between those questions which could or could not be solved with the given data. Lastly, it is fundamental that the problems are significant for the students in order to improve their performance (Planas & Iranzo, 2009). This means that problems should arouse the children’s curiosity and interest so that they are willing to solve them from the very first moment. In this sense, the teacher should give practical examples and try to relate them to family and/or motivational situations. Moreover, the teacher must ensure that the child has acquired all mathematical vocabulary, since sometimes this is the source of the problem (e.g. double or less than). Regarding problem-solving assessment, the teacher has to focus not only on the final result the child has proposed, but on the analysis of the process as a whole, so that the root of the problem can be determined. In the following example, two different, incorrect answers have been proposed for the same statement: ‘Maria has $9 and buys a book that costs $7.25; how much money does she have at the end?’ A child suggests the solution 16.25 because they have just added the quantities that appear in the statement. Another one says the solution is 2.25 as they have not done the arithmetic operation correctly, although they chose the right type, subtraction (97.25=2.25). The educational intervention would be different in each case despite the fact that both of them gave a wrong answer. For this reason, it is advisable that teachers provide two grades for the correction of mathematical problems; one referring to the representation processes and the other to the operations. As Sans (2010) points out, it is fundamental that both the teachers and the specialists treat a child together in order to adapt the curriculum to that particular student. This can be achieved by prioritizing objectives and 70 Jiménez – Overcoming Learning Difficulties in Mathematics giving the students some strategies and tools to compensate for their difficulties. They should be allowed to use an abacus, calculators or checking multiplication tables, as well as any other visual or graphic element which may help the student enhance their problem solving skills. In conclusion, an efficient method of intervention in LDM should include an explicit teaching of different strategies, which regularly strengthen the acquisition of each step; telling the child how to proceed and use strategies of problem solving; proposing significant problems to the student which have different semantic structures; doing mathematical problems which don’t always implement the same arithmetic operations; completing gaps with mathematical vocabulary and/or previous knowledge; promoting the habit of reviewing; and giving priority to tangible activities, and prioritizing the understanding of concepts and their relations over mechanical procedures. References Alsina i Pastells, À. (2007). ¿Por qué algunos niños tienen dificultades para calcular?: Una aproximación desde el estudio de la memoria humana. RELIME. Revista Latinoamericana de Investigación en Matemática Educativa, 10(3), 315-333. Artigas, J. (2011). Trastornos del Neurodesarrollo. Barcelona: Editorial Narbona. 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REDIMAT, 5(1) 73 Sowder, J. T. (1992). Estimation and number sense. In D. A. Grouws (Ed.), Handbook of research on mathematics teaching and learning (pp. 371-389). New York: Macmillan. Gracia Jiménez-Fernández es profesora contratada doctora, del departamento de Psicología Evolutiva y de la Educación, en la Universidad de Granada, España. Contact Address: La correspondencia directa sobre este artículo debe ser dirigida al autor. Dirección Postal: Facultad de Ciencias de la Educación. Campus Cartuja s/n 18071 Granada. Email: gracijf@ugr.es
https://openalex.org/W2327330019	https://escholarship.org/content/qt22x4c8gd/qt22x4c8gd.pdf?t=orwl7h	English	null	Frequency and Impact of Informant Replacement in Alzheimer Disease Research	Alzheimer disease and associated disorders	2,015	cc-by	8,822	UC Irvine Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine UC Irvine Previously Published Works Title Frequency and Impact of Informant Replacement in Alzheimer Disease Research Permalink https://escholarship.org/uc/item/22x4c8gd Journal Alzheimer Disease & Associated Disorders, 29(3) ISSN 0893-0341 Authors Grill, Joshua D Zhou, Yan Karlawish, Jason et al. Publication Date 2015-07-01 DOI 10.1097/wad.0000000000000078 Copyright Information This work is made available under the terms of a Creative Commons Attribution Li availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine UC Irvine Previously Published Works Title Frequency and Impact of Informant Replacement in Alzheimer Disease Research Permalink https://escholarship.org/uc/item/22x4c8gd Journal Alzheimer Disease & Associated Disorders, 29(3) ISSN 0893-0341 Authors Grill, Joshua D Zhou, Yan Karlawish, Jason et al. Publication Date 2015-07-01 DOI 10.1097/wad.0000000000000078 Copyright Information This work is made available under the terms of a Creative Commons Attribution Li availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed Abstract Informants serve an essential role in Alzheimer’s disease (AD) research. Were an informant to be replaced during a longitudinal study, this could have negative implications. We used data from the National Alzheimer’s Coordinating Center Uniform Data Set to examine the frequency of informant replacement among AD dementia participants, whether patient and informant characteristics were associated with replacement, and how replacement affected research outcome measures. Informant replacement was common (15.5%) and typically occurred after the first or the second research visit. Adult child (24%) and other (38%) informants were more frequently replaced than spouse informants (10%). Older spouse informant age and younger adult child informant age were associated with replacement. The between-visit change in Functional Assessment Questionnaire (FAQ) scores was greater in patients who replaced informants than in those with stable informants. Clinical Dementia Rating-Sum of Boxes, FAQ, and Neuropsychiatric Inventory scores showed greater variability in between-visit change in patients who replaced informants compared to those with stable informants. These findings suggest that informant replacement is relatively common, may have implications to study analyses, and warrant further examination in the setting of clinical trials. Author Manuscript Keywords Alzheimer’s disease; caregivers; research; informant; spouses; adult children; outcome measurements Author Manuscript Frequency and impact of informant replacement in Alzheimer’s disease research Joshua D. Grill, PhD1,, Yan Zhou, PhD1, Jason Karlawish, MD3, and David Elashoff, PhD1,2 1Mary S. Easton Center for Alzheimer’s Disease Research, Department of Neurology, David Geffen School of Medicine at UCLA, Los Angeles, CA 2Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA 3University of Pennsylvania, Perelman School of Medicine, Departments of Medicine, and Medical Ethics and Health Policy, Penn Memory Center, Philadelphia, PA Joshua D. Grill, PhD1,, Yan Zhou, PhD1, Jason Karlawish, MD3, and David Elashoff, PhD1,2 1Mary S. Easton Center for Alzheimer’s Disease Research, Department of Neurology, David Geffen School of Medicine at UCLA, Los Angeles, CA Author Manuscript Published in final edited form as: Published in final edited form as: closure: Dr. Grill serves as site investigator for clinical trials sponsored by Avanir, Biogen Idec, Eli Lilly, Genentech, Jansse heimer Immunotherapy Bristol Myers Squibb and the Alzheimer’s Disease Cooperative Study (ADCS) rresponding author: Joshua Grill, PhD, UCLA Easton Alzheimer’s Center, 10911 Weyburn Ave, Suite 200. Los Angeles, CA 095, USA. Tel.: (310) 794-2511; fax: (310) 794-3148. jgrill@mednet.ucla.edu. closure: Dr. Grill serves as site investigator for clinical trials sponsored by Avanir, Biogen Idec, Eli Lilly, Genentech, Jansse heimer Immunotherapy, Bristol-Myers Squibb, and the Alzheimer’s Disease Cooperative Study (ADCS) Alzheimer Immunotherapy, Bristol-Myers Squibb, and the Alzheimer’s Disease Cooperative Study (ADCS) Disclosure: Dr. Grill serves as site investigator for clinical trials sponsored by Avanir, Biogen Idec, Eli Lilly, Genentec Powered by the California Digital Library University of California eScholarship.org eScholarship.org Corresponding author: Joshua Grill, PhD, UCLA Easton Alzheimer’s Center, 10911 Weyburn Ave, Suite 200. Los Angeles, CA 90095, USA. Tel.: (310) 794-2511; fax: (310) 794-3148. jgrill@mednet.ucla.edu. Disclosure: Dr Grill serves as site investigator for clinical trials sponsored by Avanir Biogen Idec Eli Lilly Genentech Janssen Corresponding author: Joshua Grill, PhD, UCLA Easton Alzheimer’s Center, 10911 Weyburn Ave, Suite 200. Los Angeles, CA 90095, USA. Tel.: (310) 794-2511; fax: (310) 794-3148. jgrill@mednet.ucla.edu. Disclosure: Dr. Grill serves as site investigator for clinical trials sponsored by Avanir, Biogen Idec, Eli Lilly, Genentech, Janssen Al h i I h B i l M S ibb d h Al h i ’ Di C i S d (ADCS) HHS Public Access Author Manuscript Corresponding author: Joshua Grill, PhD, UCLA Easton Alzheimer s Center, 10911 Weyburn Ave, Suite 200. Los Angeles, CA 90095, USA. Tel.: (310) 794-2511; fax: (310) 794-3148. jgrill@mednet.ucla.edu. Disclosure: Dr. Grill serves as site investigator for clinical trials sponsored by Avanir, Biogen Idec, Eli Lilly, Genentech, Janssen Dr. Grill serves as site investigator for clinical trials sponsored by Avanir, Biogen Idec, Eli Lilly, Genentech, Janssen mmunotherapy, Bristol-Myers Squibb, and the Alzheimer’s Disease Cooperative Study (ADCS) Background Alzheimer’s disease (AD) research, especially dementia clinical trials, relies on accurate assessment of longitudinal patient change.1–2 Informants may be better at assessing disease- Grill et al. Page 2 related symptoms and changes in those symptoms than are patients themselves.3 Informant- based AD research scales assess patients’ cognitive domains, neuropsychiatric symptoms, occupational and community activities, and basic and instrumental activities of daily living. Informant-based measures of patient performance offer advantages, relative to direct patient testing. They are associated with less patient burden, may be less susceptible to cultural and educational bias, can incorporate change in performance in single time point ratings, and may be more relevant to everyday function.4 Informant-based measures of patient performance have disadvantages as well. To be effective, these scales depend on accurate and consistent ratings by informants, and some informants may provide more valid data than others. Author Manuscript Author Manuscript Factors that affect informant accuracy could bias study results and lead to error. Previous studies suggest that particular informant and patient characteristics may be associated with informant accuracy, both for subjective outcomes such as depression and quality of life,5–7 and for more objective constructs such as cognition and daily function.8–11 For example, spouses may provide more accurate data on patient cognition, compared to non-spousal relatives.8–10 Informants under greater caregiver stress may rate patient function as worse12 and may be more likely to differ from patient self-report5, 11–12 or direct patient measures.11, 13 Disease severity may also impact informant accuracy. In one study, informant reports more poorly predicted patient cognitive performance in mild (global Clinical Dementia Rating [CDR] scale=1), compared to very mild, dementia (global CDR=0.5).10 Author Manuscript Author Manuscript Another factor that might impact accuracy is replacing an informant. Replacement would require a new informant to complete research assessments, from which change scores would be calculated as if a consistent source had provided the data. Unfortunately, how often replacement occurs and how replacing an informant affects study outcome measures are unknown. Understanding the implications of switching informants is important to data interpretation in AD research, especially dementia registration trials, where one co-primary outcome to establish drug efficacy is typically informant-based. Author Manuscript We used data from the National Alzheimer’s Coordinating Center Uniform Data Set (NACC UDS) to examine the frequency of informant replacement in longitudinal AD research. Background We examined which participants most frequently experience informant turnover and assessed the impact that replacing an AD research informant has on study outcome measures. We hypothesized that, since they are at greater risk for dropout in the setting of AD trials,14 patients with nonspousal informants would be at increased risk for replacement, and that informant replacement would be associated with differences in change scores on informant- dependent research outcome measures. Author Manuscript Alzheimer Dis Assoc Disord. Author manuscript; available in PMC 2016 July 01. Initiated in 2005, the NACC UDS is a repository for longitudinal data collected from approximately 34 current or previously NIA-funded AD Centers (www.alz.washington.edu).15–16 UDS participants undergo clinical and neuropsychological Data source Initiated in 2005, the NACC UDS is a repository for longitudinal data collected from approximately 34 current or previously NIA-funded AD Centers (www.alz.washington.edu).15–16 UDS participants undergo clinical and neuropsychological Alzheimer Dis Assoc Disord. Author manuscript; available in PMC 2016 July 01. Grill et al. Page 3 assessment on an annual basis. The acceptable visit window is ±3 months, relative to the annual baseline visit anniversary. We examined data collected on or before June 1, 2013. Author Manuscript Measures Patient demographics—We examined participant age; sex; years of education; race, characterized as Caucasian or non-Caucasian; and ethnicity, categorized as Latino or non- Latino. To examine participant fatigue over time, we also included the overall UDS visit number, since some participants first enrolled in the UDS prior to meeting criteria for this study. Additionally, in some analyses we examined the visit number for this study; that is, relative to the first visit for which the participant met eligibility criteria. Author Manuscript Informant demographics—We examined informant age, sex, race, ethnicity, and the relationship of the informant to the patient, categorized as spouse, adult child, or other. Participants Eligible participants were subjects with a diagnosis of probable AD dementia, age 55–90, and a baseline CDR global score of 0.5 or 1.0 who had at least one follow-up visit. Participants were excluded if they had any subsequent neurodegenerative diagnosis other than dementia/probable AD. Study baseline was defined as the first eligible visit for which all criteria were met. The NACC UDS requires participants to have a reliable informant who accompanies them to study visits and completes a variety of outcomes. At each visit, informants are categorized based on their relationship to the participant: spouse, adult child, or other (neither spouse nor adult child) and the UDS variable NEWINF, “is this a new informant?” captures whether an informant was replaced, compared to the prior visit. We excluded data from 361 (16.5%) participants for whom (1) the informant was identified as new but the relation to the participant and the informant birth month and year were unchanged, (2) the informant relation or birth month and year were different from the previous visit but the informant was not listed as new, or (3) the relationship of the informant to the participant at baseline for this study was unknown. The final set for analysis included 1536 participants with stable informants, 218 participants who experienced informant replacement once, and 64 participants who experienced informant replacement more than once. Author Manuscript Alzheimer Dis Assoc Disord. Author manuscript; available in PMC 2016 July 01. Clinical outcomes Clinical Dementia Rating-Sum of Boxes (CDR-SB)17: The CDR is a clinical assessment of global cognitive and functional ability that documents information in six domains: memory, orientation, judgment and problem solving, community affairs, home and hobbies, and personal care. After interviewing both the patient and the informant, an investigator scores each domain as 0 (not demented), 0.5 (questionable dementia), 1.0 (mild dementia), 2.0 (moderate dementia), or 3 (severe dementia). The CDR can be used as a global score of disease state, calculated with a scoring algorithm (see http://www.biostat.wustl.edu/adrc/) or by summing the totals of the separate box scores (CDR-SB). Author Manuscript Alzheimer Dis Assoc Disord. Author manuscript; available in PMC 2016 July 01. Grill et al. Page 4 Page 4 Functional Assessment Questionnaire (FAQ)18: The FAQ is a 10-item tool based solely on informant assessment of the patient’s ability to complete activities of daily living. Items are scored as 0 (normal), 1 (has difficulty but does by self), 2 (with assistance), or 3 (in a dependent manner). Scores range from 0 to 30, with higher scores representing greater functional dependence. In the UDS, missing FAQ values are not permitted but “not applicable” (e.g., never did) is an alternative response. We replaced not-applicable responses with the mean of available items, if at least five items were completed.19 Author Manuscript Author Author Manuscript Neuropsychiatric Inventory (NPI-Q)20–21: The NPI is an informant-based instrument that assesses delusions, hallucinations, dysphoria, anxiety, euphoria, aggression, apathy, irritability, disinhibition, aberrant motor behaviors, nighttime behaviors, and eating and appetite. The informant rates each item as present or absent. If present, the item is rated for its frequency, severity, and the distress it causes the informant.20 The UDS collects the NPI- Q, a brief version of the NPI that only assesses the presence and severity of behavioral symptoms.21 Present symptoms are scored as mild (1 point), moderate (2 points), or severe (3 points), providing an NPI-Q total score ranging from 0 to 36. Author Manuscript Mini-Mental State Exam (MMSE)22: The MMSE is the most widely used cognitive assessment tool in dementia research. It uses a 30-point design to assess orientation, short- term and delayed recall, calculations, language interpretation, naming, and praxis. Higher scores represent greater cognitive performance. The researcher administers the scale to the patient participant. The MMSE was used as an objective covariate in analytic models (see below). Alzheimer Dis Assoc Disord. Author manuscript; available in PMC 2016 July 01. Statistical analyses We examined the frequency of informant replacement in UDS among AD participants in three groups based on baseline informant relationship to the patient participant. Within each informant type, we compared demographic and clinical characteristics at baseline in participants who replaced informants versus those with stable informants, using two-sample t-tests for continuous variables and Chi-square (X2) tests or Fisher’s exact tests for categorical variables. The NPI total score had a highly skewed distribution whereby the majority (69%) of participants had scores of zero, so we used Chi-square tests for the frequency of NPI-Q total scores greater than zero. Author Manuscript We used logistic regression with stepwise selection, using significance levels of 0.05 as criteria to add or remove variables, to identify the variables associated with the outcome of informant replacement. Due to systematic differences among informant groups in demographic characteristics (e.g., participants with adult child informants are older than participants with spouse informants, and adult child informants are younger than spouse informants23) and potentially different mechanisms that drive the replacement, we constructed models separately within each informant group. The pool of candidate variables included patient race and ethnicity, overall UDS visit number, baseline MMSE, CDR, FAQ, and NPI scores, and informant age and sex. For the spouse and adult child groups, patient and informant age were highly correlated (r=0.75 and r=0.78, respectively), so only informant age was included. Similarly, patient and informant sex are associated for the Author Manuscript Grill et al. Page 5 spouse group, so only informant sex was included. For participants who experienced informant replacement more than once, we used their first replacement. spouse group, so only informant sex was included. For participants who experienced informant replacement more than once, we used their first replacement. Author Manuscript Author Manuscript The next series of analyses examined change in informant-based outcome measures and associated factors. We calculated the change in each outcome measure by subtracting scores for the visit immediately preceding informant replacement from the scores for the first visit with the new informant. To select two comparable visits in the group with stable informants, we performed a visit number matching procedure so that the two groups had equal proportions of participants examined at each visit number relative to baseline. This was made possible by first matching participants at the longest duration time points. Ethics All participants and informants in the NACC UDS sign an informed consent document approved by an Institutional Review Board (IRB). The current project was approved the UCLA IRB (#13-001015). Author Manuscript Statistical analyses Participant data from the two consecutive visits selected by the matching procedure were used to compute the change scores. Because of the need to match based on visit number, these analyses excluded participants who experienced informant replacement more than once. To ensure that the comparison was not biased by the difference in the time elapsed between visits, we used actual visit dates and computed a precise time interval for every participant. Two sample t-tests were used to compare the mean of CDR, FAQ, and NPI-Q change scores between the new and stable informant groups. Tests for the equality of variances were used to compare the variance of the change scores for each outcome. In the setting of equal variance, pooled t-tests were performed. In the setting of unequal variance, Satterthwaite t- tests were performed. The same analysis was carried out among those with informant changes to compare those participants whose new informant was an adult child to those with any other type of new informant. Author Manuscript Linear regression models were used to further examine whether change in these informant- based outcome scores was associated with informant replacement when adjusting for covariates of interest. Models were run for each outcome measure and covariates included: whether replacement occurred, sex, patient age, precise time since the previous visit, and type of informant and MMSE score at the visit prior to replacement (or the matched visit in the group with stable informants). An interaction term informant replacementbaseline informant type was included to examine whether the effect of informant replacement differed by baseline informant type. All analyses were performed in SAS v.9.3 (Cary, NC). All statistical analyses are reported with a significance level of 0.05. Author Manuscript Alzheimer Dis Assoc Disord. Author manuscript; available in PMC 2016 July 01. Frequency of informant replacement 1,818 UDS AD dementia participants met study eligibility criteria; 282 (15.5%) had experienced informant replacement at least once. Among the 218 participants who experienced replacement once, most (73%) informant replacement occurred between the baseline and first follow-up visits (42%) or between the first and second follow-up visits Alzheimer Dis Assoc Disord. Author manuscript; available in PMC 2016 July 01. Alzheimer Dis Assoc Disord. Author manuscript; available in PMC 2016 July 01. Grill et al. Page 6 (31%). Fifteen percent of informant replacement occurred after the second follow-up visit, 7% after the third visit, and 3% after the fourth visit. The pattern of informant replacement based on overall UDS visit number was similar. Sixty-five percent of replacement occurred prior to the first or second follow-up visit (data not shown). Author Manuscript Among patients with a spouse informant at baseline, 9.7% experienced informant replacement (Table 1). Participants with adult child (23.7%) and other informants (37.9%) at baseline more frequently replaced informants than those with spouses (X2=113, p<0.001). New informants were typically adult children (68%). Adult children replaced 80% of spouse informants, 64% of adult child informants, and 53% of other informants. Participants with adult child informants at baseline We found no demographic or clinical differences at baseline between patient participants with adult child informants who were replaced and those whose informants were stable (Table 1). Replaced adult child informants were younger than their stable counterparts (Table 2; t-test, p=0.02). Logistic regression showed that informant age was a significant predictor of adult child informant replacement (OR=0.97, 95% CI: 0.95 – 0.999), but in the opposite direction from that seen for spouses. No other variable reached statistical significance. Participants with spouse informants at baseline Table 1 shows the patient participants’ demographic and clinical characteristics at baseline and Table 2 shows their informants’ characteristics at baseline. Patients whose spouse informants were replaced were older (t-test, p<0.001), more often female (X2, p<0.001), more frequently Latino ethnicity (Fisher’s exact test, p<0.001), and had lower education (t- test, p=0.009) than their counterparts with stable informants. Spouse informants who were replaced were older (t-test, p<0.001), more often male (X2, p<0.001), and were more frequently minority race (X2, p=0.02) and Latino ethnicity (Fisher’s exact test, p=0.002) than their consistent counterparts. Author Manuscript The final logistic regression model resulting from the stepwise procedure showed that Latino ethnicity (OR=3.67, 95% CI: 1.39–9.69), older informant age (OR=1.07, 95% CI: 1.04–1.10), and male informant sex (OR=1.60, 95% CI: 1.03–2.50) were significantly associated with replacing a spouse informant. Author Manuscript Alzheimer Dis Assoc Disord. Author manuscript; available in PMC 2016 July 01. Outcome measure change scores Among all participants, regardless of informant type, there was no difference in the mean change in CDR-SB or the NPI-Q for those who did, compared to those who did not, have a new informant. The change in FAQ score, however, was significantly greater for the group with a new informant (4.48 vs 3.45, p=0.02). The new informant group also demonstrated significantly greater variance for change in all three informant-based outcome measures examined (Table 3). The time elapsed between visits was longer on average and exhibited greater variation for the new informant group than for the stable informant group (1.23 ± 0.51 years vs. 1.13 ± 0.36 years). Author Manuscript Table 3 displays the mean between-visit changes in outcomes for those who replaced versus those with stable informants for the different baseline informant type groups. In the group with spouses at baseline (n=97 replaced; n=1100 stable), the results were similar to those for the entire population; FAQ change scores were greater in the replaced group (5.18 vs. 3.58, p=0.03) and the variance was increased for each outcome. For those with adult child (n=74 new; n=341 stable) and other informants at baseline (n=47 new; n=95 stable), no differences were observed in the mean change scores for any outcome measure; the variance associated with between-visit change was greater for the replaced adult child informant group for the NPI-Q and for the replaced other informant group for the FAQ. The time elapsed between visits was different between replaced versus stable informant groups in those with adult child (1.33 ± 0.65 years vs. 1.14 ± 0.33 years) but not those with spouse (1.20 ± 0.44 years vs. 1.13 ± 0.38 years) or other informants (1.15 ± 0.39 years vs. 1.07 ± 0.23 years) at baseline. When we compared those participants whose new informant was an adult child to those with another type of new informant, we found no differences between the groups in the mean between-visit change for any outcome and a difference in the variances only for the CDR- SB (data not shown). In regression models adjusting for the time elapsed between visits, MMSE score at the visit prior to replacement, and participant age and gender (see Table 4), the main effect of replacement held for change scores in FAQ (p=0.02). Participants with other informants at baseline Participants who replaced an informant who was neither spouse nor adult child were more frequently Latino (X2, p=0.01), had lower education (t-test, p=0.01), and had milder scores on the global CDR (t-test, p=0.03), CDR-SB (t-test, p=0.02), and FAQ (t-test, p=0.001) compared to those with stable other informants (Table 1). Other informants who were replaced were more often Latino (X2, p<0.01) and had lower education (t-test, p<0.05) than their stable counterparts (Table 2). Author Manuscript Alzheimer Dis Assoc Disord. Author manuscript; available in PMC 2016 July 01. Grill et al. Page 7 Among participants with other informants, lower scores on the FAQ (OR=0.88, 95% CI: 0.83–0.93) and higher scores on the NPI-Q (OR=1.64, 95% CI: 1.09 – 2.45) at baseline were associated with replacing the informant in the logistic regression. Among participants with other informants, lower scores on the FAQ (OR=0.88, 95% CI: 0.83–0.93) and higher scores on the NPI-Q (OR=1.64, 95% CI: 1.09 – 2.45) at baseline were associated with replacing the informant in the logistic regression. Author Manuscript Alzheimer Dis Assoc Disord. Author manuscript; available in PMC 2016 July 01. Discussion Author Manuscript These findings suggest that the replacement of informants in longitudinal AD research may be a common occurrence. In the NACC UDS, 15% of dementia participants replaced their informants. The majority of replacement occurred early in participation, after the first or second research visit, suggesting that factors other than research fatigue resulted in replacement. In fact, particular patient and informant characteristics were associated with replacement. Replacement was less frequent for those with spouse informants than for those with nonspouse informants. Among spouse informants, however, Latino ethnicity, male informant sex, and older informant age were associated with informant replacement. Latinos and male informants are less common than non-Latino Caucasians and female informants in AD research.14, 24–26 The aspects of caregiving and research participation that act as barriers to enrollment in these spouses27–28 may also increase the risk of their replacement mid- study. The age-associated risk of spouse replacement suggests that death or disability in an aging spouse may necessitate a new informant. In contrast, younger age was associated with adult child informant replacement. Nonspousal informants may have different attitudes toward AD research than do spouses.29 AD patients lacking a spouse participate in research at lower rates14, 24 and may be at increased risk for dropping out when they do.14 These results add to a literature that suggests that careful consideration of which patient-informant dyads to enroll and methods to retain particular dyads especially may be needed to reduce informant replacement and study dropout. Author Manuscript These results may also suggest that informant replacement could affect study data. We observed more than 1-point greater decline between visits on the FAQ in those who replaced, compared to those with stable, informants, after adjusting for covariates (Table 4). Informant replacement, however, was not associated with change in CDR-SB or NPI-Q. Whereas the FAQ is based entirely informant report, the CDR-SB incorporates both informant interview and patient observation. This difference may account for the discrepancy in observations for these two scales, though in the regression model the effect of informant replacement did approach significance for the CDR-SB (p=0.06). The NPI-Q, like the FAQ, is entirely informant-dependent. Though behavioral symptoms become increasingly common with disease progression, NPI-Q measures over time also include fluctuations and reoccurrence.30 In fact, our regression model suggested that only patient sex was associated with change in NPI-Q between visits. Outcome measure change scores The interaction term informant replacementbaseline informant type was not significant for change scores in any of the three outcomes, suggesting that the effect of replacement did not differ by baseline informant type. In addition, greater change in CDR-SB and FAQ were significantly associated with longer time between visits (p<0.0001); both were also associated with MMSE scores at the visit before replacement, but in opposite directions. Those with spouse informants at baseline experienced greater change in FAQ than those with other informants at baseline (p=0.01). Change in NPI-Q was only associated with gender, with females showing greater increase in neuropsychiatric symptoms (p=0.02). Author Manuscript Alzheimer Dis Assoc Disord. Author manuscript; available in PMC 2016 July 01. Page 8 Page 8 Grill et al. Alzheimer Dis Assoc Disord. Author manuscript; available in PMC 2016 July 01. Discussion In our models, greater time between visits was also associated with greater change in FAQ and CDR. Interestingly, the total time elapsed between visits was greater for those replacing versus those with stable adult child informants. Differences in time between visits were not observed for those replacing spouse or other informants, further suggesting (in addition to the regression model) that time alone does not drive the observed effect of replacement on FAQ change scores. Author Manuscript Descriptive statistics suggested that the effect of replacement on FAQ change scores was present in those replacing spouse informants, but absent in those replacing adult child Alzheimer Dis Assoc Disord. Author manuscript; available in PMC 2016 July 01. Alzheimer Dis Assoc Disord. Author manuscript; available in PMC 2016 July 01. Grill et al. Page 9 informants. Because of this, we included the baseline informant type and an interaction term for informant replacementbaseline informant type in our models. Neither baseline informant type nor this interaction term, however, reached significance for any outcome (data not shown), suggesting that the effect of replacement is not unique to patients who replace particular informants. The model did suggest that those with spouse informants demonstrated greater between-visit change on the FAQ than those with other informants, a group not examined in a previous study that found no difference in the rate of change between those with spouse versus those with adult child informants.23 Author Manuscript For each outcome measure, the variability associated with between-visit change scores was greater in those replacing informants than those with consistent informants. Increased variance was also observed for each outcome when limiting analyses to those with spouse informants at baseline. Increased variance was observed for the NPI-Q for those replacing adult child informants, and for the FAQ for those replacing nonspouse, non-adult child informants. Author Manuscript The observed differences in changes in outcome measures and the increased variance for those replacing informants could reduce statistical power to observe differences between research study groups, such as those receiving an intervention versus a control. Alzheimer Dis Assoc Disord. Author manuscript; available in PMC 2016 July 01. Acknowledgments This study was sponsored by a Junior Investigator Award from the National Alzheimer’s Coordinating Center (2012-JI-09); Research using the NACC database is also supported by UO1 AG016976. Drs. Grill, Zhou, and Elashoff receive additional funding from NIA AG016570. Dr. Grill receives additional funding from the Sidell- Kagan Foundation and from the P. Gene and Elaine Smith Endowed Term Chair in Alzheimer’s Disease Research. This study was sponsored by a Junior Investigator Award from the National Alzheimer’s Coordinating Center (2012-JI-09); Research using the NACC database is also supported by UO1 AG016976. Drs. Grill, Zhou, and Elashoff receive additional funding from NIA AG016570. Dr. Grill receives additional funding from the Sidell- Author Manuscript g g Kagan Foundation and from the P. Gene and Elaine Smith Endowed Term Chair in Alzheimer’s Disease Researc Limitations It is likely that for many AD dementia research participants, the loss of a primary caregiver results in study drop out. Participants who dropped out were not included in the current analyses. We did, however, observe that the most common new informants were adult children. This may suggest that participants who have adult children, in addition to their informant, may be at reduced risk to drop out. Author Manuscript Although the NACC UDS is an ample data source, some of the subgroup analyses included low numbers of cases (e.g., other informant replacement). These analyses may have been underpowered. Our data do not offer insight into the reasons that informant replacement occurred. The UDS does not collect information on caregiver burden or stress, but previous results suggest that measures of patient function and discrepancies between informant and patient ratings of patient function may be predicted by caregiver burden.12–13 It is possible that burden due to changes in participant function resulted in replacement, rather than replacement resulting in inflated scoring of patient worsening. Additionally, baseline MMSE score was not predictive of replacement and MMSE change scores were no different in those replacing versus those with stable informants (data not show). These findings may suggest that informant replacement is not due to disease progression. Author Manuscript Whereas lower MMSE scores were associated with greater change in the CDR-SB, higher MMSE scores were associated with greater change in the FAQ between visits. This is likely due to a ceiling effect in the population studied for the FAQ, as has been previously observed.19, 23, 31 Additionally, among those with non-spouse, non-adult child informants, Alzheimer Dis Assoc Disord. Author manuscript; available in PMC 2016 July 01. Grill et al. Page 10 milder scores on the FAQ were associated with replacement. While intriguing, further study will be necessary to elucidate this relationship. Author Manuscript Finally, given that the UDS includes annual follow-up over many years, it is unclear to what extent these results will generalize to other research studies, in particular dementia clinical trials, which are frequently 18 or 24 months in total length and have much more frequent visits and evaluations. Though the outcome measures we examined are commonly used in clinical trials, others, such as the AD Cooperative Study-Activities of Daily Living scale32 or the AD Assessment Scale-cognitive subscale,33 are not collected in the UDS. Conclusions These findings suggest that informant replacement is common and that particular research participants may be at risk for loss of informant. The loss of informant may result in inconsistent data reporting and exploratory analyses suggest that replacement may result in inflated measures of change in patient function and increased outcome measure variance. Such effects could have implications in data analyses and study interpretation. These observations warrant further study in AD dementia treatment trials. Investigators need to know the frequency of informant replacement, whether similar characteristics predict its occurrence, and if replacement impacts trial data. In addition to potential implications to outcome data, informant replacement could impact study completion rates, adverse event reporting, and other variables critical to trial success. Author Manuscript Alzheimer Dis Assoc Disord. Author manuscript; available in PMC 2016 July 01. References 2004; 18(1):11–6. 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References Baseline informant: Spouse Adult child Other Follow-up informant: Stable New Stable New Stable New N (%) 1100 (90.3) 118 (9.7) 341 (76.3) 106 (23.7) 95 (62.1) 58 (37.9) Age, mean years ± SD 73.3 ± 8.2 76.0 ± 7.1 78.9 ± 6.9 77.7 ± 7.6 75.2 ± 9.2 77.5 ± 8.5 Female, n (%) 387 (35.2) 63 (53.4)* 291 (85.3) 88 (83.0) 71 (74.7) 46 (79.3) Non-Caucasian race, n (%) 101 (9.2) 14 (12.4) 71 (21.3) 21 (21.7) 22 (23.4) 13 (26.5) Latino ethnicity, n (%) 26 (2.4) 12 (10.2)* 39 (11.6) 18 (17.1) 9 (9.6) 14 (24.6)* Education, mean years ± SD 15.4 ± 2.9 14.5 ± 3.5* 13.1 ± 3.6 13.0 ± 4.1 14.5 ± 3.7 12.5 ± 5.0* Baseline global CDR, mean ± SD 0.8 ± 0.2 0.8 ± 0.2 0.8 ± 0.2 0.8 ± 0.2 0.8 ± 0.2 0.8 ± 0.3* Baseline CDR-SB, mean ± SD 4.5 ± 1.8 4.7 ± 1.9 5.0 ± 2.0 4.6 ± 1.6 5.0 ± 2.0 4.1 ± 2.3* Baseline MMSE, mean ± SD 22.6 ± 4.1 22.7 ± 3.6 21.7 ± 4.2 22.0 ± 3.7 21.6 ± 4.4 22.4 ± 5.5 Baseline FAQ, mean ± SD 13.6 ± 7.2 14.0 ± 7.0 15.0 ± 7.3 15.0 ± 7.3 16.2 ± 7.2 11.9 ± 8.4* Baseline NPI-Q, mean ± SD 0.48 ± 0.86 0.56 ± 1.02 0.66 ± 1.10 0.64 ± 1.02 0.42 ± 0.89 0.65 ± 1.04 Table 1 statistics of patient participants who did and did not experience informant replacement, categorized by baseline informant type (p<0.05 l t t t X2 t t Fi h ’ t t t l ti t t bl ) Author Manuscript Author Manuscript Author Manuscript Table 1 Descriptive statistics of patient participants who did and did not experience informant replacement based on two-sample t-test or X2 test or Fisher’s exact test, relative to stable group). References Page 13 Author Manuscript Author Manuscript Author Manuscript Author Manuscript Table 1 Descriptive statistics of patient participants who did and did not experience informant replacement, categorized by baseline informant type (p<0.05 based on two-sample t-test or X2 test or Fisher’s exact test, relative to stable group). Baseline informant: Spouse Adult child Other Follow-up informant: Stable New Stable New Stable New N (%) 1100 (90.3) 118 (9.7) 341 (76.3) 106 (23.7) 95 (62.1) 58 (37.9) Age, mean years ± SD 73.3 ± 8.2 76.0 ± 7.1* 78.9 ± 6.9 77.7 ± 7.6 75.2 ± 9.2 77.5 ± 8.5 Female, n (%) 387 (35.2) 63 (53.4)* 291 (85.3) 88 (83.0) 71 (74.7) 46 (79.3) Non-Caucasian race, n (%) 101 (9.2) 14 (12.4) 71 (21.3) 21 (21.7) 22 (23.4) 13 (26.5) Latino ethnicity, n (%) 26 (2.4) 12 (10.2)* 39 (11.6) 18 (17.1) 9 (9.6) 14 (24.6)* Education, mean years ± SD 15.4 ± 2.9 14.5 ± 3.5* 13.1 ± 3.6 13.0 ± 4.1 14.5 ± 3.7 12.5 ± 5.0* Baseline global CDR, mean ± SD 0.8 ± 0.2 0.8 ± 0.2 0.8 ± 0.2 0.8 ± 0.2 0.8 ± 0.2 0.8 ± 0.3* Baseline CDR-SB, mean ± SD 4.5 ± 1.8 4.7 ± 1.9 5.0 ± 2.0 4.6 ± 1.6 5.0 ± 2.0 4.1 ± 2.3* Baseline MMSE, mean ± SD 22.6 ± 4.1 22.7 ± 3.6 21.7 ± 4.2 22.0 ± 3.7 21.6 ± 4.4 22.4 ± 5.5 Baseline FAQ, mean ± SD 13.6 ± 7.2 14.0 ± 7.0 15.0 ± 7.3 15.0 ± 7.3 16.2 ± 7.2 11.9 ± 8.4* Baseline NPI-Q, mean ± SD 0.48 ± 0.86 0.56 ± 1.02 0.66 ± 1.10 0.64 ± 1.02 0.42 ± 0.89 0.65 ± 1.04 Alzheimer Dis Assoc Disord. Author manuscript; a Author Manuscript Author Manuscript Author Manuscript Author Manuscript Table 1 Descriptive statistics of patient participants who did and did not experience informant replacement, categorized by baseline informant type (p<0.05 based on two-sample t-test or X2 test or Fisher’s exact test, relative to stable group). References Baseline informant: Spouse Adult child Other Follow-up informant: Stable New Stable New Stable New N (%) 1100 (90.3) 118 (9.7) 341 (76.3) 106 (23.7) 95 (62.1) 58 (37.9) Age, mean years ± SD 73.3 ± 8.2 76.0 ± 7.1 78.9 ± 6.9 77.7 ± 7.6 75.2 ± 9.2 77.5 ± 8.5 Female, n (%) 387 (35.2) 63 (53.4)* 291 (85.3) 88 (83.0) 71 (74.7) 46 (79.3) Non-Caucasian race, n (%) 101 (9.2) 14 (12.4) 71 (21.3) 21 (21.7) 22 (23.4) 13 (26.5) Latino ethnicity, n (%) 26 (2.4) 12 (10.2)* 39 (11.6) 18 (17.1) 9 (9.6) 14 (24.6)* Education, mean years ± SD 15.4 ± 2.9 14.5 ± 3.5* 13.1 ± 3.6 13.0 ± 4.1 14.5 ± 3.7 12.5 ± 5.0* Baseline global CDR, mean ± SD 0.8 ± 0.2 0.8 ± 0.2 0.8 ± 0.2 0.8 ± 0.2 0.8 ± 0.2 0.8 ± 0.3* Baseline CDR-SB, mean ± SD 4.5 ± 1.8 4.7 ± 1.9 5.0 ± 2.0 4.6 ± 1.6 5.0 ± 2.0 4.1 ± 2.3* Baseline MMSE, mean ± SD 22.6 ± 4.1 22.7 ± 3.6 21.7 ± 4.2 22.0 ± 3.7 21.6 ± 4.4 22.4 ± 5.5 Baseline FAQ, mean ± SD 13.6 ± 7.2 14.0 ± 7.0 15.0 ± 7.3 15.0 ± 7.3 16.2 ± 7.2 11.9 ± 8.4* Baseline NPI-Q, mean ± SD 0.48 ± 0.86 0.56 ± 1.02 0.66 ± 1.10 0.64 ± 1.02 0.42 ± 0.89 0.65 ± 1.04 Alzheimer Dis Assoc Disord. Author manuscript; a Author Manuscript Author Manuscript Author Manuscript Author Manuscript Table 1 Descriptive statistics of patient participants who did and did not experience informant replacement, categorized by baseline informant type (p<0.05 based on two-sample t-test or X2 test or Fisher’s exact test, relative to stable group). Alzheimer Dis Assoc Disord. Author manuscript; available in PMC 2016 July 01. Alzheimer Dis Assoc Disord. Author manuscript; available in PMC 2016 July 01. References Baseline informant: Spouse Adult child Other Follow-up informant: Stable New Stable New Stable New Age, mean years ± SD 70.6 ± 8.8 75.7 ± 9.3 51.4 ± 8.4 49.2 ± 8.7* 62.8 ± 12.8 58.7 ± 14.8 Female, n (%) 708 (64.4) 54 (45.8)* 260 (76.3) 72 (67.9) 75 (79.0) 43 (74.1) Non-Caucasian race, n (%) 76 (7.0) 15 (13.2)* 77 (23.0) 29 (28.4) 22 (23.4) 21 (36.8) Latino ethnicity, n (%) 29 (2.7) 10 (8.9)* 42 (12.5) 18 (17.7) 7 (7.5) 14 (24.1)* Education, mean years ± SD 15.1 ± 2.8 14.8 ± 3.1 15.9 ± 2.7 15.6 ± 2.6 15.6 ± 2.8 14.5 ± 3.1* l h d h i il bl i 6 l Author Manuscript Author Manuscript Author Manuscript Author Manuscript Grill et al. Table 2 Descriptive statistics of the baseline informants who were and were not replaced, categorized by baseline informant type (p<0.05 based on two-sample t- test or X2 test or Fisher’s exact test, relative to stable group). Baseline informant: Spouse Adult child Other Follow-up informant: Stable New Stable New Stable New Age, mean years ± SD 70.6 ± 8.8 75.7 ± 9.3 51.4 ± 8.4 49.2 ± 8.7* 62.8 ± 12.8 58.7 ± 14.8 Female, n (%) 708 (64.4) 54 (45.8)* 260 (76.3) 72 (67.9) 75 (79.0) 43 (74.1) Non-Caucasian race, n (%) 76 (7.0) 15 (13.2)* 77 (23.0) 29 (28.4) 22 (23.4) 21 (36.8) Latino ethnicity, n (%) 29 (2.7) 10 (8.9)* 42 (12.5) 18 (17.7) 7 (7.5) 14 (24.1)* Education, mean years ± SD 15.1 ± 2.8 14.8 ± 3.1 15.9 ± 2.7 15.6 ± 2.6 15.6 ± 2.8 14.5 ± 3.1* Alzheimer Dis Assoc Disord. Author manuscript; available in PMC 201 Page 14 Grill e Table 2 Descriptive statistics of the baseline informants who were and were not replaced, categorized by baseline informant type (p<0.05 based on two-sample t- test or X2 test or Fisher’s exact test, relative to stable group). Grill Table 2 e statistics of the baseline informants who were and were not replaced, categorized by baseline informant type (p<0.05 based on two-sample t- t Fi h ’ t t t l ti t t bl ) Page 14 Grill et al. References Baseline informant: Spouse Adult child Other Follow-up informant: Stable New Stable New Stable New N (%) 1100 (90.3) 118 (9.7) 341 (76.3) 106 (23.7) 95 (62.1) 58 (37.9) Age, mean years ± SD 73.3 ± 8.2 76.0 ± 7.1* 78.9 ± 6.9 77.7 ± 7.6 75.2 ± 9.2 77.5 ± 8.5 Female, n (%) 387 (35.2) 63 (53.4)* 291 (85.3) 88 (83.0) 71 (74.7) 46 (79.3) Non-Caucasian race, n (%) 101 (9.2) 14 (12.4) 71 (21.3) 21 (21.7) 22 (23.4) 13 (26.5) Latino ethnicity, n (%) 26 (2.4) 12 (10.2)* 39 (11.6) 18 (17.1) 9 (9.6) 14 (24.6)* Education, mean years ± SD 15.4 ± 2.9 14.5 ± 3.5* 13.1 ± 3.6 13.0 ± 4.1 14.5 ± 3.7 12.5 ± 5.0* Baseline global CDR, mean ± SD 0.8 ± 0.2 0.8 ± 0.2 0.8 ± 0.2 0.8 ± 0.2 0.8 ± 0.2 0.8 ± 0.3* Baseline CDR-SB, mean ± SD 4.5 ± 1.8 4.7 ± 1.9 5.0 ± 2.0 4.6 ± 1.6 5.0 ± 2.0 4.1 ± 2.3* Baseline MMSE, mean ± SD 22.6 ± 4.1 22.7 ± 3.6 21.7 ± 4.2 22.0 ± 3.7 21.6 ± 4.4 22.4 ± 5.5 Baseline FAQ, mean ± SD 13.6 ± 7.2 14.0 ± 7.0 15.0 ± 7.3 15.0 ± 7.3 16.2 ± 7.2 11.9 ± 8.4* Baseline NPI-Q, mean ± SD 0.48 ± 0.86 0.56 ± 1.02 0.66 ± 1.10 0.64 ± 1.02 0.42 ± 0.89 0.65 ± 1.04 Alzheimer Dis Assoc Disord. Author manuscript; available in PMC 2016 July 01. Author Manuscript Alzheimer Dis Assoc Disord. Author manuscript; available in PMC 2016 July 01. Author Manuscript Author Manuscript Author Manuscript Author Manuscript Grill et al. Page 14 Table 2 Descriptive statistics of the baseline informants who were and were not replaced, categorized by baseline informant type (p<0.05 based on two-sample t- test or X2 test or Fisher’s exact test, relative to stable group). References Author Manuscript Author Manuscript Author Manuscript Author Manuscript Table 2 Descriptive statistics of the baseline informants who were and were not replaced, categorized by baseline informant type (p<0.05 based on two-sample t- test or X2 test or Fisher’s exact test, relative to stable group). Baseline informant: Spouse Adult child Other Follow-up informant: Stable New Stable New Stable New Age, mean years ± SD 70.6 ± 8.8 75.7 ± 9.3* 51.4 ± 8.4 49.2 ± 8.7* 62.8 ± 12.8 58.7 ± 14.8 Female, n (%) 708 (64.4) 54 (45.8)* 260 (76.3) 72 (67.9) 75 (79.0) 43 (74.1) Non-Caucasian race, n (%) 76 (7.0) 15 (13.2)* 77 (23.0) 29 (28.4) 22 (23.4) 21 (36.8) Latino ethnicity, n (%) 29 (2.7) 10 (8.9)* 42 (12.5) 18 (17.7) 7 (7.5) 14 (24.1)* Education, mean years ± SD 15.1 ± 2.8 14.8 ± 3.1 15.9 ± 2.7 15.6 ± 2.6 15.6 ± 2.8 14.5 ± 3.1* Author Manuscript Author Manuscript Alzheimer Dis Assoc Disord. Author manuscript; available in PMC 2016 July 01. Grill et al. Page 15 Page 15 Author Manuscript Manuscript Author Manuscript Author Manuscript Author Manuscript Table 3 Between-visit change in informant-based outcome measure scores (p<0.05). Group Outcome measure Stable informant, mean±SD New informant, mean±SD t-test, p value Test of equal variance, p value All participants CDR-SB 2.01±2.57 2.37±3.07 0.10 0.0002 FAQ 3.45±5.02 4.48±6.39 0.02* <0.0001* NPI-Q 0.11±1.14 0.13±1.38 0.81 <0.0001* Spouse informant CDR-SB 2.06±2.56 2.74±3.43 0.06 <0.0001* FAQ 3.58±5.03 5.18±6.74 0.03* <0.0001* NPI-Q 0.11±1.08 0.24±1.33 0.34 0.002* Adult child informant CDR-SB 1.96±2.65 1.95±2.90 0.95 0.27 FAQ 3.40±5.23 3.97±5.14 0.39 0.97 NPI 0.12±1.28 0.09±1.58 0.87 0.01* Other informant CDR-SB 1.52±2.37 2.38±2.56 0.06 0.53 FAQ 1.99±3.72 4.09±7.51 0.08 <0.0001* NPI-Q 0.13±1.27 −0.02±1.08 0.48 0.23 Alzheimer Dis Assoc Disord. Author manuscript; available in PMC 2016 July 01. nuscript Author Manuscript Author Manuscript Author Manuscript Table 3 Between-visit change in informant-based outcome measure scores (p<0.05). Alzheimer Dis Assoc Disord. Author manuscript; available in PMC 2016 July 01. Alzheimer Dis Assoc Disord. Author manuscript; available in PMC 2016 July 01. References Group Outcome measure Stable informant, mean±SD New informant, mean±SD t-test, p value Test of equal variance, p value All participants CDR-SB 2.01±2.57 2.37±3.07 0.10 0.0002 FAQ 3.45±5.02 4.48±6.39 0.02* <0.0001* NPI-Q 0.11±1.14 0.13±1.38 0.81 <0.0001* Spouse informant CDR-SB 2.06±2.56 2.74±3.43 0.06 <0.0001* FAQ 3.58±5.03 5.18±6.74 0.03* <0.0001* NPI-Q 0.11±1.08 0.24±1.33 0.34 0.002* Adult child informant CDR-SB 1.96±2.65 1.95±2.90 0.95 0.27 FAQ 3.40±5.23 3.97±5.14 0.39 0.97 NPI 0.12±1.28 0.09±1.58 0.87 0.01* Other informant CDR-SB 1.52±2.37 2.38±2.56 0.06 0.53 FAQ 1.99±3.72 4.09±7.51 0.08 <0.0001* NPI-Q 0.13±1.27 −0.02±1.08 0.48 0.23 Alzheimer Dis Assoc Disord Author manuscript Author Manuscript Alzheimer Dis Assoc Disord. Author manuscript; available in PMC 2016 July 01. Grill et al. Page 16 Table 4 Results of regression models for change in each informant-based outcome measure. Author Manuscript Results of regression models for change in each informant-based outcome measure. Variable Estimate (95% C.I.) p<0.05 CDR-SB FAQ NPI-Q Time (years) 1.63 (1.32, 1.95) 1.87 (1.21, 2.53)* 0.003 (−0.14, 0.15) MMSE score −0.12 (−0.15, −0.10)* 0.07 (0.02, 0.12)* 0.008 (−0.003, 0.018) Female gender −0.07 (−0.20, 0.34) 0.13 (−0.45, 0.70) 0.15 (0.02, 0.28)* Age (years) 0.008 (−0.014, 0.016) −0.0003 (−0.033, 0.032) 0.001 (−0.006, 0.008) Replacement 0.50 (−0.04, 1.04) 1.35 (0.20, 2.50)* 0.05 (−0.21, 0.30) Informant type (reference=spouse): child −0.19 (−0.56, 0.17) −0.22 (−0.99, 0.54) −0.05 (−0.22, 0.12) Informant type (reference=spouse): other −0.42 (−0.99, 0.15) −1.53 (−2.73, −0.33)* −0.09 (−0.36, 0.17) Informant type × Replacement (reference=spouse): child −0.48 (−1.31, 0.36) −1.63 (−3.39, 0.14) −0.07 (−0.46, 0.32) Informant type × Replacement (reference=spouse): other 0.16 (−0.90, 1.22) 0.85 (−1.40, 3.10) −0.18 (−0.68, 0.31) Author Manuscript Author Manuscript Author Manuscript
https://openalex.org/W4312190590	https://www.zora.uzh.ch/id/eprint/226605/1/ZORA_s12888_022_04440_x.pdf	English	null	Validation of a new emotion regulation self-report questionnaire for children	BMC psychiatry	2,022	cc-by	14,029	Zurich Open Repository and Archive Zurich Open Repository and Archive University of Zurich University Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch University of Zurich University Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2022 © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Objective: To examine and validate the self‑report Questionnaire on the Regulation of Unpleasant Moods in Children (FRUST), which is a modified and shortened version of the Questionnaire for the Assessment of Emotion Regulation in Children and Adolescents (FEEL‑KJ). Methods: The data comprised child and parent ratings of a community‑screened sample with differing levels of affective dysregulation (AD) (N = 391, age: M = 10.64, SD = 1.33, 56% male). We conducted latent factor analyses to establish a factor structure. Subsequently, we assessed measurement invariance (MI) regarding age, gender, and AD level and evaluated the internal consistencies of the scales. Finally, we examined the convergent and divergent valid‑ ity of the instrument by calculating differential correlations between the emotion regulation strategy (ERS) scales and self‑ and parent‑report measures of psychopathology. Results: A four‑factor model, with one factor representing Dysfunctional Strategies and the three factors Distrac- tion, Problem-Solving and Social Support representing functional strategies provided the best fit to our data and was straightforward to interpret. We found strong MI for age and gender and weak MI for AD level. Differential correlations with child and parent ratings of measures of psychopathology supported the construct validity of the factors. Conclusions: We established a reliable and valid self‑report measure for the assessment of ERS in children. Due to the reduced number of items and the inclusion of highly specific regulatory behaviors, the FRUST might be a valuable contribution to the assessment of ER strategies for diagnostic, therapeutic, and research purposes. Keywords: Emotion regulation, Affective dysregulation, Children, Adolescents, Assessment Validation of a new emotion regulation self-report questionnaire for children Michaela Junghänel1†, Hildegard Wand1†, Christina Dose1, Ann‑Kathrin Thöne1, Anne‑Katrin Treier1, Charlotte Hanisch2, Anne Ritschel2, Michael Kölch3,4,5, Lena Lincke4, Veit Roessner6, Gregor Kohls6, Ulrike Ravens‑Sieberer7, Anne Kaman7, Tobias Banaschewski8, Pascal‑M. Aggensteiner8, Anja Görtz‑Dorten1,9, Manfred Döpfner1,9* and on behalf of the ADOPT Consortium Validation of a new emotion regulation self-report questionnaire for children Junghänel, Michaela ; Wand, Hildegard ; Dose, Christina ; et al ; Brandeis, Daniel DOI: https://doi.org/10.1186/s12888-022-04440-x Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-226605 Journal Article Published Version The following work is licensed under a Creative Commons: Attribution 4 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-226605 Journal Article Published Version Originally published at: Junghänel, Michaela; Wand, Hildegard; Dose, Christina; et al; Brandeis, Daniel (2022). Validation of a new emo- tion regulation self-report questionnaire for children. BMC Psychiatry, 22:820. DOI: https://doi.org/10.1186/s12888-022-04440-x Junghänel et al. BMC Psychiatry (2022) 22:820 https://doi.org/10.1186/s12888-022-04440-x Correspondence: Manfred.doepfner@uk‑koeln.de Correspondence: Manfred.doepfner@uk‑koeln.de 1 School of Child and Adolescent Cognitive Behavior Therapy (AKiP), Faculty of Medicine and University Hospital Cologne, University of Cologne, Pohligstraße 9, 50969 Cologne, Germany Full list of author information is available at the end of the article Background Given that childhood and adolescence is accompanied by many challenges that elicit intense emotions [1], there has been a growing interest in understanding emotion regulation strategies (ERS) and their development in childhood and adolescence [2]. One way to classify ERS is to differentiate between maladaptive or dysfunctional and adaptive or functional strategies based on their immediate effects on affect, cognition, and behavior [3]. An adequate use of functional ERS allows “for monitoring, †Michaela Junghänel and Hildegard Wand shared first authorship and contributed equally to this work. Correspondence: Manfred.doepfner@uk‑koeln.de © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Junghänel et al. BMC Psychiatry (2022) 22:820 Page 2 of 15 evaluating, and modifying emotional reactions, especially their intensive and temporal features” ([4], pp. 27–28). By comparison, the limited use of functional ERS and the use of dysfunctional ERS have been linked to psycho- pathology (e.g. [3]). For example, the dysfunctional ERS rumination is common in depressive disorders (e.g. [5]), whereas in most anxiety disorders, the object of fear is avoided in order to prevent negative emotions (e.g. [6]). Avoidance has also been shown to be associated with higher levels of attention-deficit/hyperactivity disorder (ADHD [7];). Moreover, studies have demonstrated that the limited use of the functional ERS problem-solving, acceptance, and reappraisal is associated with internal- izing and externalizing symptoms in general [8, 9]. Spe- cifically, Braet and colleagues [9] found that internalizing symptomatology in school-aged children was negatively associated with the functional ERS problem-oriented action, distraction, acceptance, forget, and revaluation and that externalizing symptomatology was negatively associated with the functional ERS problem-oriented action and acceptance. apply passivity, avoidance and suppression more fre- quently [11]. The term emotion dysregulation is often used synony- mously with the terms affective dysregulation (AD) and irritability, which all refer to a highly similar and strongly overlapping construct [16–18]. However, as opposed to irritability, the concepts of emotion dysregulation and AD generally include an irritable and an impulsive com- ponent [16, 19]. Moreover, it is consensus that emotion (dys-)regulation primarily describes the process of cop- ing with all kinds of emotional challenges whereas AD describes an emotional state that may result from emo- tion dysregulation [19–21]. Conversely, the expression of negative emotions may also be one of several dys- functional coping processes [22]. This being said, it is important to emphasize that the expression of negative emotions per se is not negative as an adequate expression of negative emotions can serve important social func- tions (e.g. the expression of sadness can lead other people to comfort us; the expression of anger can help set neces- sary boundaries in social contexts). Importantly, emotion dysregulation is not synonymous with any single mental disorder defined in the 5th edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5 [23];) but has been shown to be transdiagnostic and consequently associated with various externalizing and internalizing disorders [2]. At its most extreme it may reflect the new DSM-5 diagnosis of “disruptive mood dysregulation dis- order” (DMDD [24];). DMDD is characterized by severe, recurrent temper outbursts and chronic irritability or angry mood. By comparison, the International Statistical Classification of Diseases and Related Health Problems (ICD-11 [25];) takes a different approach to describing a similar pathology by adding a specifier for chronic irri- tability to the diagnosis of oppositional defiant behavior (ODD), thus reflecting the difficulties to disentangle irri- tability and ODD symptomatology [16, 26]. p Numerous studies have found associations of ERS with gender and age, albeit with inconsistent results. For instance, some studies found that ERS steadily increase with age (e.g. [2, 10, 11]), which is consistent with the cognitive maturation model [12]. In addition to this gen- eral increase in functional ERS, it was found that the kinds of strategies applied also change significantly dur- ing childhood and adolescence [2, 11, 13]. By contrast, other studies (e.g. [11, 13]) reported evidence support- ing the maladaptive shift model [12], which assumes a decrease in functional ERS and an increase in dysfunc- tional ERS in (early) adolescence. Taken together, these results suggest that efficacy and flexibility in the use of ERS increase with age, with an interruption of a few years during adolescence, in which emotion regulation (ER) temporarily deteriorates [12, 13]. Aim of study In light of the shortcomings of the FEEL-KJ, especially its length and repetitive elements, and to be able to monitor therapeutic processes, we revised and adapted this ques- tionnaire, resulting in the newly developed 48-item self- report “Questionnaire on the Regulation of Unpleasant Moods in Children” (German: “Fragebogen zur Regula- tion unangenehmer Stimmungen” [FRUST]) by [masked for blind review], unpublished manuscript). The aim of the present study was to examine the factor structure of the FRUST in a sample consisting of children with either no or pronounced AD symptomatology. Furthermore, we aimed to assess whether the observed factor structure was invariant to gender, age, and AD level. In addition, we examined the internal consistencies of the result- ing scales as well as their correlations with one another. To examine the convergent and divergent validity of the FRUST, we considered the associations of its scales with measures of AD, symptoms of ADHD, symptoms of ODD, anxious/depressed symptoms, and aggressive symptoms. Finally, we analyzed the associations of the FRUST scales with gender and age of the participants. ( Q Q Q) The self-report Questionnaire for the Assessment of Emotion Regulation in Children and Adolescents (FEEL-KJ [33, 34];), for children between the ages of 10;0 and 19;11 years, is a well-established and fre- quently used instrument to assess the application of ERS in children and adolescents [35–37]. With a total of (15x2x3=) 90 items, the FEEL-KJ assesses 15 differ- ent ERS, with each strategy assessed independently with two items for the three emotions anger, sadness, and anxiety. Accordingly, the FEEL-KJ allows for an emo- tion-specific evaluation as well as a comprehensive eval- uation of ERS across the three emotions. As reported by Grob and Smolenski [33], the different strategies can be combined into the higher-order scales adaptive emotion regulation strategies and maladaptive emotion regulation strategies. However, Cracco and colleagues [34] suggested a more complex factor structure, subdi- viding the maladaptive factor into the three lower-level factors avoidance, dysfunctional thoughts, and aggres- sion. The FEEL-KJ overcomes the disadvantage of sev- eral ER measures that assess only a limited number of strategies, although due to the detailed assessment of various ERS, each rated for the three different emo- tions anger, sadness, and anxiety, the 90-item FEEL-KJ is quite lengthy and repetitive. Regarding the effect of gender on ER, numerous studies suggest that girls show fewer functional and more dysfunctional ERS than do boys [12, 13]. For example, in a sample of 8 to 14-year- olds, girls showed significantly decreased functional ERS and significantly increased dysfunctional ERS compared to boys, and this effect was particularly pronounced in females in grades five and six (corresponding to age 10–12 [13];). This finding may potentially be attributable to higher stress levels [13] and a more intense experience of emotions [14] in girls than in boys, or different sociali- zation processes of girls and boys [15]. Other findings offer a more nuanced view on the association between gender and ER, suggesting that boys and girls apply dif- ferent ERS. In particular, functional social support seeking and dysfunctional rumination seem to be more promi- nent in girls [11, 13], whereas boys have been found to In sum, difficulties in ER are associated with a vari- ety of externalizing and internalizing psychiatric dis- orders. Moreover, ERS are especially important for the examination of the newly introduced diagnostic entities of DMDD (DSM-5 [23];) or ODD with chronic irritabil- ity (ICD-11 [25];), both introduced to better capture AD symptomatology. Furthermore, open questions remain with regard to associations of ER with gender and age. Therefore, a valid and reliable assessment of a range of functional and dysfunctional ERS for diagnostic, thera- peutic, and research purposes seems crucial. There are a number of internationally used question- naires assessing ERS in children and adolescents. A meta- analysis by Compas and colleagues [2] found 87 distinct measures of ERS, with the most commonly used being the parent-reported Emotion Regulation Checklist Junghänel et al. BMC Psychiatry (2022) 22:820 Junghänel et al. BMC Psychiatry (2022) 22:820 Page 3 of 15 Junghänel et al. BMC Psychiatry (2022) 22:820 (ERC [27];), which assesses the two dimensions of Neg- ativity and ER with a total of 24 items, and the 36-item self-report Cognitive Emotion Regulation Question- naire (CERQ [28];), which assesses nine cognitive coping strategies. Other frequently used questionnaires include the self-report Emotion Regulation Questionnaire for Children and Adolescents (ERQ-CA [29];), which is an adapted version of the Emotion Regulation Questionnaire for adults [30], and the Children’s Response Style Ques- tionnaire (CRSQ [31, 32];). Both of these measures assess a limited number of strategies (ERQ-CA: ten items, Cog- nitive Reappraisal and Expressive Suppression; CRSQ: 25 items, Rumination, Distraction, Problem-Solving). The main advantages of all of the aforementioned question- naires lie in their reasonable number of items (10–36) as well as their sound psychometric quality. However, they all come with problems, such as the lack of a self- report measure (ERC) or the limited number of strategies assessed (CERQ, ERQ-CA, CRSQ). Aim of study The distinction between the ERS in response to the three emotions seems par- ticularly questionable as the internal consistency and the test-retest reliability for most primary scales for the emotion-specific evaluation are weak [38]. Furthermore, three of the strategies (expression, social support, con- trol of emotions) could not be unequivocally assigned to either of the secondary scales, as associations with psy- chological well-being were not unambiguously positive or negative [38]. Participants and procedure Data collection took place within the ongoing mul- ticenter research project [masked for blind review], which encompasses [masked for blind review]. The multicenter research project aims to optimize the diag- nostic investigation, prevention, and treatment of AD and includes several measurement time points. For the present study, we used the baseline data (T1) of a com- munity-screened sample, which was recruited through the local residents’ registration office and was one of various different samples considered in the research project. The data were collected between August 2018 and September 2019 (for further information see the study protocol; [masked for blind review]). The sample includes n = 391 children aged 8 to 12 years (M = 10.64, SD = 1.33; 56% males). The mothers of 342 (88%) and the fathers of 46 (12%) of these children completed the parent questionnaires used for the current analy- ses. Main inclusion criteria were the age of the child (8;0–12;11 at T1), child living with at least one biologi- cal or adoptive parent, status of AD symptomatology of the child, as well as willingness and ability to partici- pate in the study. Prior to the baseline measurement, participants were assigned to an AD or a NoAD group, based on a predefined cut-off score on a parent screen- ing questionnaire ([masked for blind review; for further details regarding the screening procedure see [masked for blind review]). Children with AD symptom scores Junghänel et al. BMC Psychiatry (2022) 22:820 Junghänel et al. BMC Psychiatry (2022) 22:820 Junghänel et al. BMC Psychiatry (2022) 22:820 Page 4 of 15 Page 4 of 15 FRUST combines the response to these three emotions and assesses the strategies for the regulation of “unpleas- ant emotions” in general. In addition to the 30 items originating from the FEEL-KJ, the FRUST questionnaire includes 18 new items, which refer to interventions used in the scope of the Treatment Program for Children with Aggressive Behavior (THAV; German: “Therapie- programm für Kinder mit aggressivem Verhalten” [39];). These items target very specific behaviors taught in the THAV, that can be applied as ERS when feeling bad (e.g. “When I feel bad, I count to ten” or “When I feel bad, I do a relaxation exercise”), as the THAV program has been shown to reduce aggressive behaviors and increase prosocial behavior in children with peer-related aggres- sive behaviors [40, 41], which is closely associated to AD symptomatology. Participants and procedure The items are rated on a five-point Lik- ert-type scale ranging from 0 (almost never) to 4 (almost always). in the top 10% of the sample were allocated to the AD group and children with scores in the bottom 10% were allocated to the NoAD group. All families from the AD group and a random sample from the NoAD group were asked to participate further in the study. Partici- pating families underwent clinical child and parent interviews [masked for blind review], with the latter being used to confirm the child’s AD status. As shown in Table 1, a total of 244 (62%) study participants were assigned to the AD group. Clinical interviews with the parents ([masked for blind review] by [masked for blind review], in press) were conducted to examine whether the children and adolescents met DSM-5/ICD-10 cri- teria for a mental disorder. All diagnoses can be found in Table 1. DADYS-p/DADYS-c Child AD symptoms were assessed using the self- and parent report forms of the Diagnostic System for Affec- tive Dysregulation (DADYS; German: “Diagnostikum für Affektive Dysregulation”; unpublished manuscript [42];). The self-report form (DADYS-C) comprises 28 items and the parent report form (DADYS-P) comprises 38 items. The DADYS items originate from several existing ques- tionnaires, that is the Emotion Regulation Checklist [27], the German Symptom Checklist for Disruptive Behavior Disorder (FBB/SBB-SSV; German: “Fremd-/Selbstbeur- teilungsbogen für Störungen des Sozialverhaltens” [43];), and the Affective Reactivity Index [44]. These question- naires assess irritability/anger and/or affective dysregu- lation/emotion regulation, leading to a rather broad conceptualization of AD in the present study. All items of the DADYS-C and DADYS-P are rated on a four- point Likert-type scale ranging from 0 (not at all/never) to 3 (very much/always), with higher scores indicating higher symptom severity. A total score was computed by summing up all item scores and dividing this sum by the number of items, resulting in a total score rang- ing between 0 and 4. The internal consistencies of the total scale scores in the present sample were good, with α = .94 (M = .89, SD = .50) for the DADYS-C and α = .96 (M = .89, SD = .56) for the DADYS-P. Table 1 Sample and descriptive statistics SD Standard deviation, M Mean, AD Affective dysregulation, ADHD Attention- deficit/hyperactivity disorder, DMDD Disruptive mood dysregulation disorder, ODD oppositional defiant disorder, AD Affective dysregulation, CD Conduct disorder, MD Major depressive episode a Although not possible in the DSM-5 due to hierarchical rules, simultaneous DMDD and ODD diagnoses are reported here if all diagnostic criteria were met, due to interest in the diagnostic overlap Sample Statistics Total sample n = 391 Age: mean (SD) 10.64 (1.33) Male: n (%) 220 (56) Group: n (%) AD 244 (62) NoAD 147 (38) Diagnoses: n (%) DMDDa 41 (11) ODDa 93 (24) ADHD 62 (16) ADHD, combined type 19 (5) ADHD, predominantly inattentive type 30 (8) ADHD, predominantly hyperactive‑impulsive type 13 (3) CD 6 (2) MD 5 (1) No Diagnosis 262 (67) Measures FRUST The Questionnaire on the Regulation of Unpleasant Moods in Children (FRUST; [masked for blind review], unpublished manuscript) is an adaptation of the FEEL- KJ questionnaire [33], which assesses ERS in children and adolescents. The originally developed version of the FRUST comprises 48 items and is thus shorter than the FEEL-KJ. While the FEEL-KJ assesses the same ERS in response to anger, sadness, and anxiety separately, the SBB−/FBB-ADHS To examine the factor structure of the FRUST, we fol- lowed both an exploratory and a subsequent confirma- tory factor analytic approach. The respective analyses were performed using Mplus version 8.4 [51]. For both analyses, we used the weighted least square mean and variance adjusted (WLSMV) estimator (delta param- eterization), which is recommended for ordinal data [52]. The percentage of missing data per item was ≤0.3% for all items; missing data were handled using pairwise dele- tion. Covariance coverage was ≥ .995 for all items. We based our evaluation of model fit on the following fre- quently used global model fit indices: the comparative fit index (CFI), the Tucker-Lewis index (TLI), the root mean square error of approximation (RMSEA), and the stand- ardized root mean square residuals (SRMR). Good model fit was indicated by CFI and TLI values ≥ .95 [53] as well as RMSEA and SRMR values ≤ .05 [54, 55]. For model fit to be considered as adequate, RMSEA and SRMR values should be ≤ .08 and CFI and TLI values ≥ .90 [54–56]. The children completed the self-report (SBB-ADHS) and the mothers or fathers the parent report (FBB-ADHS) form of the German Symptom Checklist for Atten- tion-Deficit/Hyperactivity Disorder (German: “Selbst-/ Fremdbeurteilungsbogen für Aufmerksamkeitsdefizit-/ Hyperaktivitätsstörungen”) from the German Diagnos- tic System for Mental Disorders in Children and Ado- lescents based on the ICD-10 and DMS-5 (DISYPS-III [43];). The two forms comprise 20 items each, which are rated on a four-point Likert scale ranging from 0 (not at all) to 3 (very much). The two subscales Inatten- tion (nine items) and Hyperactivity/Impulsivity (eleven items) as well as a total score can be computed by averag- ing the associated item scores. The FBB-ADHS has dem- onstrated a stable factor structure. Moreover, previous research yielded good reliability and validity of the scale scores [49]. Internal consistencies in the present sample were good, with α = .91 (M = .60, SD = 0.47) for the SBB- ADHS total score and α = .95 (M = .73, SD = 0.65) for the FBB-ADHS total score. We first applied an exploratory principal axis factor analysis (estimator: WLSMV; delta parameterization). We chose an exploratory approach as we had no clear hypothesis about the underlying factor structure. In this study, we were interested in extracting factors that distin- guish between rather functional versus rather dysfunc- tional ERS. CBCL/6-18R The German version of the Child Behavior Checklist for Ages 6–18 (CBCL/6-18R [45];), originally developed by Achenbach [46], comprises 120 items assessing a range of behavioral and emotional problems in children and ado- lescents. Parents rate the items on a three-point Likert scale ranging from 0 (not true) to 2 (very true). The items Junghänel et al. BMC Psychiatry (2022) 22:820 Page 5 of 15 (CD), and 11 items assess callous-unemotional (CU) symptoms. All items are based on DSM-5 and ICD-10 symptom criteria. Following the questionnaire’s instruc- tion to assess CD and CU symptoms only in children aged 11 years or older and as the DMDD items were included in the DADYS questionnaires assessing AD, we only used the items assessing ODD for the present study. Three of the eight items assessing ODD were already included in the DADYS questionnaire and were there- fore excluded, resulting in five items assessing the defi- ant dimension of ODD (ODDD) in the current sample. All items are rated on a four-point Likert scale ranging from 0 (not at all) to 3 (very much), and a scale score can be derived by averaging the item scores. A stable factor structure of the FBB-SSV as well as good reliability of the scale scores and diagnostic accuracy have been demon- strated [50]. Internal consistencies in the present sample were good, with α = .85 (M = .68, SD = 0.53) for the SBB- ODDD scale and α = .91 (M = .89, SD = 0.73) for the FBB- ODDD scale. can be aggregated to eight syndrome scales (Anxious/ Depressed, Withdrawn/Depressed, Somatic Complaints, Social Problems, Thought Problems, Attention Problems, Rule-Breaking Behavior, Aggressive Behavior) and three broadband scales (Externalizing Problems, Internalizing Problems, Total Problems). In this study, we assessed scales of the CBCL dysregulation profile [47, 48], which includes the scales Anxious/Depressed (part of the Inter- nalizing Problems scale), Attention Problems (part of the total problems scale) and Aggressive Behavior (part of the Externalizing Problems scale). The scale scores were computed by averaging the associated item scores. The syndrome scale scores of these three scales as well as the broadband scales have shown good psychometric properties [45]. SBB−/FBB-ADHS As we did not expect all of the variance to be explained by the extracted factors and assumed correla- tions between the extracted factors, we used an oblique GEOMIN rotation. To determine a suitable number of factors to extract, we conducted a parallel analysis in Mplus, which is based on eigenvalues from the observed CBCL/6-18R In the present sample, all assessed syn- drome scales demonstrated good internal consistencies, with α = .83 for the Anxious/Depressed scale (M = .31, SD = 0.31), α = .87 for the Attention Problems scale (M = .44, SD = 0.43), and α = .93 (M = .45, SD = 0.40) for the Aggressive Behavior scale. Results Factor structure and internal consistency of the FRUST The parallel analysis suggested a four-factor solution, which yielded a good model fit (CFI = .96, TLI = .95, RMSEA = .045 [90% CI: .042–.049], SRMR = .048) and was clearly interpretable (Table 2). Factors 1 to 3 comprised functional ERS, differentiated into Distrac- tion (factor 1), Problem-Solving (factor 2) and Social Support (factor 3). Factor 4 encompassed Dysfunc- tional ERS. All four factors together explained 49% of the variance. Six items (items 8, 10, 31, 33, 34, 48) were excluded from further analyses due to substan- tial factor loadings on both a functional factor and the dysfunctional factor (see Table 2). In a next step, the model with four correlated factors derived from the EFA was tested using CFA, including the 42 remain- ing items. In this model, all cross-loadings were fixed to zero. All fit indices indicated an adequate fit of this more restricted model (CFI = .93, TLI = .93, RMSEA: .058 [90% CI: .055; .062], SRMR = .062). The factor loadings of items 25 (“When I feel bad, I withdraw”; fac- tor Dysfunctional Strategies) and 26 (“When I feel bad, I do not show my bad mood”, factor Problem-Solving) were below .30 (.25 and .16, respectively). These items were therefore excluded, resulting in a final number of 40 items (Fig. 1). As item 5 (“When I feel bad, I keep my feelings to myself”) showed a negative factor load- ing on the functional Social Support factor, the scores for this item were recoded. This slightly adapted model was then tested in another CFA and yielded a some- what improved fit (CFI = .94, TLI = .94, RMSEA: .058 [90% CI: .054–.062], SRMR = .059). The factor loadings for this final model can be found in Table S1. All items demonstrated a substantial loading on their respec- tive factor (a ≥ .36). Moreover, all functional factors Following current recommendations [59–61], we assessed measurement invariance (MI) of our final model across different ages (8–10 years vs. 11–12 years), gender (male vs. female), and AD level (AD vs. NoAD). The dif- ferent levels of invariance include an increasing number of restrictions. For configural invariance, the item-fac- tor configuration is required to be equal across groups, that is, the same items have to belong to the same fac- tor. SBB−/FBB-SSV The children completed the self-report (SBB-SSV) and the mothers or fathers the parent report (FBB-SSV) form of the German Symptom Checklist for Disruptive Behav- ior Disorders (German: “Selbst-/Fremdbeurteilungs- bogen für Störungen des Sozialverhaltens”) from the DISYPS-III [43]. The SBB−/FBB-SSV comprises a total of 37 items, of which eight items assess symptoms of oppositional defiant disorder (ODD), three items assess DMDD, 15 items assess symptoms of conduct disorder Junghänel et al. BMC Psychiatry (2022) 22:820 Junghänel et al. BMC Psychiatry (2022) 22:820 Page 6 of 15 To examine the convergent and divergent validity of the corresponding scales derived from our latent factor analyses, we calculated Pearson correlation coefficients between the scales of the FRUST as well as between the FRUST scales and the CBCL/6-18R scales Anxious/ Depressed, Attention Problems, Aggressive Behavior, the SBB−/FBB-ADHS, the SBB−/FBB-SSV ODDD and the DADYS-C/−P. Moreover, correlations of the FRUST dimensions with age and gender were examined. With gender as a dichotomous variable, the point-biserial cor- relation was calculated. In accordance with Cohen [66] a correlation coefficient between .10 and .29 was consid- ered small, a correlation coefficient between .30 and .49 as moderate, and a correlation coefficient > .50 as large. SPSS version 26 was used to calculate the internal con- sistency and the correlations. correlation matrix and compares these with eigenvalues of random variables. Factors that have larger eigenvalues than the random variables are chosen for further factor analytic examination [57]. We excluded items from fur- ther analysis if they showed substantial cross-loadings (> .30) on factors that were not compatible in terms of con- tent (i.e. positive loadings on a factor otherwise defined through items describing dysfunctional ERS and a fac- tor otherwise defined through items describing func- tional ERS) or if they only demonstrated factor loadings < .30, which is in accordance with Kline [58]. If sub- stantial loadings (> .30) emerged on two factors that did not directly oppose each other (e.g., two factors defined through functional ERS), the item was assigned to one of the factors based on theoretical considerations. Subsequently, we performed a confirmatory fac- tor analysis (CFA) including all items that had not been excluded in the preceding exploratory analysis. In the CFA, we restricted items to load only onto a certain fac- tor, thus eliminating cross-loadings. Items loading nega- tively on their respective factor were recoded such that higher item scores indicated a higher manifestation on this factor. Results Metric invariance can be assumed if the item load- ings are additionally equivalent across groups. For scalar invariance, the item thresholds have to be equal across groups. The same goodness-of-fit indices as mentioned above (CFI, TLI, RMSEA, SRMR; theta parameteriza- tion) were used, and difference tests between the fit indi- ces on different levels of invariance were computed to examine MI [60]. For the assessment of configural invari- ance, the same cut-off values as mentioned above can be applied. Additionally, a change in CFI of ≤ − .01 [62], a change in SRMR of ≤ + .03 for testing metric invariance and a change of CFI of ≤ − .01 and a change in SRMR of ≤ + .01 for testing scalar invariance [63], as well as equal or better fit of TLI and RMSEA [64] have been suggested to indicate invariance across groups under the imposed constraints. To examine the internal consistency of the scales derived from the factor analyses, we computed Cron- bach’s α. An α coefficient ≥ .70 was considered as ade- quate [65]. Junghänel et al. BMC Psychiatry (2022) 22:820 Page 7 of 15 Page 7 of 15 Table 2 Item statistics and exploratory factor analysis of the FRUST – Four‑Factor Solution M Mean, SD Standard deviation The extraction method was principal axis factoring with oblique (GEOMIN) rotation. Results Loadings marked in bold indicate the factor to which an item was assigned Items M (SD) Distraction Problem-Solving Social Support Dysfuncti Strategies When I feel bad, I … 1 … try to change what made me feel bad 2.27 (1.22) .08 .43 .08 −.04 2 … tell someone about how I feel 2.09 (1.31) −.04 .45* .59* −.08 3 … think about things that make me happy 2.45 (1.31) .70* .09 .02 −.06 4 … do something fun 2.82 (1.18) .69* .13 −.08* −.05 5 … keep my feelings to myself 2.08 (1.28) .09 −.02 −.64* .17* 6 … make the best of it 2.22 (1.30) .35* .53* −.22* −.05 7 … do not want to see anyone 1.34 (1.31) .01 −.26* −.06 .59* 8 … think that this is my own problem 1.81 (1.22) −.05 .41* −.33* .34* 9 … do not feel like doing anything 147 (1.30) −.10 −.12 −.09 .59* 10 … keep thinking about why I feel bad, without ﬁnding a solution 1.25 (1.20) −.06 .27* .02 .37* 11 … think about what I could do 2.39 (1.28) .35* .47* .12* −.05 12 … tell myself that the problem is not that bad 1.75 (1.20) .12 .54* .02 .12* 13 … start a fight 0.80 (1.08) .00 −.40* −.01 .48* 14 … say that I am in a bad mood 1.84 (1.34) −.06 .37* .51* .09 15 … try to forget what put me in a bad mood 2.14 (1.35) .24* .44* .01 .01 16 … try to find the mistake in my own behavior 2.04 (1.28) .06 .66* −.06 .11* 17 … remember happy things 2.46 (1.38) .70* .16* −.02 −.08 18 … try to make the best of a situation myself 2.26 (1.30) .53* .39* −.05 −.14* 19 … go to someone who might be able to help me 2.08 (1.33) .14* .51* .44* .02 20 … think that it will pass 2.26 (1.28) .10 .57* −.03 .02 21 … accept what makes me feel bad 2.02 (1.22) .07 .54* −.01 .04 22 … show my bad mood without annoying others 185 (1.29) −.03 .53* .22* .15* 23 … take my bad mood out on others 0.98 (1.16) −.06 −.44* −.01 .52* 24 … cannot get it out of my head 1.90 (1.33) −.21* .17* −.10* .54* 25 … withdraw 2.10 (1.30) .00 .01 −.04 .43* 26 … do not show my bad mood 1.55 (1.20) −.06 .42* −.36* .09 27 … do something I enjoy 2.51 (1.30) .71* .21* −.04 −.11* 28 … think about a solution 2.33 (1.31) .26* .58* .17* −.09* 29 … tell myself that it is not important 1.38 (1.18) −.10* .60* .06 .19* 30 … cannot do anything against my bad mood 1.34 (1.34) −.30* .01 −.12* .47* 31 … tell myself that I have to blow oﬀ steam to react cooler 1.47 (1.32) .07 .37* .01 .38* 32 … do a relaxation exercise 0.87 (1.24) .46* .06 .34* .18* 33 … hit a pillow/go for a run 1.36 (1.39) .06 .06 .46* .59* 34 … squeeze something in my hand or make a ﬁst in my pocket 1.44 (1.34) .09 −.06 .32* .64* 35 … count to ten 0.46 (0.93) .36* −.01 .26* .20* 36 … go to a nice place in my mind where I can relax 1.61 (1.46) .65* .06 .11* .09 37 … listen to music/a story/watch a movie 2.09 (1.42) .54* .01 −.02 .19* 38 … hurt myself 0.55 (1.00) −.14 −.11 .04 .58* 39 … play/paint/do handicrafts 1.77 (1.45) .54* .02 .16* .07 40 … solve riddles/read 1.84 (1.45) .60* −.08 .20* .08 41 … talk to someone about it 1.74 (1.36) .02 .47* .60* −.09* 42 … try to stay cool and see the problem from another perspective 1.81 (1.30) .13* .66* .01 −.01 43 … try to think smart instead of obsessing about it 2.10 (1.30) .24* .65* −.03 −.13* 44 … leave the situation 2.25 (1.29) .28* .58* .00 .02 45 … try to recognize the situation early that made me feel bad 2.08 (1.31) .00 .76* .07 .05 46 … eat something tasty 1.68 (1.38) .57* −.03 .01 .24* 47 … lie on my bed and dream about something nice/try to sleep 1.38 (1.33) .53* −.04 .08 .14* 48 … do something on my computer/smartphone/tablet 1.60 (1.48) .14* .07 −.36* .19* Table 2 Item statistics and exploratory factor analysis of the FRUST – Four‑Factor Solution The extraction method was principal axis factoring with oblique (GEOMIN) rotation. Loadings marked in bold indicate the factor to which an item was assigned. Items that were excluded due to substantial factor loadings (> .30) on an functional and the dysfunctional factor are marked in italics M Mean, SD Standard deviation The extraction method was principal axis factoring with oblique (GEOMIN) rotation. Loadings marked in bold indicate the factor to whic that were excluded due to substantial factor loadings (> .30) on an functional and the dysfunctional factor are marked in italics Junghänel et al. BMC Psychiatry (2022) 22:820 Page 8 of 15 Fig. 1 Final Four‑Factor Structure of the FRUST. Correlated factors model with three functional factors (distraction, problem‑solving, social support) and one dysfunctional factor after the exclusion of items 25 and 26 (due to low factor loadings). Item numbers are displayed in the boxes and residuals are not shown for clarity of presentation Fig. 1 Final Four‑Factor Structure of the FRUST. Correlated factors model with three functional factors (distraction, problem‑solving, social support) and one dysfunctional factor after the exclusion of items 25 and 26 (due to low factor loadings). Item numbers are displayed in the boxes and residuals are not shown for clarity of presentation or Structure of the FRUST. Correlated factors model with three functional factors (distraction, problem‑solving, social support) factor after the exclusion of items 25 and 26 (due to low factor loadings). Item numbers are displayed in the boxes and n for clarity of presentation p < .01) and moderate negative correlations with the scale Dysfunctional Strategies (r = −.38 to r = −.40; all p < .01). The three functional ER scales showed significant (p < .01 or p < .05) small to large negative correlations with par- ent and child ratings of AD, ADHD, ODDD, and the three CBCL scales Anxious/Depressed, Attention Problems, and Aggressive Behavior (r = −.12 to r = −.57). The dys- functional ER scale showed significant (all p < .01) small to large positive correlations with parent and child rat- ings of AD, ADHD, ODDD, and the three CBCL scales (r = .23 to r = .71). All FRUST scales demonstrated higher correlations with child ratings of AD, ADHD, and ODDD symptomatology than with parent ratings of these variables on a descriptive level. There were no sig- nificant correlations between age and the FRUST scales. The functional strategies Distraction and Social Support showed small (r = .14 and r = .17, respectively) positive correlations with gender (Table 4). correlated positively with each other (r = .64 to .84, all p < .001) and negatively with the dysfunctional factor (r = −.50 to −.54, all p < .001). Measurement invariance of the four-factor model of the FRUST across age, gender, and AD level Regarding gender and age, the global model fit param- eters as well as their change between the different lev- els of MI indicates that invariance can be assumed on a configural, metric, and scalar level. This suggests an equal factor organization, equivalent loading patterns, and equivalent item thresholds across age- and gender- related groups (see Table 3). For AD level, MI could be assumed on the configural and metric level, but not on the scalar level, as the change in CFI of −.26 was larger than the recommended cut-off of −.01. This suggests an equal factor organization and equivalent loading pat- terns, but differences in item thresholds (Table 3). Internal consistencies, scale Intercorrelations, and associations of the FRUST scales with psychopathology, gender, and age Internal consistencies of the corresponding scales were adequate to good, with α = .78 for Dysfunctional Strat- egies (M = 1.19, SD = 0.80), α = .83 for Social Support (M = 1.94, SD = 1.01), α = .87 for Distraction (M = 1.86, SD = 0.83), and α = .91 for Problem-Solving (M = 2.05, SD = 0.84). Discussion Th The present study examined the factor structure, inter- nal consistencies, and validity of the newly developed, self-report FRUST questionnaire for the assessment of ERS in a community-screened sample of children and adolescents aged 8 to 12 years. The results of an explora- tory principal axis analysis suggest a four-factor struc- ture, comprising one factor of Dysfunctional ERS and the three functional factors Distraction, Problem-Solving, and Social Support. (Dys-)functionality of the factors was supported by correlations with measures of externalizing Internal consistencies of the corresponding scales were adequate to good, with α = .78 for Dysfunctional Strat- egies (M = 1.19, SD = 0.80), α = .83 for Social Support (M = 1.94, SD = 1.01), α = .87 for Distraction (M = 1.86, SD = 0.83), and α = .91 for Problem-Solving (M = 2.05, SD = 0.84). The positive scales of the FRUST demonstrated high positive correlations with each other (r = .51 to r = .71; all Page 9 of 15 Junghänel et al. BMC Psychiatry (2022) 22:820 Table 3 Measurement Invariance for the Four‑Factor Model of the FRUST AD Affective dysregulation, CFI Comparative fit index, TLI Tucker-Lewis index, RMSEA Root mean square error of approximation, CI confidence interval, SRMR Standardized root mean square residual female (n = 170) vs. male (n = 221) AD (n = 244) vs. NoAD (n = 147) 8–10 years (n = 231) vs. 11–12 years (n = 160) Level of invariance χ2 (df) CFI TLI RMSEA [90% CI] SRMR χ2 (df) CFI TLI RMSEA [90% CI] SRMR χ2 (df) CFI TLI RMSEA [90% CI] SRMR configural 2358.247* (1468) .943 .939 .056 [.052, .060] .070 2239.890* (1318) .932 .927 .060 [.056, .064] .072 2339.512* (1468) .945 .941 .055 [.051, .059] .071 metric 2274.546* (1504) .950 (∆ = .007) .948 .051 [.047, .055] .076 (∆ = .006) 2155.655* (1352) .940 (∆ = .008) .938 .055 [.051, .059] .079 (∆ = .007) 2380.652* (1504) .944 (∆ = −.001) .942 .055 [.050, .059] .081 (∆ = .010) scalar 2481.617* (1660) .947 (∆ = −.003) .950 .050 [.046, .054] .077 (∆ = .001) 2812.709* (1500) .914 (∆ = −.026) .920 .067 [.063, .071] .095 (∆ = .016) 2571.871* (1660) .942 (∆ = −.002) .946 .053 [.049, .057] .081 (∆ = .000) Junghänel et al. Discussion Th BMC Psychiatry (2022) 22:820 Page 10 of 15 Table 4 Scale intercorrelations, and associations of the FRUST scales with AD, ADHD, ODDD, the CBCL Scales and Age and Gender DADYS Diagnostic System for Affective Dysregulation (DADYS; German: “Diagnostikum für Affektive Dysregulation”), P Parent rating, C Child rating, ADHD Attention- deficit/hyperactivity disorder, ODDD Oppositional defiant disorder – only defiant dimension, CBCL Child Behavior Checklist, AD Anxious/depressed, AP Attention problems, AB Aggressive behavior a 1=male, 2=female Distraction Problem-Solving Social Support Dysfunctional Distraction .71 .51 −.38 Problem‑Solving .61 −.40 Social Support −.38 DADYS‑P −.25 −.30 −.28 .38 DADYS‑C −.47 −.57 −.48 .71 FBB‑ADHS −.15 −.18 −.21 .24 SBB‑ADHS −.28 −.34 −.32 .57 FBB‑ODDD −.16 −.24 −.24 .37 SBB‑ODDD −.37 −.47 −.40 .70 CBCL‑AD −.12* −.12* −.12* .23** CBCL‑AP −.13* −.18 −.19 .23** CBCL‑AB −.18* −.25 −.26 .36 Age .10 .01 .04 .01 Gendera .14 .00 .17** .00 Table 4 Scale intercorrelations, and associations of the FRUST scales with AD, ADHD, ODDD, the CBCL Scales and Age and Gender Di t ti P bl S l i S i l S t D f ti Table 4 Scale intercorrelations, and associations of the FRUST scales with AD, ADHD, ODDD, the CBCL Scales and Age and Gender and associations of the FRUST scales with AD, ADHD, ODDD, the CBCL Scales and Age and Gender DADYS Diagnostic System for Affective Dysregulation (DADYS; German: “Diagnostikum für Affektive Dysregulation”), P Parent rating, C Child rating, ADHD Attention- deficit/hyperactivity disorder, ODDD Oppositional defiant disorder – only defiant dimension, CBCL Child Behavior Checklist, AD Anxious/depressed, AP Attention problems, AB Aggressive behavior and internalizing psychopathology. This structure yielded an adequate model fit in the following CFA. The four- factor structure of the FRUST was invariant across dif- ferent age groups and gender. However, the analyses did not support scalar invariance across AD/NoAD children, suggesting that the FRUST is not ideal to discriminate between these groups. factors. Thus, it comes as no surprise that the one dys- functional factor could not be differentiated further. However, the differentiation of the functional factors is interesting, and it remains to be investigated in future studies whether this more detailed assessment of func- tional ERS has additional predictive value and/or use in therapeutic settings. g p Similar to the findings of Cracco and colleagues [34], our results suggest a more complex structure than would be depicted by the mere differentiation between func- tional and dysfuntional ERS. Discussion Th Interestingly, we found that item 16 ( “… try to ﬁnd the mistake in my own behavior”) grouped with items representing problem-solving strat- egies, suggesting that it might be an functional strategy in the present sample, though it was originally found to represent a maladaptive ERS in the FEEL-KJ. The dif- ferent assignment of this item might be due to the fact that children with externalizing symptomatology were overrepresented in our sample, suggesting that the strat- egy of finding mistakes in their own behavior might be rather functional in these children. This points at the important issue of whether a strategy should be classi- fied as functional or dysfunctional. It has been suggested that the functionality of a certain strategy depends on the individual’s symptom background [67], the combination with other strategies (e.g. distraction has been found to be functional when combined with acceptance strategies and dysfunctional when combined with avoidance strat- egies [68];, the extent and flexibility of use of a strategy (e.g. more expression regulation might lead to overregu- lation and inhibition [69];), as well as the context [4]. It is important to keep this in mind when using question- naires to assess ERS profiles, as the functionality of a strategy depends on a number of aspects that need to be considered for each individual case. If, for example, a child scores very high on the factor Distraction but low on all other factors, this may indicate a lack of flexibility and an extensive use of this strategy, which might even be dysfunctional for this particular child. In the present analyses, we found no significant cor- relations between any of the FRUST scales and age, and only a small correlation of the two functional scales Dis- traction and Social Support with gender, suggesting that girls use these strategies more often than boys do. With regard to social support seeking, the findings are in line with previous studies demonstrating a higher use of the strategy Social Support in girls than in boys [70]. The fre- quent finding that girls generally show a more dysfunc- tional ER [13] could not be replicated in our sample. This may be explained at least in part by the young age of the children in our sample (8–12 years), as some previous studies found that gender effects only emerged later in adolescence [36]. Discussion Th Whereas Cracco and col- leagues [34] proposed a three- or four-factor structure with one functional and two or three dysfunctional fac- tors (three-factor solution: avoidance, dysfunctional thoughts, and aggression; two-factor solution: avoidance and approach), our final structure of the FRUST com- prises three functional factors and only one dysfunctional factor. In the FEEL-KJ, five dysfunctional strategies, seven functional strategies, and three ambiguous strategies were identified. The dominance of the functional factors in our sample might be due to the comparatively larger number of functional items in the FRUST compared to the FEEL-KJ, as the 18 newly conceptualized items were primarily intended to represent functional techniques whose acquisition could be targeted in therapy. Seven- teen of these 18 items grouped accordingly with other functional items. Similarly, the six items from the three ambiguously (mal-)adaptive strategies in the FEEL-KJ mostly grouped with items of one of the functional ER p g In the course of the analysis, two items were excluded due to low factor loadings on all factors, and six addi- tional items were excluded due to similarly high factor loadings on one of the functional factors and the dys- functional factor. This seemingly puzzling finding may be explained by the specific symptomatology of each indi- vidual child. For instance, item 8 ( “… think that this is my own problem”) or item 10 ( “… keep thinking about why I feel bad without ﬁnding a solution”) can be an functional strategy for a child with externalizing symptomatology, who might benefit from looking at their own misbehavior before acting out. By contrast, it might be a dysfunctional strategy for a child with depression, who already focuses on his own behavior in a dysfunctional way. Item 10 might also entail the aspect of “thinking” on the functional side and of “not finding a solution” on the dysfunctional side. Items 31, 33, and 34 were originally thought of as func- tional items, but include a wording (“blow oﬀ steam”, “hit”, “make a ﬁst”) that can evoke associations with aggressive behavior in some children and may therefore potentially explain the cross-loadings on the functional and dysfunc- tional factors. Item 48 ( “… do something on my computer/ Junghänel et al. Discussion Th Furthermore, the small age range in the present sample might also explain the lack of associations found between age and ERS. As we examined a restricted and not a representative community sample (children were selected based on their AD symptomatology and categorized into a noAD/AD group), the results have to be interpreted with caution, since potential gender and/ or age effects might have been masked by an overrepre- sentation of a certain group [71]. Longitudinal studies observing the use of ERS during childhood and adoles- cence in more representative samples are needed in order to better understand the impact of age and gender on ER. Discussion Th BMC Psychiatry (2022) 22:820 Page 11 of 15 smartphone/tablet”) can be a functional regulation of a negative emotion, but when used excessively it might be a dysfunctional way to regulate one’s emotions that even has addictive potential. Interestingly, we found that item 16 ( “… try to ﬁnd the mistake in my own behavior”) grouped with items representing problem-solving strat- egies, suggesting that it might be an functional strategy in the present sample, though it was originally found to represent a maladaptive ERS in the FEEL-KJ. The dif- ferent assignment of this item might be due to the fact that children with externalizing symptomatology were overrepresented in our sample, suggesting that the strat- egy of finding mistakes in their own behavior might be rather functional in these children. This points at the important issue of whether a strategy should be classi- fied as functional or dysfunctional. It has been suggested that the functionality of a certain strategy depends on the individual’s symptom background [67], the combination with other strategies (e.g. distraction has been found to be functional when combined with acceptance strategies and dysfunctional when combined with avoidance strat- egies [68];, the extent and flexibility of use of a strategy (e.g. more expression regulation might lead to overregu- lation and inhibition [69];), as well as the context [4]. It is important to keep this in mind when using question- naires to assess ERS profiles, as the functionality of a strategy depends on a number of aspects that need to be considered for each individual case. If, for example, a child scores very high on the factor Distraction but low on all other factors, this may indicate a lack of flexibility and an extensive use of this strategy, which might even be dysfunctional for this particular child. transdiagnostic (AD, CBCL Attention Problems scale), and internalizing (CBCL – Anxious/Depressed scale) corre- lates, the observed correlations with ER can thus be inter- preted as an indication of construct validity. smartphone/tablet”) can be a functional regulation of a negative emotion, but when used excessively it might be a dysfunctional way to regulate one’s emotions that even has addictive potential. Strengths and limitations On the other hand, it might also be that this more global description of negative emotions is more appro- priate for children in this age range, who might have difficulty in clearly distinguishing between a range of negative emotions. Depending on the research or clini- cal goal, a decision should be made as to whether a time- efficient approach or a more detailed measure of ERS in the specific case is more useful. Fifth, the FRUST only assesses the regulation of negative emotions and not of positive emotions. Though this is common in instru- ments measuring ER, future research should addition- strategies in the regulation of positive emotions. These strategies might also constitute an important starting point for therapeutic interventions or, in the case of functional strategies, an important resource. Sixth, as we were not able to confirm scalar invariance of the model with four correlated factors across children with differ- ent AD levels, the FRUST in its current form cannot be used to differentiate between these groups. Seventh, as a result of the limited sample size and the large number of items of the FRUST, we decided against splitting the sample in half and first performing an EFA in one half in order to then cross-validate the observed structure in the other half. This approach would have been methodo- logically stronger and as we did not apply it in this study, future work will have to cross-validate the observed structure to gain information regarding its stability. In addition to the aforementioned limitations regard- ing the general construction and validation of the FRUST, there are limitations regarding the (non-)inclu- sion of certain sociodemographic variables. First, we only considered correlations of ERS with age and gen- der. The correlations with age are also limited, as we only included children between the ages of 8–12 in our sample and have not assessed the structure of the FRUST in a sample of adolescents. Future research should take further sociodemographic variables as well as age groups into account. For example, Greuel and colleagues [72] found that an immigration background and a lower social status of the parents were related to a higher use of functional strategies in their children, whereas no associations with the use of dysfunctional strategies emerged. Strengths and limitations This study comes with a number of strengths and limita- tions. In terms of strengths, we developed a shortened, more time-efficient and likely less repetitive version of the FEEL-KJ [33] by assessing the regulation of “unpleas- ant emotions” in general instead of considering dif- ferential responses for dealing with anger, anxiety, and sadness. Moreover, we demonstrated the reliability and validity of this instrument. The present study is the first to validate a shortened version of the self-report FEEL- KJ, as only the parent short version has previously been validated [72]. With the newly conceptualized items in the FRUST, which refer to specific ERS, it is possible to quantitatively capture the therapeutic success regard- ing the mastery of these specific ERS by administering the questionnaire at different stages of the therapeutic process. The inclusion and description of very specific behaviors might also be advantageous for younger chil- dren, as they are less abstract and thus easier to compre- hend. Another strength of our study is that – in contrast to other studies on the FEEL-KJ – we assessed the MI of the FRUST factor structure, suggesting an overall stable factor structure of the FRUST. With regard to results on the group level, we found that the positive correlations between the functional scales as well as the negative correlations between the functional scales and the dysfunctional scale supported the construct validity of the FRUST. In terms of external correlates, there was a clear pattern of negative correlations of the primarily functional scales Distraction, Problem-Solving, and Social Support with AD, ADHD, ODDD, and the three assessed scales of the CBCL (Anxious/Depressed, Attention Prob- lems, and Aggressive Behavior). Moreover, positive corre- lations emerged between the primarily dysfunctional scale and AD, ADHD, ODDD and the three assessed scales of the CBCL. These correlations between the ER scales and measures of psychopathology support the classification of the four factors as generally either functional or dysfunc- tional in the present sample. As emotion dysregulation is assumed to be strongly related to AD [16–18], the posi- tive correlation of AD with the dysfunctional scale and the negative correlation of AD with the functional scales sup- port convergent validity. Given that we assessed external- izing (ADHD, ODDD, CBCL Aggressive Behavior scales), Junghänel et al. BMC Psychiatry (2022) 22:820 Junghänel et al. Strengths and limitations BMC Psychiatry (2022) 22:820 Page 12 of 15 Page 12 of 15 Nevertheless, several limitations of the study need to be mentioned. First, the cross-sectional design does not allow for any predictive assumptions about the devel- opment of ER or any causal interpretations. The few existing longitudinal studies on ER strategies suggest smaller correlations between ER and later psychopathol- ogy than found in cross-sectional studies [2]. Second, the FRUST assesses ERS retrospectively, which limits the ecological validity of the ratings. The FRUST does attempt to reduce this problem through the addition of items assessing very specific strategies (it is poten- tially easier to recall counting to ten or doing a relaxa- tion exercise in a challenging situation than it is to recall trying “… to make the best of a situation”). However, the problem of lacking ecological validity still remains. A combination of the FRUST with other methods such as ecological momentary assessment [9, 14], in which participants are asked to rate their current feelings and thoughts over a longer time period in a naturalistic set- ting, might be valuable to obtain a more accurate picture of ERS. Third, the FRUST conceptualizes ERS indepen- dently of the context, which is opposed to Thompson’s [4] understanding of ER as being strongly depend- ent on the current situation. Previous findings suggest that the degree of adaptiveness of functional strategies tends to depend on the situation (e.g. problem solving can only be functional if there is an actual problem to solve), whereas dysfunctional strategies like rumination are indeed dysfunctional in most cases [3]. Fourth, the combination of the regulation of sadness, anger, and anxiety into “unpleasant emotions” can be criticized, as there is evidence to suggest that different strategies are used depending on the emotion to be regulated. For instance, social support and avoidance have been shown to be used more frequently for the regulation of sadness, whereas the strategies suppression and rumination are employed more often when attempting to regulate anxi- ety or anger [11]. Moreover, it is possible that children or adolescents interpret the introductory phrase “When I feel bad” in different ways, or that individual children interpret it differently depending on the specific emo- tion. Strengths and limitations Second, as we solely considered German-speaking children, the factor structure, psy- chometric properties, and the age- and gender-related results cannot be generalized to other countries and cultures. This is particularly problematic as there is evi- dence that ERS might differ between individualistic and collectivistic cultures in terms of the expression or sup- pression of emotions [73]. Future research should there- fore validate the FRUST in other languages, cultures and age-groups (e.g. adolescents). Conclusion The FRUST, which was developed to assess ERS in a more time-efficient and less repetitive manner than the FEEL-KJ [33], demonstrates a stable, well-interpretable factor structure consisting of three functional factors and one dysfunctional factor, which was strongly invariant to age and gender and weakly invariant to AD level. Moreo- ver, the measure showed good psychometric properties in terms of internal consistency and validity. The inclu- sion of very specific ERS allows for a continuous moni- toring of the therapeutic process and might make the Junghänel et al. BMC Psychiatry (2022) 22:820 Page 13 of 15 Page 13 of 15 questionnaire more accessible for younger children. We believe that due to these features, the FRUST is a valu- able contribution to the assessment of ERS for diagnostic, therapeutic, and scientific purposes. Treier, Anne Kaman and Pascal Aggensteiner coordinated the study and were responsible for data management. Analysis were performed by Michaela Junghänel and Hildegard Wand. Statistical analyses were reviewed by Ann‑ Kathrin Thöne. The first draft of the manuscript was written by Michaela Junghänel and Hildegard Wand and revised by Ann‑Kathrin Thöne, Christina Dose, Gregor Kohls, Lena Lincke and Manfred Döpfner and all authors critically reviewed and approved the final manuscript. Acknowledgements The dataset and the code used during the current study are available from the corresponding author upon reasonable request. We thank all other members of the ADOPT Consortium for their contribution to the project: We thank all other members of the ADOPT Consortium for their contribution to the project: Dorothee Bernheim3, Stefanie Bienioschek5, Maren Boecker7, Daniel Brandeis8,10,11, Nina Christmann8, Andrea Daunke4, Jörg M. Fegert3, Franziska Frenk1, Franziska Giller6, Claudia Ginsberg1, Carolina Goldbeck1, Monja Groh8, Martin Hellmich12, Sarah Hohmann8, Nathalie Holz8, Christine Igel8, Anna Kaiser8, Betül Katmer‑Amet3, Josepha Katzmann1, Sabina Millenet8, Kristina Mücke1, Anne Schreiner8, Jennifer Schroth5, Anne Schüller5, Marie‑Therese Steiner8, Marion Steiner3, Anne Uhlmann6, Matthias Winkler8 and Sara Zaplana1. Consent for publication Not applicable. 2Department of Special Education, Faculty of Human Sciences, University of Cologne, Cologne, Germany. Funding The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s12888‑ 022‑ 04440‑x. Open Access funding enabled and organized by Projekt DEAL. This work was supported by the research consortium on affective dysregulation, ADOPT and was funded by the German Federal Ministry of Education and Research (FKZ 01GL1741D). Additional file 1: Table S1. Standardized Factor Loadings (Standard Error) for the Confirmatory Factor Analyses of the Correlated Factors Models with Four Factors. The parents of all participating children and adolescents provided informed consent. Competing interests The present work 3Department of Child and Adolescent Psychiatry/Psychotherapy, University of Ulm, Ulm, Germany. 4Department of Child and Adolescent Psychiatry, Neurology, Psychosomatics, and Psychotherapy, University Medical Center Rostock, Rostock, Germany. 5Department of Child and Adolescent Psychiatry, Psychotherapy and Psycho‑ somatics, University Hospital Ruppin‑Brandenburg, Neuruppin, Germany. 6Department of Child and Adolescent Psychiatry and Psychotherapy, TU Dresden, Dresden Germany. Dresden, Dresden Germany. 7Department of Child and Adolescent Psychiatry, Psychotherapy, and Psychosomatics, Research Unit Child Public Health, University Medical Center Hamburg‑Eppendorf, Hamburg, Germany. 8Department of Child and Adolescent Psychiatry and Psychotherapy, Central Institute of Mental Health, Medical Faculty Mannheim, University of Heidel‑ berg, Mannheim, Germany. 8Department of Child and Adolescent Psychiatry and Psychotherapy, Central Institute of Mental Health, Medical Faculty Mannheim, University of Heidel‑ berg, Mannheim, Germany. 9Department of Child and Adolescent Psychiatry, Psychosomatics and Psycho‑ therapy, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany. 10Department of Child and Adolescent Psychiatry and Psychotherapy, Psychi‑ atric Hospital, University Hospital, University of Zürich, Zürich, Switzerland. 11Neuroscience Center Zürich, University and ETH Zürich, Zürich, Switzerland. 12Institute of Medical Statistics and Computational Biology (IMSB), Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany. This trial was supported by the Clinical Trials Centre Cologne (CTCC), Medical Faculty, University of Cologne, which performed the monitoring. Study data were collected and managed using REDCap electronic data capture tools hosted at the Clinical Trials Centre Cologne (https:// proje ctred cap. org/ resou rces/ citat ions/). Data preparation was conducted by Ingrid Becker from the Institute of Medical Statistics and Computational Biology (IMSB), Faculty of Medicine and University Hospital Cologne. rces/ citat ions/). Data preparation was conducted by Ingrid Becker from the Institute of Medical Statistics and Computational Biology (IMSB), Faculty of Medicine and University Hospital Cologne. Ethics approval and consent to participate Approval was obtained from the ethics committee of the University of Cologne (18–033), the University Hospital of Dresden (EK 35409218), the University Hospital of Mannheim (2018‑554 N‑MA) and the University Hospital of Ulm (297/18). The procedures used in this study adhere to the tenets of the Declaration of Helsinki. Competing interests p g Tobias Banaschewski served in an advisory or consultancy role for ADHS digital, Infectopharm, Lundbeck, Medice, Neurim Pharmaceuticals, Oberberg GmbH, Roche, and Takeda. He received conference support or speaker’s fees from Medice and Takeda. He received royalities from Hogrefe, Kohlhammer, CIP Medien, Oxford University Press; the present work is unrelated to these relation‑ ships. Daniel Brandeis served as an unpaid scientific advisor for an EU‑funded neurofeedback trial unrelated to the present work. Manfred Döpfner receives royalties from publishing companies as an author of books and treatment manuals on child behavioral therapy and of assessment manuals published by Beltz, Elsevier, Enke, Guilford, Hogrefe, Huber, Kohlhammer, Schattauer, Springer, and Wiley. He receives income as a consultant for Child Behavior Therapy at the National Association of Statutory Health Insurance Physicians. He also receives consulting income and research support from Lilly, Medice, Takeda, and eyelevel GmbH. Jörg Fegert received research funding from EU, BMG (Federal Ministry of Health), BMBF (Federal Ministry of Education and Research), BMFSFJ (Federal Ministry of Family, Senior Citizens, Women and Youth), DFG (German Research Foundation), G‑BA Innovation Fund, State Ministries of Baden‑Württemberg and Saarland, State Foundation Baden‑Württemberg, Porticus Foundation, Evangelical‑Lutheran Church in Württemberg. He received travel grants, honoraria, sponsorship for conferences and medical educational purposes from APK, Adenauer‑ and Ebertstiftung, Deutschlandfunk, DFG, DJI, DKSB, Infectopharm, med update, UNICEF, professional associations, universities and federal and state ministries. He is a consultant for APK, federal and state ministries. He has no industry‑sponsored lecture series, no shareholdings, no participation in pharmaceutical companies. Anja Görtz‑Dorten receives royalties from publishing companies as an author of books and treatment manuals on child behavioral therapy and assessment manuals, including the treatment manuals evaluated in this trial. She receives income as a consultant for Child Behavior Therapy at the National Association of Statutory Health Insur‑ ance Physicians. She also receives consulting income and research support from Medice and eyelevel GmbH. Charlotte Hanisch receives royalties from a publishing company as the author of a treatment manual. Martin Hellmich receives consulting income from eyelevel GmbH. Josepha Katzmann receives royalties from a publishing company as author of two self‑help books on child behavioral therapy. Michael Kölch receives royalties from publishing companies as an author of books. He served as PI or CI in clinical trials of Lundbeck, Pascoe and Janssen‑Cilag. He served as scientific advisor for Janssen. Affiliations 1 1 School of Child and Adolescent Cognitive Behavior Therapy (AKiP), Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany. Author details 1 1 School of Child and Adolescent Cognitive Behavior Therapy (AKiP), Faculty of Medicine and University Hospital Cologne, University of Cologne, Pohlig‑ straße 9, 50969 Cologne, Germany. 2 Department of Special Education, Faculty of Human Sciences, University of Cologne, Cologne, Germany. 3 Department of Child and Adolescent Psychiatry/Psychotherapy, University of Ulm, Ulm, Germany. 4 Department of Child and Adolescent Psychiatry, Neurology, Psy‑ chosomatics, and Psychotherapy, University Medical Center Rostock, Rostock, Germany. 5 Department of Child and Adolescent Psychiatry, Psychotherapy and Psychosomatics, University Hospital Ruppin‑Brandenburg, Neuruppin, Germany. 6 Department of Child and Adolescent Psychiatry and Psychother‑ apy, TU Dresden, Dresden, Germany. 7 Department of Child and Adolescent Psychiatry, Psychotherapy, and Psychosomatics, Research Unit Child Public Health, University Medical Center Hamburg‑Eppendorf, Hamburg, Germany. 8 Department of Child and Adolescent Psychiatry and Psychotherapy, Central Institute of Mental Health, Medical Faculty Mannheim, University of Heidel‑ berg, Mannheim, Germany. 9 Department of Child and Adolescent Psychiatry, Psychosomatics and Psychotherapy, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany. 15. 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https://openalex.org/W4233448706	https://bmcmusculoskeletdisord.biomedcentral.com/track/pdf/10.1186/s12891-020-03933-8	English	null	Comparison of three treatment methods for simple bone cyst in children	Research Square (Research Square)	2,020	cc-by	4,923	Open Access © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Comparison of three treatment methods for simple bone cyst in children Ke-Xue Zhang1,2†, Wei Chai2†, Jia-Jia Zhao3, Jun-Hao Deng2, Zhan Peng2 and Ji-Ying Chen2* Abstract Background: The unicameral bone cyst (UBC) is a kind of benign tumor whose clinical treatments and efficacy are controversial. The purpose of this study was to evaluate the efficacy of the elastic stable intramedullary nail (ESIN), the injection of autologous bone marrow (ABM), and the combination of ESIN and ABM in the treatment of bone cyst in children. Methods: Eighty-three cases with simple bone cyst were analyzed retrospectively. Twenty-eight cases were treated with ABM. Twenty-eight cases were treated with ESIN. Twenty-seven cases were treated with ABM and ESIN. All cases were diagnosed through X-ray, CT, or MRI scans. For the suspicious ones, the pathological biopsy was performed for an accurate diagnosis. X-ray examinations were carried out for the postoperative follow-up. Capanna criteria for bone cyst was used for postoperative evaluation of three methods. Results: All cases accomplished the follow-up. The effective rate of the ABM + ESIN group was significantly higher than that of the ABM group (P < 0.05), and the cure rates of the ESIN group and the ABM + ESIN group were higher than that of the ABM group (P < 0.05, respectively). The cure time in the ESIN group was lower than that of the other two groups (P < 0.05, respectively). The times for admission were 2.0 ± 0.0 in the ESIN group, 5.7 ± 1.9 in the ABM group, and 4.7 ± 2.4 in the ABM + ESIN group (P < 0.05 when compared with each other). Conclusions: The method of ABM combined with ESIN for children’s bone cyst has the highest effective rate and curative rate. For the individual method, ESIN has a higher effective rate and curative rate than that of ABM. Meanwhile, it has the fewest time of hospitalization. Keywords: Child, Bone cyst, Elastic stable intramedullary nail, Autologous bone marrow Keywords: Child, Bone cyst, Elastic stable intramedullary nail, Autologous bone marrow Background elastic stable intramedullary nail (ESIN) [9–11] et al. Among them, local injection of ABM and implantation of ESIN were used widely. However, the comparison of clin- ical efficacy among ABM, ESIN, and ABM combined with ESIN is rarely reported. In this study, we tried to analyze the three methods in a total of 83 cases retrospectively to disclose and compare their efficacy. Twenty-eight cases were treated with ABM, 28 cases were treated with ESIN, and 27 cases were treated with ABM combined with ESIN. All patients were evaluated by preoperative and postoper- ative X-ray examination. The Capanna evaluation criteria [12] was used to compare the clinical efficacy. The unicameral bone cyst (UBC) is a kind of benign tumor that usually occurs in the epiphysis of the long diaphysis [1–3]. The etiology and pathogenesis of UBC in children have not been identified [3]. There are many treatments in clinical practice for bone cysts, such as autograft or allograft bone grafting after lesion curettage [3–5], local injection of autogenous bone marrow (ABM) [6, 7] or methylprednisolone [8], and implantation of the * Correspondence: 15110245907@163.com †Ke-Xue Zhang and Wei Chai are Co-first author. 2Department of Orthopaedics, Chinese PLA General Hospital, Beijing 100853, China Full list of author information is available at the end of the article ina ll list of author information is available at the end of the article * Correspondence: 15110245907@163.com †Ke-Xue Zhang and Wei Chai are Co-first author. 2Department of Orthopaedics, Chinese PLA General Hospital, Beijing 100853, China Full list of author information is available at the end of the article (2021) 22:73 (2021) 22:73 Zhang et al. BMC Musculoskeletal Disorders https://doi.org/10.1186/s12891-020-03933-8 Ethical approval The study was approved by the ethical committee of the Chinese PLA General Hospital. Written informed consent for this study was obtained from all children’s parents. Inclusion criteria ESIN Implantation [14]: After general anesthesia with endotracheal intubation, the operative area were sterilized and laid with sterile sheets. Under the guidance of X-ray fluoroscopy, a 1 cm incision was made at the epiphysis of the long diaphysis away from the bone cyst. A hemostatic forceps was used to dissect the subcutaneous tissue to the bone cortex. An electric drill was used to drill a hole in the bone cortex. The ESIN, pre-bent into a “C” shape, was slowly inserted into the medullary cavity along the drill hole with the guidance of X-ray fluoroscopy. When the ESIN was properly implanted, the outer portion of the nail was bent and cut for complete subcutaneous embedding. Then the incision was sutured and wrapped with sterile gauze. The diagram of ESIN implantation was shown in Fig. 1b. (1) Age was under 14 years old. (2) Diagnosis was ascer- tained by X-ray, CT, or MRI images. For the suspicious ones, the pathological biopsy was performed. (3) No treatment was performed before admission. Criteria for diagnosis In X-ray plains, the simple cyst presents with a round or oval low-density area with mild plumping. Its long axis is mostly parallel to the shaft. Bone ridge separation is visible inside of the bone cyst. The boundary of the cyst is clear and sharp, mostly with the thin-wall sclerotic edge. Bone debris collapse sign can be found when pathological fracture happens [1, 13]. ESIN+ABM method: After one to three times of ABM injection with the interval of three-month in each time, the ESIN was implanted. All cases were given postoperative antibiotics of cefur- oxime sodium for 2 days. The affected limbs were fixed with plaster for 6 to 8 weeks. All cases were encouraged to early exercises after surgery. For cyst cases of lower extremities, they were encouraged to weight-free exer- cise in bed in the early period of recovery in order to avoid pathological fracture. Exclusion criteria (1) Lesions were complicated with other neoplasms. (2) Pathological fractures occurred in the processes of internal and external fixations. Follow-up and outcome appraisal indicators The therapeutic effect of treatment was evaluated by the criteria of Capanna [12] for the bone cyst. All children received preoperative X-ray, CT, or MRI examinations. The pathological biopsy was carried out if necessary. All patients were followed up with X-ray examinations. Preoperative and Postoperative images evaluation were performed for all the patients. In the process of Capanna evaluation, two experienced clinicians conducted the double-blind evaluation. When there was a difference in Page 2 of 7 Page 2 of 7 Zhang et al. BMC Musculoskeletal Disorders (2021) 22:73 (2021) 22:73 Zhang et al. BMC Musculoskeletal Disorders (2021) 22:73 Methods bone surface adjacent to the cyst. Then it was pierced into the center of the cyst slowly. When the inside core of the trocar was withdrawn, the liquid of the bone cyst outflowed. Next, ABM extracted from the Iliac crest was injected into the bone cyst cavity slowly. After the com- pletion of the ABM injection, the trocar was removed. The puncture points were covered with sterile dressings. The diagram of ABM injection was shown in Fig. 1a. Patients data Eighty-three children with bone cysts were retrospectively studied. They were admitted to the Department of Pediatric Surgery of Chinese PLA general hospital from January 2010 to December 2016. Twenty-eight cases were treated with ABM. Twenty-eight cases were treated with ESIN. Twenty- seven cases were treated with ABM combined with ESIN. The general data of patients were shown in Table 1. There was no statistical significance in age, gender constitution, weight, and height among the three groups. Results the scores of the same patient, the third specialist partic- ipated. The evaluation criteria of Capanna [12] for bone cyst: (1) Complete cure: The cyst cavity is completely filled with new bone. No residual lesion is observed. (2) Residual Cure: Lesion area is mostly replaced by newly growing bone tissue. The mixture of newly growing bone with the surrounding cyst wall bone can be seen. The cyst wall of the cortex sclerosis thickens. Small transparent areas present in the original cyst site. (3) Recurrence: In the early stage of the treatment, a good effect can be observed. Subsequently, transparent areas in the original cyst cavity emerged again. Bone cortex around the cyst becomes thinner. (4) No response: X-ray shows no favorable changes and healing tendency. Surgical procedures ABM Injection [14]: After general anesthesia with endo- tracheal intubation, the operative area were sterilized and laid with sterile sheets. Under the guidance of X-ray fluoroscopy, a core-inside trocar was punctured to the Table 1 General clinical data in 83 cases among three groups Group Cases Gender Age Weight Height Follow-up time Location of cyst Male Female (x̄ ± s,year) (x̄ ± s,kg) (x̄ ± s,cm) (x̄ ± s,month) proximal humerus proximal femur proximal tibia femoral shaft distal femur ABM 28 16 12 7.7 ± 2.0 17.6 ± 4.0 121.9 ± 7.3 32.4 ± 9.2 10 8 6 4 0 ESIN 28 18 10 7.5 ± 3.1 18.1 ± 3.0 120.8 ± 6.1 32.1 ± 3.2 11 7 5 4 1 ABM + ESIN 27 17 10 7.7 ± 2.3 18.3 ± 4.0 122.7 ± 7.0 31.7 ± 6.4 10 6 5 4 2 F – 0.3 0.1 2.2 0.5 0.7 2.5 P – 0.8 0.9 0.3 0.5 0.7 1.0 uloskeletal Disorders (2021) 22:73 Zhang et al. BMC Musculoskeletal Disorders (2021) 22:73 (2021) 22:73 Page 3 of 7 Zhang et al. BMC Musculoskeletal Disorders Fig. 1 The diagrams of autologous bone marrow injection (1a) and elastic stable intramedullary nail implantation (1b). 1a The bone marrow blood was extracted from the ilium using a bone penetration needle (short solid arrowhead) and injected into the femoral cyst with the guidance of the X-ray fluoroscopy. 1b The holding device (hollow arrowhead) was used to implant the elastic intramedullary needle (long solid arrow) into the femur with the guidance of the X-ray fluoroscopy Fig. 1 The diagrams of autologous bone marrow injection (1a) and elastic stable intramedullary nail implantation (1b). 1a The bone marrow blood was extracted from the ilium using a bone penetration needle (short solid arrowhead) and injected into the femoral cyst with the guidance of the X-ray fluoroscopy. 1b The holding device (hollow arrowhead) was used to implant the elastic intramedullary needle (long solid arrow) into the femur with the guidance of the X-ray fluoroscopy The comparison of three methods in the treatment of bone cyst in children y All the cases accomplished the follow-up. The follow-up times were 32.1 ± 3.2 months in the ESIN group, 32.4 ± 9.2 months in the ABM group and 31.7 ± 6.3 months in the ABM + ESIN group (Table 1). In the ESIN group, 25 cases were healed (23 cases cure, two cases residual cure), two cases recurred, one case did not respond to the treatment. In the ABM group, 18 cases were healed (13 cure, five re- sidual cure), eight cases recurred, and two did not respond to the treatment. In the ABM + ESIN group, 26 cases were cured and one case residual was cured (Table 2). The effective rate of the ABM + ESIN group was significantly higher than that of the ABM group (P < 0.05), and the cure rates of the ESIN group and the ABM + ESIN group were significantly higher than that of the ABM group (P < 0.05, respectively). Among three groups who were completely cured (23 in the ESIN group, 13 in the ABM group and 26 in the ABM + ESIN group), the cure period was 22.2 ± 3.3 months in the ESIN, 27.7 ± 7.8 months in ABM, and 31.3 ± 8.5 months in the ABM + ESIN group. The cure period of the ESIN group was significantly lower than that of the other two groups (P < 0.05). The frequency of hospitalization was 5.7 ± 1.9 times in the ABM group, 2.0 ± 0.0 times in ESIN group, and 4.7 ± 2.4 times in ABM + ESIN group. Pairwise comparisons between the three groups were statistically significant (P < 0.05) (Fig. 2). Effective cases included complete cure cases and re- sidual cure cases. The effective rate was calculated by the proportion of effective cases to the total number of the cases being treated. Statistical analysis SPSS 20.0 statistical analysis software was used for statistical analysis. One-way ANOVA test was used in the analysis data of admission times, age, and height. Kruskal- Wallis rank-sum test was used in the analysis of weight, follow-up duration, and therapeutic times for cure ones. Chi-square test was used in the analysis of gender compos- ition, effective rate, cure rate, and the site of cyst. P < 0.05 was considered statistical significance. Table 2 The comparison of three methods in the treatment of bone cyst in children Group Case Number of effective cases (effective rate) Cured cases (cure rate) ABM 28 18(64.3%) 13(46.4%) ESIN 28 25(89.3%) 23(82.1%)* ABM + ESIN 27 27(100%)* 26(96.3%)* X2 test was used to compare the therapeutic effect among the three methods. Compared with ABM group, P < 0.05 indicates the statistical significance Zhang et al. BMC Musculoskeletal Disorders (2021) 22:73 Page 4 of 7 Fig. 2 The comparison of the hospitalization time and the cure period in the three groups. 2a The number of admissions. 2b Cure time in the cured cases. One-way ANOVA test was used. P < 0.05, ** P < 0.01 indicates the statistical significance Fig. 2 The comparison of the hospitalization time and the cure period in the three groups. 2a The number of admissions. 2b Cure time in the cured cases. One-way ANOVA test was used. P < 0.05, * P < 0.01 indicates the statistical significance factors and mesenchymal cells for the cystic cavity. Mesenchymal cells have a variety of differentiation potentials and can differentiate into osteogenic cells, then the newly growing bone tissue may be antici- pated. This study showed that ESIN had the thera- peutic advantage of a shorter curative period and hospitalization time when compared with that of ABM. ABM provides mesenchymal cells and related growth factors locally, but the pathological condition of cyst formation remains and leaves a high risk of recurrence and refracture [16]. The treatment princi- ples of ESIN are as follows (1) Decompression and drainage of the cyst fluid. Drainage can reduce the pressure within the cyst and even remove the adverse factors that hinder the healing, such as PGE2, lyso- somes, toxic free radicals, and interleukin-2. (2) ESIN provides support inside the bone marrow cavity. (3) ESIN promotes the growth of new bone around the intramedullary nail. Many scholars also believe that Discussion The bone cyst has been studied for more than 100 years, but its pathogenesis and etiology are still un- known. Chigira et al. [15] believed that the increase of intracapsular pressure caused by the stasis of the intraosseous vein resulted in the accumulation of a large amount of exudate in the epiphysis of the di- aphysis and led to the localized osteonecrosis of the epiphysis, which eventually led to the formation of the bone cyst. Injection of ABM can provide growth Fig. 3 Male, eight years old, unicameral bone cyst localized in his right proximal humerus. 3a X-ray before the implantation of ESINs. 3b X-ray image after the implantation of ESINs. The ESINs runs through the cyst. 3c X-ray image at three months after the operation and there is new osteotylus in the cyst. 3d X-ray image at six months after the operation showed more osteotylus formed in the cyst. The bone cyst was healed Fig. 3 Male, eight years old, unicameral bone cyst localized in his right proximal humerus. 3a X-ray before the implantation of ESINs. 3b X-ray image after the implantation of ESINs. The ESINs runs through the cyst. 3c X-ray image at three months after the operation and there is new osteotylus in the cyst. 3d X-ray image at six months after the operation showed more osteotylus formed in the cyst. The bone cyst was healed (2021) 22:73 Zhang et al. BMC Musculoskeletal Disorders (2021) 22:73 usculoskeletal Disorders (2021) 22:73 (2021) 22:73 Page 5 of 7 Zhang et al. BMC Musculoskeletal Disorders Fig. 4 Male, nine years old, the bone cyst localized in his right humeral bone. 4a X-ray image before the first ABM injection therapy. 4b X-ray image prior to the second ABM treatment (3 months after the first ABM injection). 4c X-ray image before the third ABM treatment (6 months after the first ABM injection), the formation of the newly growing bone callus in the cyst can be seen. 4d X-ray image before the fourth ABM treatment (1 year after the first ABM injection), more callus bone could be seen. 4e X-ray image before the seventh ABM treatment (30 months after the 1st bone marrow blood injection), bone cyst is divided into multiple cystic spaces by the newly growing bone. 4f 42 months after the first ABM injection, the bone cyst was healed. Discussion 3 g: 56 months after the first ABM, the bone cyst was cured Fig. 4 Male, nine years old, the bone cyst localized in his right humeral bone. 4a X-ray image before the first ABM injection therapy. 4b X-ray image prior to the second ABM treatment (3 months after the first ABM injection). 4c X-ray image before the third ABM treatment (6 months after the first ABM injection), the formation of the newly growing bone callus in the cyst can be seen. 4d X-ray image before the fourth ABM treatment (1 year after the first ABM injection), more callus bone could be seen. 4e X-ray image before the seventh ABM treatment (30 months after the 1st bone marrow blood injection), bone cyst is divided into multiple cystic spaces by the newly growing bone. 4f 42 months after the first ABM injection, the bone cyst was healed. 3 g: 56 months after the first ABM, the bone cyst was cured ESIN is the best treatment for patients with simple bone cyst. Roposch et al. [11] treated 32 patients with bone cyst with ESIN, with an average follow-up time of 105 months, and found that the cure rate was as high as 94%。De Sanctiset al [10]. treated 47 patients with bone cyst treated by ESIN, with an average follow-up time of 11 years, and found that the cure rate was as high as 100%. include poor long-term treatment effect, high recurrence rate, high cumulative treatment cost, the need for multiple times of surgeries, and cumulative pain of overall treat- ment. Hence, ESIN is more worthy of recommendation in the treatment of simple bone cyst in children. It should be emphasized that there are some com- plications and risks associated with ESIN [17]. Most bone cysts occur at both ends of the long bone and close to the epiphyseal plate. In the case whose cyst is adjacent to or invades the epiphyseal periphery, im- proper implantations of the ESINs easily cause dam- age to epiphysis plate, which will result in a negative effect on the growth and development of the bone. Furthermore, when the cyst occurred in a thin bone. Zhang et al. BMC Musculoskeletal Disorders (2021) 22:73 Funding h k This work was supported by grants of Military Medical and Health Achievement Fund (19WKS09), Military Medicine Youth Fund of PLA General Hospital (QNF19056) and grants of Foundation and Strengthening Program in Technical Area (2019-JCJQ-JJ-149). The funding bodies played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. 12. Capanna R, Dal Monte A, Gitelis S, et al. The natural history of unicameral bone cyst after steroid injection. Clin Orthop Relat Res. 1982;(166):204–11. 12. Capanna R, Dal Monte A, Gitelis S, et al. The natural history of unicameral bone cyst after steroid injection. Clin Orthop Relat Res. 1982;(166):204–11. 13. Noordin S, Allana S, Umer M, et al. Unicameral bone cysts: Current concepts. Ann Med Surg (2012). 2018;34:43–9. https://doi.org/10.1016/j. amsu.2018.06.005. 13. Noordin S, Allana S, Umer M, et al. Unicameral bone cysts: Current concepts. Ann Med Surg (2012). 2018;34:43–9. https://doi.org/10.1016/j. amsu.2018.06.005. 14. Zhang K-X, Chou X-B, Li H-Y. Treatment of long bone cyst in children by autologous bone marrow blood injection and elastic intramedullary needle. Zhongguo Gu Shang. 2019;32(5):475–8. https://doi.org/10.3969/j.issn.1003- 0034.2019.05.017. 14. Zhang K-X, Chou X-B, Li H-Y. Treatment of long bone cyst in children by autologous bone marrow blood injection and elastic intramedullary needle. Zhongguo Gu Shang. 2019;32(5):475–8. https://doi.org/10.3969/j.issn.1003- 0034.2019.05.017. Consent for publication Not applicable. Consent for publication Not applicable. References 1. Zhang P, Zhu N, Du L, et al. Treatment of simple bone cysts of the humerus by intramedullary nailing and steroid injection. BMC Musculoskelet Disord. 2020;21(1):70. https://doi.org/10.1186/s12891-020-3054-6. 1. Zhang P, Zhu N, Du L, et al. Treatment of simple bone cysts of the humerus by intramedullary nailing and steroid injection. BMC Musculoskelet Disord. 2020;21(1):70. https://doi.org/10.1186/s12891-020-3054-6. 2. Hou T, Wu Z, Xing J, et al. Tissue-engineered bone treating simple bone cyst--a new strategy. J Surg Res. 2016;200(2):544–51. https://doi.org/10.1016/ j.jss.2015.09.032. 3. Noordin S, Allana S, Umer M, et al. Unicameral bone cysts: Current concepts. Ann Med Surg (Lond). 2018;34:43–9. https://doi.org/10.1016/j. amsu.2018.06.005. Discussion ESIN could easily penetrate outside of the bone cor- tex (such as case one), which will lead to the damage Our study suggested that when compared with the previously reported methods of local curettage and bone grafting [12], ESIN had a higher cure rate and shorter cure period. For the local curettage and bone grafting method, although the lesion is removed ephemerally, the pathological condition causing the bone cyst formation persists. ABM has the advantages of simplicity and safety, less pain in a single surgery and wide source of bone marrow. Its disadvantages Fig. 5 Male, eight years old, unicameral bone cyst localized in his right distal femur. 5a-5b The X-ray plains of preoperative and postoperative of ABM injection for the first time. 5c-5d: The second ABM injection was performed 5 months later. 5e-5f 5 months later, the ESIN was inserted. 5 g- 5 h: 14 months later, the ESIN was removed and the bone lesion was cured Fig. 5 Male, eight years old, unicameral bone cyst localized in his right distal femur. 5a-5b The X-ray plains of preoperative and postoperative of ABM injection for the first time. 5c-5d: The second ABM injection was performed 5 months later. 5e-5f 5 months later, the ESIN was inserted. 5 g- 5 h: 14 months later, the ESIN was removed and the bone lesion was cured Fig. 5 Male, eight years old, unicameral bone cyst localized in his right distal femur. 5a-5b The X-ray plains of preoperative and postoperative of ABM injection for the first time. 5c-5d: The second ABM injection was performed 5 months later. 5e-5f 5 months later, the ESIN was inserted. 5 g- 5 h: 14 months later, the ESIN was removed and the bone lesion was cured Zhang et al. BMC Musculoskeletal Disorders (2021) 22:73 Zhang et al. BMC Musculoskeletal Disorders (2021) 22:73 Zhang et al. BMC Musculoskeletal Disorders Page 6 of 7 of surrounding blood vessels and nerves. ESIN tail can often lead to the irritant reaction of the subcuta- neous tissue, which may cause local inflammatory hyperplasia and ulceration. For the avoidance of the epiphysis plate injury, if the implantation of ESIN be- comes superficial, it will compromise the function of supporting and draining which will lead to unsatisfac- tory therapeutic effect. Hence, we propose the ABM method for this kind of patients in the early stage of therapy. Acknowledgements Not applicable. Not applicable. Received: 5 May 2020 Accepted: 28 December 2020 Received: 5 May 2020 Accepted: 28 December 2020 Authors’ contributions 10. de Sanctis N, Andreacchio A. Elastic stable intramedullary nailing is the best treatment of unicameral bone cysts of the long bones in children?: Prospective long-term follow-up study. J Pediatr Orthop. 2006;26(4):520–5. https://doi.org/10.1097/01.bpo.0000217729.39288.df. 10. de Sanctis N, Andreacchio A. Elastic stable intramedullary nailing is the best treatment of unicameral bone cysts of the long bones in children?: Prospective long-term follow-up study. J Pediatr Orthop. 2006;26(4):520–5. https://doi.org/10.1097/01.bpo.0000217729.39288.df. KXZ and WC wrote the manuscript and did the study work. JJZ, JHD and ZP did the study work. JYC designed and conducted the study. All authors read and approved the final manuscript. 11. Roposch A, Saraph V, Linhart WE. Flexible intramedullary nailing for the treatment of unicameral bone cysts in long bones. J Bone Joint Surg Am. 2000;82(10):1447–53. https://doi.org/10.2106/00004623-200010000-00011. Discussion When a new callus in the cyst presents, ESIN combined with ABM was recommended to re- duce the following surgical manipulations. Our study showed that the combination of ABM and ESIN has the advantages of two methods, especially for the bone cysts adjacent to the epiphyseal plate. It has the characteristics of high efficiency and a high cure rate. imageology acquisitions were undertaken at the Department of Diagnostic Radiology, Chinese PLA General Hospital. The datasets used and analysed during the current study available from the corresponding author on reasonable request. imageology acquisitions were undertaken at the Department of Diagnostic Radiology, Chinese PLA General Hospital. The datasets used and analysed during the current study available from the corresponding author on reasonable request. Conclusion ABM combined with ESIN has a definite efficacy for the treatment of children’s bone cyst with a high cure rate and controllable treatment process, especially for the cases of cysts adjacent to the epiphysis periphery or even evade the epiphysis plate. This method is worthy of clinical application. For the individual method, ESIN is better than that of ABM with less hospitalization time and a shorter cure period. 4. Zhang K, Wang Z, Zhang Z. Comparison of curettage and bone grafting combined with elastic intramedullary nailing vs curettage and bone grafting in the treatment of long bone cysts in children. Medicine (Baltimore). 2019;98(25):e16152. https://doi.org/10.1097/MD. 0000000000016152. 5. Mavcic B, Saraph V, Gilg MM, et al. Comparison of three surgical treatment options for unicameral bone cysts in humerus. J Pediatr Orthop B. 2019; 28(1):51–6. https://doi.org/10.1097/BPB.0000000000000518. 6. Kadhim M, Thacker M, Kadhim A, et al. Treatment of unicameral bone cyst: systematic review and meta analysis. J Child Orthop. 2014;8(2):171–91. https://doi.org/10.1007/s11832-014-0566-3. Abbreviations UBC U i l 7. Kadhim M, Sethi S, Thacker MM. Unicameral Bone Cysts in the Humerus: Treatment Outcomes. J Pediatr Orthop. 2016;36(4):7. UBC: Unicameral bone cyst; ABM: Autogenous bone marrow; ESIN: Elastic stable intramedullary nail; CT: Computerized tomography; MRI: Magnetic Resonance Imaging; ANOVA: Analysis of variance; PLA: Chinese People’s Liberation Army 8. Zhao JG, Wang J, Huang WJ, et al. Interventions for treating simple bone cysts in the long bones of children. Cochrane Database Syst Rev. 2017;2: CD010847. https://doi.org/10.1002/14651858.CD010847.pub3. 9. Pogorelic Z, Kadic S, Milunovic KP, et al. Flexible intramedullary nailing for treatment of proximal humeral and humeral shaft fractures in children: A retrospective series of 118 cases. Orthop Traumatol Surg Res. 2017;103(5): 765–70. https://doi.org/10.1016/j.otsr.2017.02.007. 9. Pogorelic Z, Kadic S, Milunovic KP, et al. Flexible intramedullary nailing for treatment of proximal humeral and humeral shaft fractures in children: A retrospective series of 118 cases. Orthop Traumatol Surg Res. 2017;103(5): 765–70. https://doi.org/10.1016/j.otsr.2017.02.007. Ethics approval and consent to participate Ethics approval and consent to participate The study was approved by the ethical committee of Chinese PLA General Hospital. Written informed consents for study were obtained from all children‘s parents. Author details 1 1Medical School of Chinese PLA, Beijing 100853, China. 2Department of Orthopaedics, Chinese PLA General Hospital, Beijing 100853, China. 3 3Department of Anesthesiology, Shun Yi District Hospital, Beijing 101300, China. The limitation of this study was that it was a retro- spective study with a short follow-up period. The aver- age age of patients was around 7 years old in all 3 groups and the overall follow-up was 32 months (aver- age) in all groups. The follow-up period needs to be ex- tended in the future. Meanwhile, further mechanism study of the combination therapy of ABM and ESIN should be continued. Availability of data and materials Data associated with this study is retained at a central repository at the Department of Pediatric Surgery, Chinese PLA General Hospital. All Page 7 of 7 Zhang et al. BMC Musculoskeletal Disorders (2021) 22:73 Zhang et al. BMC Musculoskeletal Disorders (2021) 22:73 15. Chigira M, Maehara S, Arita S, et al. The aetiology and treatment of simple bone cysts. J Bone Joint Surg Br. 1983;65(5):633–7. 16. Lokiec F, Ezra E, Khermosh O, et al. Simple bone cysts treated by percutaneous autologous marrow grafting. A preliminary report. J Bone Joint Surg Br. 1996;78(6):934–7. https://doi.org/10.1302/0301-620x78b6.6840. 17. Neer CS 2nd, Francis KC, Marcove RC, et al. Treatment of unicameral bone cyst. A follow-up study of one hundred seventy-five cases. J Bone Joint Surg Am. 1966;48(4):731–45. 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.
W3208816000.txt	https://economic-bulletin.com/index.php/journal/article/download/796/804	en		Accounting and analytical support for the management of monetary assets in forestry enterprises	Ekonomìčnij vìsnik unìversitetu	2,021	cc-by	5,020	ЕКОНОМІКА ТА УПРАВЛІННЯ УДК 657.42 https://doi.org/10.31470/2306-546X-2021-50-76-83 ОБЛІКОВО-АНАЛІТИЧНЕ ЗАБЕЗПЕЧЕННЯ УПРАВЛІННЯ МОНЕТАРНИМИ АКТИВАМИ НА ПІДПРИЄМСТВАХ ЛІСОВОГО ГОСПОДАРСТВА Перчук О. В., Яременко Л. М., Бородіна Т. Г. Предмет дослідження: теоретичні та практичні проблеми формування системи обліковоаналітичного забезпечення управління монетарними активами. Метою статті є розроблення наукових підходів і методичних рекомендацій щодо розробки обліковоаналітичного забезпечення управління монетарними активами на лісогосподарських підприємствах. Методологія проведення роботи. Методологічною основою дослідження є загальнотеоретичні методи наукового пізнання, основні підходи та положення обліково-аналітичного забезпечення системи управління, що висвітлені у працях вітчизняних та зарубіжних науковців. Для виконання поставлених завдань використано методи: діалектичний метод пізнання – для аналізу законодавчо-нормативних документів та наукових праць вчених, що стосуються проблематики формування дієвої системи обліковоаналітичного забезпечення управління монетарними активами; аналізу та синтезу - при дослідженні та узагальненні зарубіжного та вітчизняного досвіду теоретико-методичних основ обліку та аналізу монетарних активів; абстрактно-логічний – для теоретичних узагальнень та формулювання висновків. Галузь застосування результатів. Результати дослідження можуть бути використанні в практичній діяльності підприємств лісогосподарського комплексу для підвищення ефективності управлінської діяльності. Результати і висновки роботи. На основі проведеного дослідження розкрито сутність монетарних активів, визначено їх роль у забезпеченні функціонування підприємств лісової галузі. Обґрунтовано важливість побудови системи обліково-аналітичного забезпечення управління монетарними активами, охарактеризовано її складові та визначено проблемні моменти реалізації облікової та аналітичної функцій при формуванні інформації для потреб управління монетарними активами та грошовими потоками лісогосподарських підприємств. Підприємства лісогосподарського комплексу потребують збалансованості грошових потоків та їх поповнення для більш ефективного фінансування своєї діяльності. Управління підприємством повинно ґрунтуватись на аналізі й оцінці грошових потоків з використанням інформації, яку формує система обліку та аналізу. Таким чином, використання інформації про монетарні активи повинно стати важливим елементом оцінки якості управління підприємством і привести до підвищення ефективності використання і прогнозування грошових потоків. Ключові слова: аналіз, грошові кошти, монетарні активи, облік, обліково-аналітичне забезпечення, підприємства лісового господарства, управління. УЧЕТНО-АНАЛИТИЧЕСКОЕ ОБЕСПЕЧЕНИЕ УПРАВЛЕНИЕ МОНЕТАРНЫМИ АКТИВАМИ НА ПРЕДПРИЯТИЯХ ЛЕСНОГО ХОЗЯЙСТВА Перчук О. В., Яременко Л. Н., Бородина Т. Г. Предмет исследования: теоретические и практические проблемы формирования системы учетно-аналитического обеспечения управления монетарными активами. Целью статьи является разработка научных подходов и методических рекомендаций по формированию учетно-аналитического обеспечения управления монетарными активами на лесохозяйственных предприятиях. Методология проведения работы. Методологической основой исследования являются общетеоретические методы научного познания, основные подходы и положения учетноаналитического обеспечения системы управления, которые освещены в работах отечественных и зарубежных ученых. Для выполнения поставленных задач использованы методы: диалектический метод познания – для анализа законодательно-нормативных документов и научных трудов ученых, касающиеся проблематики формирования действенной системы учетно-аналитического обеспечения управления монетарными активами; анализа и синтеза – при исследовании и обобщении зарубежного и отечественного опыта теоретико-методических основ учета и анализа монетарных активов; абстрактно-логический – для теоретических обобщений и формулирование выводов. Область применения результатов. Результаты исследования могут быть использованы в практической деятельности предприятий лесного комплекса для повышения эффективности управленческой деятельности. Результаты и выводы работы. На основе проведенного исследования раскрыта сущность монетарных активов, определены их роль в обеспечении функционирования предприятий лесной отрасли. Обоснована важность построения системы учетно-аналитического обеспечения управления 76 Економічний вісник університету \| Випуск № 50 © Перчук О. В., Яременко Л. М., Бородіна Т. Г., 2021 ЕКОНОМІКА ТА УПРАВЛІННЯ монетарными активами, охарактеризованы ее составляющие и определены проблемные моменты реализации учетной и аналитической функций при формировании информации для нужд управления монетарными активами и денежными потоками лесохозяйственных предприятий. Предприятия лесного комплекса потребует сбалансированности денежных потоков и их пополнения для более эффективного финансирования своей деятельности. Управление предприятием должно основываться на анализе и оценке денежных потоков с использованием информации, которую формирует система учета и анализа. Таким образом, использование информации о монетарных активах должно стать важным элементом оценки качества управления предприятием и привести к повышению эффективности использования и прогнозирования денежных потоков. Ключевые слова: анализ, денежные средства, монетарные активы, учет, учетно-аналитическое обеспечение, предприятия лесного хозяйства, управление. ACCOUNTING AND ANALYTICAL SUPPORT FOR MONETARY ASSETS MANAGEMENT IN FORESTRY ENTERPRISES Perchuk Oksana, Yaremenko Liudmila, Borodyna Tetiana The subject of research: theoretical and practical problems of forming a system of accounting and analytical support for monetary assets management. The purpose of the study is to develop scientific approaches and methodical recommendations for the development of accounting and analytical support for monetary assets management in forestry enterprises. Method or methodology for conducting research. The methodological basis of the study is general theoretical methods of scientific knowledge, the main approaches and framework of accounting and analytical support of the management system, which are highlighted in the works of domestic and foreign scientists. To reach the research tasks the following methods were used: dialectical method of cognition – to analyze legislative and regulatory documents and scientific works of scientists concerning the formation of an effective system of accounting and analytical support for monetary assets management; analysis and synthesis – to study and generalize the foreign and domestic experience of theoretical and methodological fundamentals of the accounting and analysis of the monetary assets; abstract logical – for theoretical generalizations and formulation of conclusions. The field of application of results. The results of the study can be used in the practice of forestry enterprises to improve management efficiency. The results of research and conclusions. Based on the study, the essence of monetary assets is revealed, their role in ensuring the functioning of forestry enterprises is determined. The importance of building a system of accounting and analytical support for monetary assets management is substantiated; its components are characterized, and the problematic aspects of accounting and analytical functions implementation in generating the information for managing forestry enterprises’ monetary assets and cash flows are determined. The enterprises of the forestry complex need the cash flows balancing and replenishment for more efficient financing of their activities. Enterprise management should be based on the analysis and evaluation of cash flows using the information generated by the accounting and analysis system. Thus, the use of information about monetary assets should become an important element in assessing the quality of enterprise management, and lead to improving the efficiency of cash flow use and forecasting. Key words: analysis, cash, monetary assets, accounting, accounting and analytical support, forestry enterprises, management. JEL Classіfіcatіon: М41, Q19. Постановка проблеми. Сучасні умови функціонування підприємств лісової галузі в Україні створюють особливу необхідність в обґрунтованості прийнятих управлінських рішень. На сьогодні лісове господарство – складна галузь, яка впливає на розвиток національної економіки. Однак поки що у ній ще не вироблено чіткої концепції щодо розвитку недержавних підприємницьких структур, на відміну від європейських країн, де можемо зустріти приклади високої продуктивності приватних лісів. На даному етапі в Україні лісогосподарська галузь утримується за рахунок державного бюджету, тобто вона знаходиться в державній формі власності. Це зумовлено також тим, що основна продукція – деревина – дозріває десятки, а в регіонах Карпат України – навіть 100-140 років при щоденній потребі в ній. Тому найнебезпечнішим є вузькогалузевий, споживацький, лісоексплуатаційний підхід до лісо- і природокористування. На думку багатьох вчених та практиків в галузі лісового господарства, реформування галузі потребує вдосконалення механізму фінансового забезпечення лісового господарства з урахуванням регіональних особливостей, вдосконалення системи оподаткування, оновлення системи продажу необробленої деревини тощо [1]. Пріоритетним завданням є пошук шляхів самостійного виживання державних лісогосподарських підприємств на основі реальної оцінки їх конкурентної позиції на ринку та обґрунтування адекватних рішень відповідно до поточної та перспективної ринкової кон'юнктури. У свою чергу, поглиблення ринкових відносин та глобалізаційних процесів, зумовлює розширення кола проблем, що покликані вирішувати бухгалтерський облік та економічний аналіз. У таких умовах питання створення ефективної системи контролю та управління інформацією щодо руху фінансових потоків на лісогосподарських підприємствах набуває пріоритетного значення в загальній системі Економічний вісник університету \| Випуск № 50 77 ЕКОНОМІКА ТА УПРАВЛІННЯ управління суб’єктом господарювання. Раціональна організація процедур спостереження, реєстрації, контролю, аналізу грошових коштів та їх потоків повинна здійснюватися на всіх рівнях системи управління підприємством та сприяти ефективному їх використанню. Адже, втрата лісогосподарськими підприємства фінансової стабільності носить не лише фінансово-економічні наслідки, але й соціальні, екологічні та негативно відображається на цінності деревини та погіршені товарної і асортиментної структури продукції. Фінансові засоби (кошти) часто без достатніх підстав називаються монетарними, а матеріальні і нематеріальні активи – немонетарними. Монетарні активи відносяться до тих особливих категорій, які завжди були найбільш актуальними в економічній думці, оскільки в процесі грошового руху найбільшою мірою проявляються і реалізуються інтереси суб’єктів ринку. Під монетарними об’єктами загалом розуміються об’єкти, вартісна оцінка яких фіксована їх статусом або договором і не залежить від змін купівельної спроможності відповідної грошової одиниці. Загальним об’єктом у системі управління фінансовими ресурсами виступають саме монетарні активи, основою яких є гроші виражені в різних формах. Аналіз останніх досліджень і публікацій. Удосконалення управління лісогосподарським підприємством в цілому за допомогою обліково-аналітичного забезпечення досліджено в працях Макаренко А. [2]. Питання теорії та практики обліку, аналізу та контролю грошових коштів досліджували такі автори як А. Андросова, С. Береза, М. Білуха, Н. Бондаренко, Ф. Бутинець, С. Голов, Н. Грабова, Н. Гудзь, В. Завгородній, З, Задорожній, Є. Калюга, Г. Кірейцев, М. Кужельний, Л. Кулаковська, Н. Малюга, Ю. Піч, А. Поддєрьогін, Н. Попадинець, В. Таран, О.Терещенко, Л. Сук, Р. Хом’як, М. Чумаченко та інші. Проблеми теорії та методології обліку та контролю монетарних активів досліджувала в своїх працях В. Скоробагатова [3] Постановка завдання. Метою статті є розроблення наукових підходів і методичних рекомендацій щодо удосконалення обліково-аналітичного забезпечення управління монетарними активами на лісогосподарських підприємствах. Виклад основного матеріалу. Обліково-аналітичне забезпечення управління монетарними активами перш за все передбачає реалізацію таких його функцій, як облік та аналіз, останні знаходяться у тісному взаємозв’язку та утворюють обліково-аналітичну систему з метою забезпечення менеджерів усіх ланок управління підприємств необхідною інформацією задля впровадження ефективних рішень. Індикаторами ефективності використання коштів можна розглядати динаміку певних показників, які характеризують ефективність управління. Підсистема облікового забезпечення управління монетарними активами складається з трьох взаємопов’язаних елементів: ланки первинного обліку, аналітичного обліку та підсумкового обліку. Невіддільними елементами бухгалтерського обліку монетарних активів є великий потік первинних документів щодо оформлення руху грошових коштів, постійне виконання арифметичних операцій, перевірка правильності розрахунків та законності операцій, зіставлення даних з інформацією на суміжних ділянках обліку тощо. Первинне оформлення операцій з монетарними активами забезпечується. наступними документами, які переважно є уніфікованими формами: прибутковий та видатковий касові ордери, грошовий чек, супровідна відомість, платіжне доручення, виписка банку, фіскальний чек, розрахункова квитанція, платіжна відомість, фіскальний касовий чек. Підсистема аналітичного забезпечення управління монетарними активами через реалізацію функції аналізу досліджує наявність, структуру, динаміку монетарних активів та окремих їх складових за допомогою аналітичних та економіко-математичних методів, вивчає ефективність їх використання, вивчає їх вплив на ліквідність, платоспроможність та загалом фінансовий стан підприємства. До базових методів аналізу монетарних активів можна віднести: - горизонтальний та вертикальний аналіз руху монетарних активів; - трендовий аналіз руху монетарних активів; - коефіцієнтний аналіз. Аналіз звітів держлісгоспів і показників їх фінансово-економічного стану створює досить велику інформаційну базу про рівень їх фінансового становища та ефективність управління грошовими потоками та монетарними активами. У господарській діяльності будь-якого суб’єкта грошові кошти є базою фінансового добробуту та є основою для забезпечення сучасної схеми здійснення розрахункових операцій. Наявність на лісогосподарському підприємстві обігових коштів у достатній мірі є запорукою нарощення активів, підвищення рівня рентабельності, ліквідності і, як результат, забезпечення фінансової рівноваги та платоспроможності. Введення вище наведеного поділу активів на монетарні і немонетарні в практику бухгалтерського обліку України пов’язано з рядом проблем, але основним фактором стало закріплення монетарно-немонетарного методу оцінки в Міжнародних стандартах фінансової звітності, а згідно ст. 1 Закону України «Про бухгалтерський облік та фінансову звітність в Україні» національні стандарти не повинні суперечити міжнародним. Зважаючи на вище сказане, важливим елементом системи обліково-аналітичного забезпечення управління монетарними активами є нормативно-правова складова, яка формується на декількох рівнях, зокрема: 1 рівень. Міжнародні норми права (МСБО 21 «Вплив змін валютних курсів», МСБО 29 «Фінансова звітність в умовах гіперінфляції»). 2 рівень Законодавці акти України (Цивільний кодекс, Господарський кодекс, Податковий кодекс, Кодекс про адміністративні правопорушення, Кримінальний кодекс, Закони України «Про бухгалтерський облік і фінансову звітність», «Про банки і банківську діяльність», «Про платіжні системи і переказ коштів в Україні» тощо). 78 Економічний вісник університету \| Випуск № 50 ЕКОНОМІКА ТА УПРАВЛІННЯ 3 рівень. Укази Президента та Постанови НБУ (Указ Президента України «Про застосування штрафних санкцій за порушення норм з регулювання обігу готівки»). 4 рівень. Підзаконні нормативні документи (НП(С)БО 1 «Загальні вимоги до фінансової звітності», П(С)БО 21 «Вплив змін валютних курсів», П(С)БО 22 «Вплив інфляції», Інструкція «Про безготівкові розрахунки в Україні в національній валюті», Інструкція НБУ «Про безготівкові розрахунки в господарському обороті України», Інструкція НБУ «Про порядок відкриття, використання і закриття рахунків в національній та іноземній валютах» та ін.). 5 рівень. Децентралізовані нормативні документи (Рішення і постанови органів місцевого самоврядування, локальні нормативно-правові документи, положення про облікову політику що стосуються монетарних активів). Загородній А.Г. до немонетарних активів відносить всі активи, крім грошових коштів та їх еквівалентів, дебіторської заборгованості у фіксованій (або визнаній) сумі грошей [4, с. 23]. Американські дослідники Е.С. Хендріксен та М.Ф. Ван Бред розуміють під монетарними активами права (вимоги) на фіксовану кількість грошових одиниць (наприклад, доларів), що представляє загальну купівельну спроможність. Хоча ціни на товари та послуги можуть змінюватися, вимоги (права), виражені в певній кількості доларів, залишаються незміненими, але купівельна спроможність, або здатність конвертувати ці права в товари та послуги, змінюється. Крім того, Е.С. Хендріксен та М.Ф. Ван Бред вважають, що до монетарних активів слід відносити грошові кошти; обумовлені договором права на певні суми грошових коштів у майбутньому, наприклад дебіторська заборгованість; а також вкладення, які передбачають отримання доходів у вигляді відсотків і дивідендів і будуть відшкодовані у фіксованій сумі, хоча дата відшкодування може бути не визначена, наприклад, як для привілейованих акцій [3]. Згідно з П(С)БО 21 «Вплив змін валютних курсів» монетарні статті – це статті балансу про грошові кошти, а також про такі активи й зобов’язання, які будуть отримані або сплачені у фіксованій (або визначеній) сумі грошей або їх еквівалентів [5]. Тобто за зазначених умов, монетарними статтями можуть бути кредиторська та дебіторська заборгованість. Термін «монетарні статті» більш широкий у порівнянні з терміном «монетарні активи», оскільки він включає в себе як активи, так і зобов’язання. У відповідності до п. 8 МСФЗ (IAS) 38 «Нематеріальні активи» монетарні активи – наявні грошові кошти та активи, що підлягають отриманню у вигляді фіксованих або визначених сум грошових коштів. МСФЗ (IAS) 21 «Вплив змін обмінних курсів валют» п. 8 визначає монетарні статті як одиниці готівкової валюти, а також активи і зобов’язання до отримання чи виплати, виражені фіксованою або визначеною кількістю валютних одиниць [6]. Якщо виходити з того, що термін «одиниці готівкової валюти» не охоплює кошти на депозитних рахунках, то тоді цим терміном позначені грошові (платіжні) засоби. Термін «активи, до отримання» можна визначити як право на вимогу, тобто являють собою дебіторську заборгованість. Основою монетарності активів є функція грошей як «засобу платежу», яка, як зазначає В.Д. Базилевич, передбачає обслуговування грошима, погашення різноманітних боргових зобов’язань між суб’єктами економічних відносин [7, с. 159.] В.В. Скоробагатова визначає два критерії віднесення активів до монетарних [3]: 1) монетарність, який означає, що актив виступає у вигляді грошових коштів, як у готівковій формі, так і у безготівковій або їх еквівалентів; 2) в основі певної операції з активами має лежати така функція грошей як «засіб платежу», тобто маються на увазі певні розрахунки. Відповідно до даних критеріїв до монетарних активів відносяться: грошові кошти в касі національній та іноземній валюті; кошти на рахунках в банках в національній та іноземній валюті, як поточні, так і інші; електронні гроші, які емітуються у відкрито циркулюючій системі, депозити до запитання та кошти в дорозі. Фінансові інвестиції (довгострокові та поточні), дебіторська заборгованість (довгострокова та поточна) та векселі одержані (довгострокові та короткострокові) відносяться до змішаних активів, при цьому якщо буде виконуватися умова погашення їх грошовими коштами, то вони можуть вважатися монетарними активами. Також зазначені активи можуть вважатися монетарними за умови, якщо вони обліковуватимуться на балансі підприємства до моменту їх погашення, а також виступають засобом капіталізації. Тому постає питання щодо відображення на рахунках бухгалтерського обліку кожної із складових монетарних активів. Грошові кошти виражені в різних формах є основою монетарних активів. Щодо вибору рахунків для облікового відображення саме грошових коштів не виникає ніяких сумнівів, адже це визначено на законодавчому рівні. Чого не можна сказати про інші складові монетарних активів, а саме: депозити до запитання та електронні гроші у відкрито циркулюючій системі. Електронні гроші є особливим та новим видом грошей, що потребує як правового регулювання так і фінансового контролю з боку держави та обумовлює проблематику ідентифікації як об’єкта бухгалтерського обліку. Підтримуючи думку Лукашової І.О. та І.С. Несходовського [8] доцільно для синтетичного обліку електронних коштів використовувати зарезервований рахунок 32 з назвою «Електронні кошти», а аналітичний облік здійснювати за їх видами. У фінансовій звітності сальдо по рахунку 32 «Електронні кошти» пропонується відображати в балансі у складі статті «Грошові кошти та їх еквіваленти». Слід відмітити, що перевагою такої форми розрахунків є відсутність обмеження суми розрахунків за день, на відміну від розрахунків готівкою, що встановлено Положенням про ведення касових операцій у національній валюті в Україні [9], але поряд з цим Економічний вісник університету \| Випуск № 50 79 ЕКОНОМІКА ТА УПРАВЛІННЯ існує інший обмежуючий фактор, зокрема наявність комісії у відсотках від суми переказу, яка стягується з відправника в момент проведення кожної транзакції в середині системи. Важливою характеристикою монетарних активів є їх ліквідність, тобто здатність брати участь в придбанні товару чи інших благ. Відповідно суттєвою обставиною, що відрізнятиме монетарні активи від всіх інших є те, що премія за ліквідність набагато перевищує витрати їх на утримання. Ліквідність монетарних активів є максимальною, а тривалість періоду трансформації для них рівна нулю. Оскільки ефективне управління монетарними активами на лісогосподарських підприємствах та контроль за ними вимагають від бухгалтерського обліку високого ступеня деталізації інформації, важливу роль відіграє аналітика, яку може забезпечити розробка робочого плану рахунків. При цьому, що стосується зокрема грошових коштів, то деталізація можлива за наступними критеріями: форма розрахунків, валюта платежів, тривалість формування, вид господарської діяльності, центр фінансової відповідальності, місце зберігання грошових коштів та економічний зміст господарської операції. Основними проблемами бухгалтерського обліку монетарних активів є: по-перше, відсутність нормативних документів, що регулюють облікове відображення монетарних активів; по-друге, недосконалість нормативного регулювання бухгалтерського обліку складових монетарних активів; неоднозначність думок науковців щодо складових монетарних активів, зокрема видів грошових коштів; по-третє, повнота та своєчасність відображення монетарних активів у системі бухгалтерського обліку. Адже, якщо грошові кошти не будуть повністю та своєчасно оприбутковані, то не буде чіткого відображення наявності коштів, що призведе до нарахування штрафних санкцій контролюючими органами. Обсяг фінансових ресурсів є одним з найважливіших обмежень при плануванні масштабу та напрямків діяльності підприємства. Згідно статті 98 Лісового кодексу України [10] видатки на підвищення продуктивності, поліпшення якісного складу лісів, їх відтворення і охорони здійснюються за рахунок: а) державного бюджету та власних коштів підприємств, установ і організацій лісового господарства – щодо лісів державної власності; б) місцевого бюджету та власних коштів підприємств, установ і організацій лісового господарства – щодо лісів комунальної власності; в) власних коштів власників лісів – щодо лісів приватної власності. Видатки на зазначені заходи можуть здійснюватися за рахунок інших джерел, не заборонених законом. Видатки на підвищення продуктивності, поліпшення якісного складу лісів державної і комунальної власності, їх охорону, захист і відтворення фінансуються шляхом цільового виділення коштів державного та місцевих бюджетів для реалізації загальнодержавних, державних і регіональних (місцевих) програм ведення лісового господарства, головною з яких є державна цільова програма «Ліси України». Отже, державні підприємства лісового господарства можуть отримувати кошти з місцевих бюджетів для проведення лісокультурних робіт, в тому числі це можуть бути надходження від фондів охорони навколишнього природного середовища районних та обласних бюджетів. Дані надходження в обліку відображаються в складі інших операційних доходів. Різноманітні програми фінансування передбачають грошові надходження коштів загального та спеціального фонду державного бюджету. Так, зокрема у 2019 році була передбачена бюджетна програма КПКВ 2805060 «Ведення лісового і мисливського господарства, охорона і захист лісів в лісовому фонді». Для державних підприємств лісової галузі характерною є залежність від зовнішніх грошових надходжень, адже при їх скороченні можуть виникнути ризики при веденні фінансовогосподарської діяльності, які будуть пов’язані із недостатністю обігових коштів та можуть спричинити збитки. Протягом останніх років частка видатків лісового господарства, яка фінансується з державного бюджету постійно знижувалась. Розподіл цих коштів є дуже нерівномірний. Більшість успішних, з фінансової точки зору лісових підприємств Полісся та Карпат, частина підприємств Лісостепової зони повністю відмовились від бюджетного фінансування і ведуть лісове господарство за рахунок власних коштів, отриманих від продажу лісових продуктів [10] На сьогодні основним джерелом фінансування лісового господарства в цілому по Україні стали власні кошти підприємств, які формуються від реалізації продукції лісозаготівель, їх частка становить більше 90%. Необхідно відмітити, що продукції лісогосподарських підприємств реалізується на експорт, що є зумовлює необхідність оцінки та обліку монетарних активів виражених в іноземній валюті. Після заборони експорту деревини в круглому вигляді, значно (біля 120 тис. м³ в рік) зросли об’єми експорту пилопродукції. Рівень експортних цін на пиломатеріали залежить від логістики, умов поставки, фізичних розмірів, вологості та породи та адміністративних витрат держлісгоспів. Для прикладу при умові поставки і валюті – ціни коливаються протягом 2016-2018 років за 1 м³: • пиломатеріали Дуб – FCA-EUR – 150–260–300 $/ м³; • пиломатеріали Сосна – FCA-EUR – 90–105–110–130 $/ м³; • паркетні заготовки Дуб – FCA-EUR – 6–8 $/ м³; • пиломатеріали Ялина – FCA-EUR – 100–130 $/ м³; • заготовка для піддонів Сосна – FCA-EUR – 90–95 $/ м³ [11]. Зважаючи на вище зазначені умови фінансування діяльності лісогосподарських підприємств, дуже важливою частиною аналізу є аналіз грошових потоків. Аналіз грошових потоків передбачає проведення чотирьох послідовних етапів дослідження. 80 Економічний вісник університету \| Випуск № 50 ЕКОНОМІКА ТА УПРАВЛІННЯ 1) Аналіз обсягу та динаміки грошових потоків, метою проведення якого є визначення загальних обсягів грошових потоків, основних тенденцій та пропорцій їх розвитку. 2) Аналіз джерел формування монетарних активів (грошових коштів), що передбачає вивчення структури позитивного грошового потоку за видами діяльності, ранжирування джерел надходження грошових коштів, визначення місця (вагомості) кожного з них у грошовому забезпеченні функціонування лісогосподарського підприємства. 3) Аналіз напрямів використання монетарних активів (грошових коштів), об'єктом якого є вихідний грошовий потік підприємства. 4) Аналіз рівномірності грошових потоків, метою якого є ідентифікація та оцінка основних видів грошових потоків за ступенем регулярності здійснення, виявлення факторів коливань грошових потоків, наявності та причин затримок у надходженні монетарних активів (грошових коштів) та відкладенні запланованих видатків. Основними та найпоширенішими прогнозними документами надходження та витрачання грошових коштів на підприємствах мають стати касовий бюджет та платіжний календар. Поділ активів на монетарні та немонетарні спрямований на підвищення інвестиційної привабливості підприємства. Ступінь деталізації інформації щодо обсягу монетарних активів дозволяє менеджерам аналізувати конкретні заходи щодо поліпшення результатів діяльності. Тому вартість підприємства, виражена в монетарних статтях, на дату балансу потребує достовірної оцінки. Відповідно, монетарні статті виражені в іноземній валюті мають бути перераховані за валютним курсом на дату балансу, а також необхідно враховувати темп інфляції. Умови формування повноцінного обліково-аналітичного забезпечення управління монетарними активами залежать від ряду факторів впливу, до яких можна віднести налагодженість системи збору інформації про грошові кошти, узагальнення та обробки її потоків, оперативність реагування, чіткість та координація управлінських дій та рішень як результат. Значущість аналітичного забезпечення підкреслюється ще й тим, що завдяки йому виконується функція візуалізації фінансової картини даних по підприємству та виникає можливість створення максимально комфортних та перспективних умов для економічного зростання та розвитку підприємства. Можемо запропонувати можливі варіанти вдосконалення обліково-аналітичного забезпечення управління монетарними активами для лісогосподарських підприємств, що в майбутньому зможе призвести до покращення процесів обліку та аналізу й досягнення високого рівня фінансової незалежності за рахунок власних коштів: - удосконалення методики обліку монетарних активів; - модернізація форм ведення внутрішньої документації; - використання окремих рахунків для обліку елементів монетарних активів; - відкриття додаткових субрахунків для аналітичного обліку монетарних активів. - впровадження сучасного автоматизованого облікового забезпечення. Висновки. Проведені дослідження показали, що для лісогосподарських підприємств притаманна безсистемність обліково-аналітичного забезпечення управління монетарними активами (зокрема, превалювання забезпечення інформацією тільки поточної діяльності без орієнтації на перспективні тенденції змін рівня ліквідності та платоспроможності). Отже, можна сказати, що безперечними залишається факт актуальності обліково-аналітичного забезпечення в управлінні підприємством. Встановлено, що цей процес не можливий без дотримання ряду положень та принципів облікового та аналітичного характеру. Якість управлінських рішень, які формуються на основі обліково-аналітичного забезпечення, залежить від оперативності та точності інформаційного забезпечення даного процесу та швидкості реагування на зміни в ньому. Для більш ефективної процедури налагодження системи обліково-аналітичного забезпечення управління монетарними активами мають створюватися всі умови на підприємстві, адже наслідком роботи системи є якість управлінських дій та фінансова незалежність підприємства в кінцевому результаті. Список використаних джерел 1. Мостепанюк В. А., Гайдучок Т. С. Фінансування лісового господарства України в контексті європейського досвіду. URL: http://ir.znau.edu.ua/bitstream/123456789/4166/1/ANOV__2015_619-622.pdf 2. Макаренко А. Обліково-аналітичне забезпечення як фактор підвищення результативності управління раціональним лісокористуванням. Agricultural and Recourse Economics: International Scientific EJournal. 2017. Vol. 3. № 2. Pp. 109-121. URL: www.are-journal.com. 3. Скоробагатова В. В. Бухгалтерський облік та контроль монетарних активів: проблеми теорії та методології: монографія. Житомир : ЖДТУ. 2012. 520 с. 4. Загородній А. Г., Партин Г. О. Бухгалтерський облік: основи теорії та практики: навч. посіб. 2-ге вид., перероб. і доп. Київ: Т-во «Знання», КОО, 2003. 327 с. 5. Положення (стандарт) бухгалтерського обліку 21 «Вплив змін валютних курсів», затв. Наказом МФУ № 193 від 10.08.2000. URL: https://zakon.rada.gov.ua/laws/show/z0515-00 6. Міжнародний стандарт бухгалтерського обліку 21 «Вплив змін валютних курсів» від 01.01.2012. URL : http://zakon0.rada.gov.ua/laws/show/929_022 7. Економічна теорія: політекономія: підручник / За ред. В. Д. Базилевича. Київ: Знання, 2006. 631 с. 8. Лукашова І. О. Усунення протиріч у методології бухгалтерського обліку коштів. URL: http://www.nbuv.gov.ua/old_jrn/Soc_ Gum/Tiru/2010_30_1/Lukash.pdf. 9. Положення «Про ведення касових операцій у національній валюті в Україні», затверджене постановою правління НБУ від 15.12.2004 р. № 637 зі змінами та доповненнями URL : http://zakon2.rada.gov.ua/laws/show/z0040-05 Економічний вісник університету \| Випуск № 50 81 ЕКОНОМІКА ТА УПРАВЛІННЯ 10. Лісовий кодекс України. URL: http://zakon5.rada.gov.ua/laws/show/3852-12⁎8 11. Мостепанюк В. А., Гайдучок Т. С. Фінансування лісового господарства України в контексті європейського досвіду. URL: http://ir.znau.edu.ua/bitstream/123456789/4166/1/ANOV__2015_619-622.pdf 12. Аналітичний звіт про стан лісових ринків в Україні та їх регулювання – рекомендації по удосконаленню регулювання. URL: https://www.apd-ukraine.de/images/2019/Agrarpolitische_Berichte/ Forst_Marchuk/APD_FPB_Marchuk_UKR_final.pdf References 1. Mostepanyuk V. A., Hayduchok T. S. (2015). 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[in Ukr.] ДАНI ПРО АВТОРА Перчук Оксана Володимирівна, к.е.н., доцент кафедри фінансів, обліку і оподаткування, Університет Григорія Сковороди в Переяславі e-mail: oksi011279@gmail.com http://orcid.org/0000-0002-64844-7011 Researcher ID: 55052018 Яременко Людмила Михайлівна, к.е.н., доцент, доцент кафедри фінансів, обліку і оподаткування, Університет Григорія Сковороди в Переяславі e-mail: lludayaremenko@gmail.com http://orcid.org/0000-0003-3127-5181 Researcher ID: 40622017 Бородіна Тетяна Григорівна, здобувач 2 року навчання другого (магістерського) рівня вищої освіти, спеціальність 071 Облік і оподаткування, Університет Григорія Сковороди в Переяславі ДАННЫЕ ОБ АВТОРЕ Перчук Оксана Владимировна, к.э.н., доцент кафедры финансов, учета и налогообложения, Университета Григория Сковороды в Переяславе e-mail: oksi011279@gmail.com Яременко Людмила Михайловна, к.э.н., доцент, доцент кафедры финансов, учета и налогообложения, Университет Григория Сковороды в Переяславе e-mail: lludayaremenko@gmail.com Бородина Татьяна Григорьевна, соискатель 2 года обучения второго (магистерского) уровня высшего образования, специальность 071 Учет и налогообложение, Университет Григория Сковороды в Переяславе 82 Економічний вісник університету \| Випуск № 50 ЕКОНОМІКА ТА УПРАВЛІННЯ DATA ABOUT THE AUTHOR Perchuk Oksana, PhD in Economics, Associate Professor, Hryhorii Skovoroda University in Pereiaslav, Pereiaslav e-mail: oksi011279@gmail.com Yaremenko Liudmila, PhD in Economics, Associate Professor, Hryhorii Skovoroda University in Pereiaslav, Pereiaslav e-mail: lludayaremenko@gmail.com Borodyna Tetiana, applicant for 2 years of study of the second (master's) level of higher education, specialty 071 Accounting and Taxation, Hryhorii Skovoroda University in Pereiaslav, Pereiaslav Подано до редакції 02.08.2021 Прийнято до друку 28.08.2021 УДК 378.14.015.62 https://doi.org/10.31470/2306-546X-2021-50-83-96 ПРИКЛАДНІ АСПЕКТИ ПІДВИЩЕННЯ ЕФЕКТИВНОСТІ ДІЯЛЬНОСТІ МОЛОКОПЕРЕРОБНИХ ПІДПРИЄМСТВ НА ОСНОВІ КОМПЛЕКСНОЇ ЕКОНОМІЧНОЇ ДІАГНОСТИКИ Рибакова Т. О., Семененко О. Г., Черняєв О.С. Актуальність теми дослідження. Одним з дієвих методів вибору стратегій підвищення ефективності господарської діяльності підприємств різних галузей може стати комплексна економічна діагностика як засіб, що дозволяє корегувати стратегію і тактику виробників та визначити ефективні шляхи їх подальшого розвитку. Постановка проблеми. Складні економічні умови сьогодення вимагають від підприємств не тільки стабілізації комерційної діяльності, але й підвищення ефективності виробництва, конкурентоспроможності на ринку продукції та послуг. Одним із засобів, що дозволяють підвищити ефективність діяльності підприємств, є проведення комплексної економічної діагностики. Особливо це актуально для молокопереробних підприємств країни у зв’язку зі специфікою організації постачання та збуту, швидким псуванням продукції та кризою ринку молочної продукції в умовах сьогодення. Аналіз останніх досліджень і публікацій. Вивченню даної проблеми присвятили праці багато закордонних та українських вчених, серед яких: І. Фаріон, Л. Савчук, С. Жукевич, І. Кривов’язюк, Т. Божидарнік, А. Кірієнко, Н. Зимин, І. Зеленчук, Т. Городня, І. Мойсеєнк., Н. Кушнір, Н. Сарай, О. Гетьман, Т. Загорна, Н. Бобко, Ю. Коваль, В. Забродський, В. Зінченко, А. Вартанов, О. Гадзевич, Т. Костенко та інші. Виділення недосліджених частин загальної проблеми. В більшості наукових праць увага зосереджена на дослідженні окремих складових елементів економічної діагностики. Розробка методологічних підходів щодо здійснення та запровадження повноцінної методики комплексної економічної діагностики також немає достатнього відображення в економічній літературі. Постановка завдання, мети дослідження. Теоретичне обґрунтування та розробка методичних та практичних рекомендацій щодо удосконалення процесу комплексної економічної діагностики господарської діяльності підприємств молокопереробної промисловості (на матеріалах молокопереробного підприємства ТОВ «Переяслав-Молпродукт»), з метою підвищення ефективності бізнесу. Метод або методологія проведення дослідження. Для досягнення мети, вирішення поставлених у дослідженні завдань використано комплекс загальнонаукових та специфічних методів дослідження, а також неформалізованих та формалізованих методів. Результати дослідження. Обґрунтовано науково-методичні підходи та практичні рекомендацій щодо удосконалення процесу комплексної діагностики підприємства та запропоновано шляхи підвищення ефективності господарської діяльності підприємства молокопереробної промисловості. Галузь застосування результатів. Отримані результати дослідження можуть бути використані підприємствами при діагностуванні поточного стану підприємств власними зусиллями на основі стратегічних міркувань, при розробці бізнес-планів, інвестиційних та інноваційних проектів, стратегічних планів розвитку, а також в навчальному процесі (при підготовці відповідних розділів підручників і навчальних посібників з курсів «Економічний аналіз», «Менеджмент», «Стратегія підприємства», «Оптимізаційні методи і моделі»). Висновки відповідно до статті. Сформульовані й обґрунтовані теоретичні положення дали змогу представити пропозиції науково-практичного характеру щодо застосування моделі комплексної економічної діагностики для підвищення ефективності діяльності підприємств. Ключові слова: діагностика, молокопереробні підприємства, інтегральна оцінка, методи дослідження, потенціал, конкурентоспроможність, ефективність. © Рибакова Т. О., Семененко О. Г., Черняєв О.С., 2021 Економічний вісник університету \| Випуск № 50 83
https://openalex.org/W4256154087	https://www.researchsquare.com/article/rs-522038/v1.pdf?c=1631882515000	English	null	Forest Fragmentation Slows the Decomposition of Coarse Woody Debris in a Subtropical Forest	Research Square (Research Square)	2,021	cc-by	10,475	Forest Fragmentation Slows the Decomposition of Coarse Woody Debris in a Subtropical Forest Chunsheng Wu Nanchang Institute of Technology Research License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Forest Science on September 17th, 2021. See the published version at https://doi.org/10.1093/forsci/fxab035. Title: 1 Forest fragmentation slows the decomposition of coarse woody debris in a subtropical 2 forest 3 4 Authors: 5 Chunsheng Wua,b, Cindy E. Prescottc, Chunjie Shua, Baoyong Lib, Zhijian Zhangb, Hankun Wangb, 6 Yi Zhangd, Yuanqiu Liub,d, G. Geoff Wangb,e 7 8 Affiliation: 9 a Jiangxi Provincial Engineering Research Center For Seed-Breeding and Utilization of Camphor Trees, School of 10 Hydraulic and Ecological Engineering, Nanchang Institute of Technology, Tianxiang Rd. 289, Nanchang 330099, 11 P.R.China. 12 b Key Laboratory of Silviculture, Co-Innovation Center of Jiangxi Typical Trees Cultivation and Utilization, College 13 of Forestry, Jiangxi Agricultural University, Zhimin Rd. 1101, Nanchang, 330045, P.R. China. 14 c Faculty of Forestry, University of British Columbia,2424 Main Mall, Vancouver, BC, Canada. 15 d Lushan Nature Reserve of Jiangxi (Lushan Mountain National Forest Ecological Station), Henan Rd. 600, Jiujiang 16 332900, P.R. China. 17 e Department of Forestry and Environmental Conservation, Clemson University, Clemson, SC, 29634, USA. 18 Co-correspondence authors: GGW: gwang@clemson.edu, 1-864-656-4864; YL: liuyq404@163.com, 19 +08615079126688. 20 Title: 1 of Forestry, Jiangxi Agricultural University, Zhimin Rd. 1101, Nanchang, 330045, P.R. China. 14 c Faculty of Forestry, University of British Columbia,2424 Main Mall, Vancouver, BC, Canada. 15 d Lushan Nature Reserve of Jiangxi (Lushan Mountain National Forest Ecological Station), Henan Rd. 600, Jiujiang 16 332900 PR China 17 e Department of Forestry and Environmental Conservation, Clemson University, Clemson, SC, 29634, USA. 8 +08615079126688. 20 23 Abstract: 24 Forest fragmentation is increasing rapidly around the world, and edge effects caused by 25 fragmented forests can influence ecosystem functions and ecological processes, including coarse 26 woody debris (CWD) decomposition. Understanding the influencing mechanisms of edge effect on 27 CWD decomposition is needed to assess the effects of forest fragmentation on C cycling and storage. 28 We measured rates of mass loss of CWD of Cinnamomum camphora and Pinus taiwanensis over 29 two years at two distances (0-5m vs. 60m) from a forest edge at two altitudes (215 and 1400 m a.s.l.), 30 in a subtropical forest. In addition, we determined the microbial community of each CWD and the 31 soil beneath via phospholipid fatty acids (PLFAs). Mass loss of CWD 60 m from the forest edge 32 was > 15 % greater than that at the edge (0-5m). Mass loss was positively correlated with the 33 abundance of microbial community and moisture content of the decaying CWD. Distance from edge 34 explained 17.4% of the total variation of the microbial abundance in CWD. The results indicated 35 that the reduced abundance of microbial communities and moisture content at forest edges reduced 36 rates of decomposition of CWD. Long-term experiments with more tree species and more forest 37 types are needed to assess the edge effect's generality. 38 Abstract: 24 Forest fragmentation is increasing rapidly around the world, and edge effects caused by 25 fragmented forests can influence ecosystem functions and ecological processes, including coarse 26 woody debris (CWD) decomposition. Understanding the influencing mechanisms of edge effect on 27 CWD decomposition is needed to assess the effects of forest fragmentation on C cycling and storage. 28 We measured rates of mass loss of CWD of Cinnamomum camphora and Pinus taiwanensis over 29 two years at two distances (0-5m vs. 60m) from a forest edge at two altitudes (215 and 1400 m a.s.l.), 30 in a subtropical forest. In addition, we determined the microbial community of each CWD and the 31 soil beneath via phospholipid fatty acids (PLFAs). Mass loss of CWD 60 m from the forest edge 32 was > 15 % greater than that at the edge (0-5m). Mass loss was positively correlated with the 33 abundance of microbial community and moisture content of the decaying CWD. Distance from edge 34 explained 17.4% of the total variation of the microbial abundance in CWD. The results indicated 35 that the reduced abundance of microbial communities and moisture content at forest edges reduced 36 rates of decomposition of CWD. Long-term experiments with more tree species and more forest 37 types are needed to assess the edge effect's generality. 38 39 Keywords: Edge effects; Decomposition; CWD; Microbial community; Subtropical forests 40 41 1. Introduction 42 Forests around the world store a large amount of carbon (C) in soils, dead and living above- 43 ground biomass (including coarse woody debris: CWD), which is regarded as a significant C sink 44 (Pan et al., 2011). Understanding the factors that influence rates of C cycling processes in forests 45 (including CWD decomposition) is necessary to quantify the role of global forests in the global C 46 cycle (Pan et al., 2011; Intergovernmental Panel on Climate Change, 2014; Tang et al., 2018). Forest 47 fragmentation due to natural and anthropogenic disturbances is a global phenomenon, affecting 48 forest ecosystem functioning (Numata et al., 2010; FAO, 2010; Haddad et al., 2015; Barlow et al., 49 2016). Globally, nearly 20 % of all forest area is situated within 100 m of a forest edge (Haddad et 50 al., 2015). Abstract: 24 Forest edges have distinct microclimates from forest interiors, including lower soil 51 moisture, lower humidity, increased light availability, and increased wind and rain impacts 52 (Laurance and Yensen, 1991; Didham & Ewers, 2012; Albiero-Júnior et al., 2020). These altered 53 microclimatic conditions at forest edges can alter the decay rates of CWD. In a temperate forest in 54 the United Kingdom, blocks of wood from European beech (Fagus sylvatica) placed at the forest 55 edge lost mass at about half the rate of blocks placed 100 m within the forest (Crockatt & Bebber, 56 2015). The slower mass loss was attributed to the lower humidity and moisture content of the 57 decaying wood at the forest edge (Crockatt & Bebber, 2015). In contrast, in a temperate forest in 58 USA, Forrester et al. (2012) reported higher respiration rates from CWD in canopy gaps than under 59 intact canopy. 60 Alterations in microclimatic conditions near forest edges may also influence soil microbial 61 community composition. Previous studies have shown that the abundance of fungi strongly 62 increased in response to changes in microclimate conditions due to edge effects (Boddy et al., 1989; 63 Boddy, 2001; Castaño et al., 2018; van der Linde et al., 2018; Boeraeve et al., 2019). The fungal 64 communities in soil and wood interact during all CWD decay phases (Mäkipää et al., 2017; 65 Purahong et al., 2019; Wu et al., 2020, 2021), so changes in soil microbial communities due to edge 66 Alterations in microclimatic conditions near forest edges may also influence soil microbial 61 community composition. Previous studies have shown that the abundance of fungi strongly 62 increased in response to changes in microclimate conditions due to edge effects (Boddy et al., 1989; 63 Boddy, 2001; Castaño et al., 2018; van der Linde et al., 2018; Boeraeve et al., 2019). The fungal 64 communities in soil and wood interact during all CWD decay phases (Mäkipää et al., 2017; 65 Purahong et al., 2019; Wu et al., 2020, 2021), so changes in soil microbial communities due to edge 66 effects could also alter fungal community dynamics within decomposing CWD. Reduced moisture 67 availability at forest edges can affect the growth and activity of saprotrophic fungi (Crockatt & 68 Bebber, 2015; Snäll & Jonsson, 2001) and mycorrhizal fungi (Boeraeve et al., 2019). In addition, 69 the abundance of white rot species, such as Xeromphalina campanella, Rigidoporus sp. Abstract: 24 and 70 Skeletocutis odora, is positively correlated with moisture content (Fukasawa et al., 2015, 2018). 71 Here, we compare rates of decomposition and microbial communities in CWD in the interior 72 and edges of a subtropical forest ecosystem. We measured two-year mass loss of CWD of Pinus 73 taiwanensis Hayata and Cinnamomum camphora (Linn.) Presl at the forest edge and 60 m into the 74 forest and characterized the microbial community in the decaying CWD. The experiment was 75 conducted at sites at two elevations (215m and 1400m a.s.l.). Specifically, we asked: (1) do CWD 76 mass loss rates differ between forest edge and forest interior positions? (2), do microbial 77 communities in decaying CWD differ between forest edge and interior positions? and (3) how do 78 characteristics of the microbial community relate to environmental conditions in the two 79 environments? Based on studies from temperate forests, we hypothesize that CWD decomposition 80 rates will be lower at the forest edge than in the forest interior and that the differences will be related 81 to changes in microbial communities and moisture. 82 the abundance of white rot species, such as Xeromphalina campanella, Rigidoporus sp. and 70 Skeletocutis odora, is positively correlated with moisture content (Fukasawa et al., 2015, 2018). 71 Here, we compare rates of decomposition and microbial communities in CWD in the interior 72 and edges of a subtropical forest ecosystem. We measured two-year mass loss of CWD of Pinus 73 taiwanensis Hayata and Cinnamomum camphora (Linn.) Presl at the forest edge and 60 m into the 74 forest and characterized the microbial community in the decaying CWD. The experiment was 75 conducted at sites at two elevations (215m and 1400m a.s.l.). Specifically, we asked: (1) do CWD 76 mass loss rates differ between forest edge and forest interior positions? (2), do microbial 77 communities in decaying CWD differ between forest edge and interior positions? and (3) how do 78 characteristics of the microbial community relate to environmental conditions in the two 79 environments? Based on studies from temperate forests, we hypothesize that CWD decomposition 80 rates will be lower at the forest edge than in the forest interior and that the differences will be related 81 to changes in microbial communities and moisture. 82 83 2. Materials and methods 84 2.1. Abstract: 24 Study area 85 This field research was conducted in a mixed coniferous-broad-leaved forest (CBF) at Lushan 86 Mountain in Jiangxi Province, China (29°31′~29°41′ N, 115°51′~116°07′ E). The area is 87 characterized as a subtropical monsoon climate with four distinct seasons. Mean annual 88 precipitation and temperature range from 1308 to 2068 mm, and from 17.1 to 11.6 °C, respectively 89 (Wu et al., 2019b). According to the FAO soil texture classification, soil types in Lushan change 90 from ferric alisols at low elevations to haplic alisols at high ones (Liu & Wang, 2010; Wu et al., 91 2018b). Mixed coniferous–broad-leaved forests are dominated by several Platycarya strobilacea 92 and Acer davidii species, and some deciduous woodland species and shrubs (Liu & Wang, 2010). 93 2.2. Sampling design 94 In December 2015, study sites were established in a mixed coniferous-broad-leaved forest at 95 two altitudes (215m and 1400m). The sites at the two altitudes had similar aspect, slope steepness 96 and position, stand age, and understory vegetation. Characteristics of the soils at the two sites are 97 provided in Appendix 1. At each site, three plots were established within 0-5 m (Plot one: 2 m, Plot 98 two: 3 m, Plots three: 2.5 m) of the forest edge, and another three plots were established 60 m (Plot 99 one: 60 m, Plot two: 59.5 m, Plots threee: 59 m) inside the forest. The two tree species selected for 100 CWD were Pinus taiwanensis Hayata and Cinnamomum camphora (Linn.) Presl. Fresh logs about 101 15 cm in central diameter were selected and cut into segments about 150 cm long (Table 1). Two 102 CWD segments of each tree species were placed flush on the ground 30 cm from one another and 103 35 cm from the boundary of each plot. Therefore, a total of 48 CWD segments (2 altitudes × 2 edge 104 distances × 3 plots × 2 CWD species × 2 segments) were tested. 105 2.3. Wood physicochemical properties analysis 106 A 2-cm-thick disk of each CWD was collected from a randomly selected place on each of the 107 48 logs at the beginning of the experiment and after 3, 6, 9, 12, 15, 18, 21, and 24 months. Disks 108 (Wu et al., 2019b). Abstract: 24 According to the FAO soil texture classification, soil types in Lushan change 90 from ferric alisols at low elevations to haplic alisols at high ones (Liu & Wang, 2010; Wu et al., 91 2018b). Mixed coniferous–broad-leaved forests are dominated by several Platycarya strobilacea 92 and Acer davidii species, and some deciduous woodland species and shrubs (Liu & Wang, 2010). 93 2.2. Sampling design 94 A 2-cm-thick disk of each CWD was collected from a randomly selected place on each of the 107 48 logs at the beginning of the experiment and after 3, 6, 9, 12, 15, 18, 21, and 24 months. Disks 108 were sealed in a plastic bag to preserve their moisture content before being transferred to the 109 laboratory (Wu et al., 2018a, 2019a). The disk samples were collected more than 80 cm from the 110 mid of wood. Each CWD sample was weighed and then oven-dried at 70°C and re-weighed, and 111 their moisture content was calculated using equation (1). 112 their moisture content was calculated using equation (1). 112 𝑀𝐶𝑊𝐷= 𝑊𝑤−𝑊𝑑 𝑊𝑑 (1) 𝑀𝐶𝑊𝐷= 𝑊𝑤−𝑊𝑑 𝑊𝑑 (1) 113 (1) where MCWD (%) was the moisture content of each CWD during each measurement, Ww (g) was the 114 where MCWD (%) was the moisture content of each CWD during each measurement, Ww (g) was the 114 wet wood weight, and Wd (g) was the dry weight. 115 wet wood weight, and Wd (g) was the dry weight. 115 The density of each CWD was calculated using equation (2): First, the weights of the disks of 116 the CWD were measured (m, g), and the disks were placed in a container with a specific amount of 117 water and wood disks in the container (𝑣1, ml), the initial volume of water (𝑣2, ml), and the density 118 of each CWD sample (ρ, g/cm3) was calculated. 119 The density of each CWD was calculated using equation (2): First, the weights of the disks of 116 the CWD were measured (m, g), and the disks were placed in a container with a specific amount of 117 water and wood disks in the container (𝑣1, ml), the initial volume of water (𝑣2, ml), and the density 118 of each CWD sample (ρ, g/cm3) was calculated. their moisture content was calculated using equation (1). 112 Abstract: 24 119 𝜌= 𝑚/(𝑣1 −𝑣2) (2) 𝜌= 𝑚/(𝑣1 −𝑣2) (2) 120 𝜌= 𝑚/(𝑣1 −𝑣2) ( 0 (2) 𝑀𝐿𝐶𝑊𝐷= 𝑀𝑖 – 𝑀𝑡 𝑀𝑖 (3) 122 𝑀𝐿𝐶𝑊𝐷= 𝑀𝑖 – 𝑀𝑡 𝑀𝑖 (3) (3) where 𝑀𝐿𝐶𝑊𝐷 (%) was the mass loss of each CWD sample during each measurement, 𝑀𝑖 (g) was 123 where 𝑀𝐿𝐶𝑊𝐷 (%) was the mass loss of each CWD sample during each measurement, 𝑀𝑖 (g) was 123 where 𝑀𝐿𝐶𝑊𝐷 (%) was the mass loss of each CWD sample during each measurement, 𝑀𝑖 (g) was 123 the initial dry mass, and 𝑀𝑡 (g) was the wood mass after 3, 6, 9, 12, 15, 18, 21 or 24 months. 124 the initial dry mass, and 𝑀𝑡 (g) was the wood mass after 3, 6, 9, 12, 15, 18, 21 or 24 months. 124 CWD temperature was measured at approximately 2 cm depth using a hand-held long-stem 125 thermometer (Model SK-250WP, Sato Keiryoki Mfg. Co. Ltd, Tokyo, Japan). Concentrations of C 126 and N in each CWD sample were determined using a TOC analyzer (Vario TOC, Elementar, 127 German), and concentrations of lignin and cellulose were determined using the ADF-sulphuric 128 method (Rowland et al., 1994) and the Kjeldahl method (K-370, Buchi Scientific Instruments, 129 Switzerland). Soil available N (sum of the ammonium nitrogen, nitrate nitrogen, amino acid and 130 readily hydrolyzed proteins nitrogen) was measured through the oxidation hydrolyzed into ammonia 131 nitrogen, then absorbed by the boric acid solution and determined by sulfuric acid and titration (Liu, 132 1996). Soil available P was extracted using HCl-NH4F solution and determined by Molybdenum- 133 Antimony Anti colorimetric assay (Liu, 1996). Relative to P. taiwanensis, C. camphora CWD had 134 higher initial concentrations of carbon and lignin, and lower cellulose (Appendix 1). Soil organic 135 matter, N, hydrolyzed N and available P significantly differed between the two sites (Appendix 2). 136 2.4. Phospholipid fatty acid (PLFA) analyses 137 Microbial biomass and the relative index of bacterial to fungal biomass were estimated by 138 PLFA analysis (Bossio & Scow, 1998; Olsson et al., 1999). CWD samples and mineral soil (0-5 cm) 139 under each CWD segment were collected every three months during the study period and stored at 140 -20℃ for PLFA analysis. Concentrations of each PLFA were calculated relative to the 19:0 internal 141 standard concentration. Total microbial biomass summed by each individual PLFAs (ng g-1 dry 142 wood material). Abstract: 24 Microbial groups of bacteria (B), fungi (F), Gram-negative bacteria (G-), Gram- 143 positive bacteria (G+), actinomycetes (ACT), and arbuscular mycorrhizal fungi (AMF) were 144 biomarkers by the characteristic fatty acids (Appendix 3) (Olsson et al., 1999). 145 2.5. Data analysis 146 The effects of month, distance from the edge, and their interactions on temperature, moisture 147 content, and mass loss of CWD were determined by repeated measures ANOVA for each species. 148 We used three-way ANOVA to test the distance from edge, tree species, and altitude gradient on 149 mass loss and microbial community changes. Due to significant interactions among altitude, edge 150 distance, and species, all comparisons among altitude, edge distance or tree species were performed 151 using one-way ANOVA of Dunnett’s post-hoc. The microbial communities in the soil (0-5 cm), 152 under each CWD and in the CWD itself were compared by simple correlation analysis, and 153 differences in the PLFA signatures of the microbial community under CWD or soil among different 154 treatments (tree species, altitude gradient or edge distance) were tested by redundancy analysis 155 (RDA). All data were analysed using SPSS 20.0 (SPSS Inc., Chicago, USA). Differences were 156 higher initial concentrations of carbon and lignin, and lower cellulose (Appendix 1). Soil organic 135 matter, N, hydrolyzed N and available P significantly differed between the two sites (Appendix 2). 136 2.4. Phospholipid fatty acid (PLFA) analyses 137 higher initial concentrations of carbon and lignin, and lower cellulose (Appendix 1). Soil organic 135 matter, N, hydrolyzed N and available P significantly differed between the two sites (Appendix 2). 136 2.4. Phospholipid fatty acid (PLFA) analyses 137 Microbial biomass and the relative index of bacterial to fungal biomass were estimated by 138 PLFA analysis (Bossio & Scow, 1998; Olsson et al., 1999). CWD samples and mineral soil (0-5 cm) 139 under each CWD segment were collected every three months during the study period and stored at 140 -20℃ for PLFA analysis. Concentrations of each PLFA were calculated relative to the 19:0 internal 141 standard concentration. Total microbial biomass summed by each individual PLFAs (ng g-1 dry 142 wood material). Microbial groups of bacteria (B), fungi (F), Gram-negative bacteria (G-), Gram- 143 positive bacteria (G+), actinomycetes (ACT), and arbuscular mycorrhizal fungi (AMF) were 144 biomarkers by the characteristic fatty acids (Appendix 3) (Olsson et al., 1999). 145 considered significant at p < 0.05. 157 158 3. Abstract: 24 Results 159 3.1. Distance from edge effects 160 During the two-year experimental period, the mean mass loss of P. taiwanensis and C. 161 camphora 60 m away from the forest edge was significantly greater than that at the forest edge at 162 both sites (Table 2 and Fig. 1). The moisture content of the CWD segments was also greater 60 m 163 from the forest edge than at the edge throughout the two-year period (Fig. 2). There was a significant 164 positive correlation between CWD's mass loss and moisture content for each altitude, edge distance, 165 and tree species (Table 3). 166 3.2. Microbial community composition 167 Concentrations of total PLFA, total fungi, total bacteria, G+ bacteria, G- bacteria, soil fungi, 168 and AMF in the soil beneath CWD of both tree species were all higher 60m from the forest edge 169 than at the forest edge (Fig. 3). Concentrations of total PLFA, total bacteria, total fungi, G+ bacteria, 170 G- bacteria, fungi, and AMF in the CWD of both tree species were also higher 60 m from the forest 171 edge than at the edge (Fig. 4). The concentrations of fungal components (total fungi, fungi, and 172 AMF) in the CWD were generally higher than that of bacteria components (total bacteria, G+ 173 bacteria, and G- bacteria) (Fig. 4). 174 3.3. Relationship between soil and CWD microbial communities 175 Correlations between microbial communities in CWD and soil (Table 4) were generally 176 positive in plots 60 m from the forest edge. There were some negative correlations between CWD 177 fungi and soil fungi at the forest edge (Table 4). 178 considered significant at p < 0.05. 157 158 3. Results 159 3.1. Distance from edge effects 160 During the two-year experimental period, the mean mass loss of P. taiwanensis and C. 161 camphora 60 m away from the forest edge was significantly greater than that at the forest edge at 162 both sites (Table 2 and Fig. 1). The moisture content of the CWD segments was also greater 60 m 163 from the forest edge than at the edge throughout the two-year period (Fig. 2). There was a significant 164 positive correlation between CWD's mass loss and moisture content for each altitude, edge distance, 165 and tree species (Table 3). 166 3.2. Abstract: 24 In our study, CWD of both tree species in the forest interior 195 had greater total PLFA, total bacteria, total fungi, G+ bacteria, G- bacteria, fungi, and AMF compared 196 to CWD at the forest edge. Other studies have reported increased activity of both wood- and litter- 197 decomposing saprotrophic fungi with distance from the forest edge (Snäll & Jonsson, 2001; Riutta 198 et al., 2012; Crockatt & Bebber, 2015; Fukasawa et al., 2015, 2018; Ruwanza, 2019). Previous 199 studies also reported that the abundance of mycorrhizal fungi was greater within forests than at 200 3.4. Effects of edge distance and tree species on CWD microbial community 179 3.4. Effects of edge distance and tree species on CWD microbial community 179 Distance to the forest edge had a larger influence on the CWD microbial community than did 180 tree species or altitude (Table 5). Distance to edge explained 17.4% of the total variation, while tree 181 species and altitude explained 3.1% and 10.9%, respectively (Table 5). 182 183 4. Discussion 184 Consistent with our hypothesis, mass loss of CWD within this studied subtropical forest was > 185 15 % greater than that at the edge, at both altitudes, which is lower to the 23 % greater mass loss of 186 woodblocks placed 100 m into the forest than at the edge in a temperate forest (Crockatt & Bebber, 187 2015). The CWD at the forest edge had lower moisture content throughout the study, which is 188 consistent with our hypothesis. Similarly, Crockatt and Bebber (2015) reported that the moisture 189 content of the woodblocks increased with distance from the edge. 190 The higher moisture content of soil and wood in the forest interior probably facilitated the 191 detection and colonization of CWD by microbes, which are largely soil-dwelling (Mäkipää et al., 192 2017; Fukasawa et al., 2018; Law et al., 2019). Malmivaara-Lämsä et al. (2008) found that fungal 193 biomass was about 30 % higher 20 m inside the forest when compared to the edge, due to the higher 194 humus moisture content inside the forest. In our study, CWD of both tree species in the forest interior 195 had greater total PLFA, total bacteria, total fungi, G+ bacteria, G- bacteria, fungi, and AMF compared 196 to CWD at the forest edge. Abstract: 24 Microbial community composition 167 Concentrations of total PLFA, total fungi, total bacteria, G+ bacteria, G- bacteria, soil fungi, 168 and AMF in the soil beneath CWD of both tree species were all higher 60m from the forest edge 169 than at the forest edge (Fig. 3). Concentrations of total PLFA, total bacteria, total fungi, G+ bacteria, 170 G- bacteria, fungi, and AMF in the CWD of both tree species were also higher 60 m from the forest 171 edge than at the edge (Fig. 4). The concentrations of fungal components (total fungi, fungi, and 172 AMF) in the CWD were generally higher than that of bacteria components (total bacteria, G+ 173 bacteria, and G- bacteria) (Fig. 4). 174 Correlations between microbial communities in CWD and soil (Table 4) were generally 176 positive in plots 60 m from the forest edge. There were some negative correlations between CWD 177 fungi and soil fungi at the forest edge (Table 4). 178 3.4. Effects of edge distance and tree species on CWD microbial community 179 Distance to the forest edge had a larger influence on the CWD microbial community than did 180 tree species or altitude (Table 5). Distance to edge explained 17.4% of the total variation, while tree 181 species and altitude explained 3.1% and 10.9%, respectively (Table 5). 182 183 4. Discussion 184 Consistent with our hypothesis, mass loss of CWD within this studied subtropical forest was > 185 15 % greater than that at the edge, at both altitudes, which is lower to the 23 % greater mass loss of 186 woodblocks placed 100 m into the forest than at the edge in a temperate forest (Crockatt & Bebber, 187 2015). The CWD at the forest edge had lower moisture content throughout the study, which is 188 consistent with our hypothesis. Similarly, Crockatt and Bebber (2015) reported that the moisture 189 content of the woodblocks increased with distance from the edge. 190 The higher moisture content of soil and wood in the forest interior probably facilitated the 191 detection and colonization of CWD by microbes, which are largely soil-dwelling (Mäkipää et al., 192 2017; Fukasawa et al., 2018; Law et al., 2019). Malmivaara-Lämsä et al. (2008) found that fungal 193 biomass was about 30 % higher 20 m inside the forest when compared to the edge, due to the higher 194 humus moisture content inside the forest. Abstract: 24 Other studies have reported increased activity of both wood- and litter- 197 decomposing saprotrophic fungi with distance from the forest edge (Snäll & Jonsson, 2001; Riutta 198 et al., 2012; Crockatt & Bebber, 2015; Fukasawa et al., 2015, 2018; Ruwanza, 2019). Previous 199 studies also reported that the abundance of mycorrhizal fungi was greater within forests than at 200 edges (Malmivaara-Lämsä et al., 2008; Kjøller et al., 2012; Erlandson et al., 2016; Boeraeve et al., 201 2019). The abundance and diversity of various soil fauna groups may also be reduced near forest 202 edges (Goosem, 2000; Laurance et al., 2002; Lehtinen et al., 2003; Watson et al., 2004; Laurance, 203 2004; Pfeifer et al., 2017). 204 2004; Pfeifer et al., 2017). 204 This study investigated CWD decomposition during a two-year experimental period, a 205 relatively short time span considering the turnover time of wood. Long-term research is needed to 206 determine decay rates and the proportion of the CWD mass that is converted into more persistent 207 organic-matter pools over a longer study period. Our sampling method-collecting a 2-cm-thick disk 208 from the end part of each CWD segment-would have increased exposure of the segment to fungal 209 invasion. This would overestimate actual rates of decomposition but should be more realistic than 210 the common practice of using woodblocks or tongue depressors. 211 212 5. Conclusion 213 Coarse woody debris of two tree species (Cinnamomum camphora and Pinus taiwanensis) 214 placed at the edge of a subtropical forest decomposed slower than that placed 60 m within the forest. 215 Rates of decay positively correlated with abundance of microorganisms in the CWD and the CWD 216 moisture content. Distance from edge (17.4%) was more important than tree species (3.1%) and 217 altitude gradient (10.9%) in determining microbial abundance in CWD. Our results indicate that the 218 lower moisture content at forest edges reduce microbial activity and CWD decomposition rates. 219 Given the increasing rate of forest fragmentation worldwide, higher rates of CWD decomposition 220 at forest edges need to be incorporated into global C models. 221 222 This study investigated CWD decomposition during a two-year experimental period, a 205 relatively short time span considering the turnover time of wood. Long-term research is needed to 206 determine decay rates and the proportion of the CWD mass that is converted into more persistent 207 organic-matter pools over a longer study period. at forest edges need to be incorporated into global C models. Abstract: 24 Our sampling method-collecting a 2-cm-thick disk 208 from the end part of each CWD segment-would have increased exposure of the segment to fungal 209 invasion. This would overestimate actual rates of decomposition but should be more realistic than 210 the common practice of using woodblocks or tongue depressors. 211 222 Acknowledgment 223 We are grateful to the Lushan Mountain National Forest Ecological Station for providing the 224 study sites. This study was financially supported by the National Natural Science Foundation of 225 China (31960303, 31901292 and 31460185). 226 We are grateful to the Lushan Mountain National Forest Ecological Station for providing the 224 study sites. This study was financially supported by the National Natural Science Foundation of 225 China (31960303, 31901292 and 31460185). 226 We are grateful to the Lushan Mountain National Forest Ecological Station for providing the 224 study sites. This study was financially supported by the National Natural Science Foundation of 225 China (31960303, 31901292 and 31460185). 226 227 Author Contributions 228 All authors contributed to the study at various phases. Specifically, C.S.W., C.J.S., B.Y.L., 229 Z.J.Z., H.K.W., Y.Z., and Y.Q.L. were responsible for study design, data collection and analysis, 230 and writing the early drafts of this research. C.S.W., C.P., Y.Q.L. and G.G.W substantially 231 contributed to interpreting and revising the manuscript. 232 233 Conflicts of Interest 234 The authors declare no conflict of interest. 235 236 References: 237 Albiero-Júnior, A., Venegas-González, A., Rodríguez-Catón, M., Oliveira, J. 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Biological Conservation 120, 311-327. 355 Wu, C.S., Ulyshen, M., Shu, C.J., Zhang, Z.J., Zhang, Y., Liu, Y.Q., Wang, G.G., 2021. Stronger effects 356 Management 493, 119263. 358 Wu, C.S., Mo, Q.F., Wang, H.K., Zhang, Z.J., Huang, G.X., Ye, Q., Zou, Q., Kong, F.Q., Liu, Y.Q., Wang, 359 G.G., 2018b. Moso bamboo (Phyllostachys edulis (Carriere) J. Houzeau) invasion affects soil 360 phosphorus dynamics in adjacent coniferous forests in subtropical China. Annals of Forest Science 361 75, 24. 362 Wu, C.S., Wang, H.K., Mo, Q.F., Zhang, Z.J., Huang, G.X., Kong, F.Q., Liu, Y.Q., Wang, G.G., 2019a. 363 Effects of elevated UV-B radiation and N deposition on the decomposition of coarse woody debris. 364 Science of the Total Environment 663, 170-176. Wu, C.S., Zhang, Z.J., Shu, C.J., Mo, Q.F., Wang, H.K., Kong, F.Q., Wang, G.G., Liu, Y.Q., 2020. The 366 response of coarse woody debris decomposition and microbial community to nutrient additions in 367 a subtropical forest. Forest Ecology and Management 460, 117799. 368 Wu, C.S., Zhang, Z.J., Wang, H.K., Huang, G.X., Shu, C.J., Kong, F.Q., Zhang, Y., Wang, G.G., Liu, 369 Y.Q., 2019b. Home-field advantage of CWD decomposition in subtropical forests varied by field 370 sites. Forest Ecology and Management 444, 127-137. 371 Wu, C.S., Zhang, Z.J., Wang, H.K., Li, C., Mo, Q.F., Liu, Y.Q., 2018a. Photodegradation accelerates 372 coarse woody debris decomposition in subtropical Chinese forests. Forest Ecology and Management 373 409, 225-232. 374 Zak, D.R., Ringelberg, D.B., Pregitzer, K.S., Randlett, D.L., White, D.C., Curtis, P.S., 1996. Soil 375 microbial communities beneath populus grandidentata grown under elevated atmospheric CO2. microbial communities in soil: a review. Biology and Fertility of Soils 29, 111-129. Acknowledgment 223 376 Ecological Applications 6, 257-262. 377 Zelles, L., 1999. Fatty acid patterns of phospholipids and lipopolysaccharides in the characterisation of 378 microbial communities in soil: a review. Biology and Fertility of Soils 29, 111-129. 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 400 Tables and Figures 401 Table 1. Initial diameter, length and density (mean ± SE, n=6) of the log segments of the two tree species at each 402 altitude (215m and 1400m) and forest edge distance (edge and 60m from the forest edge). Note: experiment material 403 was all of similar decay stage. 404 Tree species Altitude Edge distance Average diameter (cm) Average length (cm) Average density (g/cm3) Pinus taiwanensis 215m edge 16.21±2.07 147.35±4.26 0.405±0.04 60m 15.87±1.94 150.06±3.88 0.411±0.03 1400m edge 15.66±2.11 147.56±4.16 0.403±0.04 60m 16.19±1.96 150.33±3.94 0.406±0.05 Cinnamomum camphora 215m edge 15.36±2.56 149.55±4.42 0.434±0.05 60m 15.62±1.99 150.32±3.88 0.452±0.03 1400m edge 15.77±2.24 147.33±4.28 0.442±0.03 60m 15.76±1.99 151.12±3.54 0.452±0.06 Tables and Figures 401 412 Table 2. Two-year average mass loss, temperature and moisture content of CWD (mean ± SE, n=6) of the two studied 413 species at the two altitudes (215m and 1400m) and forest edge distances (edge and 60m from the forest edge) in 414 Lushan Mountain, China. Different uppercase letters indicate significant differences between edge distances for the 415 same species (e.g., Pinus taiwanensis or Cinnamomum camphora) at p < 0.05) and altitude. Different lowercase 416 letters indicate significant differences among the two species for the same edge distance and altitude (p < 0.05). 417 Different lowercase letters in brackets indicate significant differences between altitudes for the same edge distance 418 and tree species (p < 0.05). 419 Tree species Altitude Edge distance Mass loss (%) Temperature (℃) Moisture content (%) Pinus taiwanensis 215m edge 23.3±3.8 Ba (a) 16.71±1.32 Aa (a) 74.16±6.34 Ba (a) 60m 27.7±3.7 Ab (a) 16.01±1.11 Aa (a) 79.26±6.47 Aa (a) 1400m edge 15.5±3.6 Ba (c) 11.57±1.46 Aa (c) 69.74±6.54 Ba (b) 60m 20.1±3.4 Ab (c) 10.74±1.53 Aa (c) 75.36±6.25 Aa (b) Cinnamomum camphora 215m edge 29.6±4.1 Ba (a) 17.48±1.27 Aa (a) 73.60±6.13 Ba (a) 60m 34.0±4.5 Aa (a) 16.33±1.63 Aa (a) 79.03±7.22 Aa (a) 1400m edge 22.0±3.1 Ba (c) 12.20±1.38 Aa (c) 71.21±6.31 Ba (b) 60m 26.6±3.5 Aa (c) 11.04±1.75 Aa (c) 76.16±6.44 Aa (b) 424 Table 3. Acknowledgment 223 Correlations of mass loss with moisture content and temperature of CWD by altitude, forest edge distance and tree species. ns not significant, p＜0.05, p＜0.01, * p＜0.001. 425 Tree species Fixed effects 215m 1400m edge 60m edge 60m Pinus taiwanensis Mass loss × Moisture content 0.644 0.689 0.705 0.731 Mass loss × Temperature 0.035 ns 0.058 ns 0.093 ns 0.051 ns Cinnamomum camphora Mass loss × Moisture content 0.672 0.693 0.711 0.749 Mass loss × Temperature 0.078 ns 0.051 ns 0.064 ns 0.076 ns 439 Table 4. Correlation between the composition of different phospholipid fatty acid (PLFA) (ng g-1 dry mass) 440 signatures of CWD and soil for each altitude, forest-edge distance, and tree-species combination. Total, total PLFA 441 concentrations; B, bacterial PLFAs; F, fungal PLFAs; F/B, the fungal to bacterial ratio; G+, Gram-positive bacteria; 442 G-, Gram-negative bacteria; G+/G-, ratio of Gram-positive to Gram-negative bacteria; AMF, arbuscular mycorrhizal 443 fungi. ns not significant, * p＜0.05, p＜0.01, * p＜0.001. 444 Fixed effects Altitude Edge distance AMF Fungi Total Fungi G+ G- Total B Total Pinus taiwanensis × Soil 215m edge 0.643* -0.611* -0.678* 0.822* 0.662* 0.704 0.771 60m 0.801 0.778 0.791 0.767 0.683* 0.778 0.804 1400m edge 0.654* -0.647* -0.674* 0.789** 0.771* 0.782 0.801 60m 0.812 0.785 0.799 0.801 0.788* 0.784 0.797 Cinnamomum camphora ×Soil 215m edge 0.634* -0.645* -0.635* 0.811** 0.642* 0.661* 0.788 60m 0.803 0.777 0.781 0.781** 0.650* 0.789 0.804 1400m edge 0.639* -0.627* -0.647* 0.789** 0.629* 0.796 0.792 60m 0.811 0.784 0.796 0.801 0.640* 0.769 0.781 452 Table 5. Effects of tree species, altitude, and forest-edge distance on the selected phospholipid fatty acid (PLFAs) 453 signatures of CWD tested with redundancy analysis (RDA). 454 Explanatory variable Explained variance % Contribution % Pseudo-F P (< 0.05) Tree species 3.1 9.5 3.8 0.036 Altitude 10.9 37.2 18.8 0.002 Forest edge distance 17.4 45.5 20.6 0.001 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 Table 5. Effects of tree species, altitude, and forest-edge distance on the selected phospholipid fatty acid (PLFAs) 453 453 Table 5. Effects of tree species, altitude, and forest-edge distance on the selected phospholipid fatty acid (PLFAs Table 5. Effects of tree species, altitude, and forest-edge distance on the selected phospholipid fatty acid (PLFAs) 453 signatures of CWD tested with redundancy analysis (RDA). Acknowledgment 223 454 Explanatory variable Explained variance % Contribution % Pseudo-F P (< 0.05) Tree species 3.1 9.5 3.8 0.036 Altitude 10.9 37.2 18.8 0.002 Forest edge distance 17.4 45.5 20.6 0.001 signatures of CWD tested with redundancy analysis (RDA). 454 470 Appendix 1. Initial chemical characteristics of CWD (fresh wood) of the two tree species at each altitude (215m 471 and 1400m) and forest-edge distance (edge and 60m from the forest edge) in Lushan Mountain of subtropical China. 472 Different lowercase letters indicate significant differences between tree species for the same forest edge distance and 473 altitude (p < 0.05). Note: The concentrations of C, N, lignin, and cellulose in CWD of the same species did not 474 significantly differ (p < 0.05) between the two forest-edge distances (i.e., edge vs. 60m). 475 Tree species Altitude Edge distance C concentration (g/kg) N concentration (g/kg) C/N Lignin concentration (%) Cellulose concentration (%) Pinus taiwanensis 215m edge 383.41±9.11 b 5.46±0.53 a 70.22 b 18.11±1.56 b 38.04±2.31 a 60m 381.66±9.06 b 5.49±0.47 a 69.52 b 18.13±1.78 b 37.97±2.53 a 1400m edge 382.94±9.01 b 5.57±0.59 a 68.75 b 18.11±1.66 b 38.03±2.47 a 60m 383.72±9.17 b 5.49±0.45 a 69.89 b 18.12±1.69 b 37.91±2.08 a Cinnamomum camphora 215m edge 433.21±10.88 a 5.89±0.62 a 73.55 a 25.77±1.47 a 33.04±2.49 b 60m 430.74±11.29 a 5.91±0.58 a 72.88 a 25.82±1.62 a 33.02±2.31 b 1400m edge 432.24±12.11 a 5.91±0.58 a 73.14 a 25.75±1.61 a 33.05±2.21 b 60m 430.97±11.73 a 5.88±0.68 a 73.29 a 25.71±1.58 a 33.02±1.98 b 481 Appendix 2. Initial physicochemical properties (± SE, n=24) of the upper 15 cm of the soil at the two sites. Different 482 lowercase letters indicate significant differences among the altitudes for the same index (p < 0.05). 483 Altitude Organic matter (g/kg) N (g/kg) Hydrolyzed N (mg/kg) P (g/kg) Available P (mg/kg) Available K (mg/kg) pH Bulk density (g/cm3) Soil ty 215m 38.62±3.91 c 1.74±0.12 b 192.94±18.88 c 0.26±0.05 a 3.66±0.29 b 41.78±3.21 b 4.7±0.5 a 0.98±0.14 a Ferric a 1400m 51.36±4.56 a 2.01±0.22 a 215.17±21.02 a 0.29±0.05 a 4.09±0.32 a 47.35±4.86 a 4.8±0.4 a 0.93±0.13 a Haplic a 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 Appendix 2. Initial physicochemical properties (± SE, n=24) of the upper 15 cm of the soil at the two sites. Differen Appendix 2. Acknowledgment 223 Initial physicochemical properties (± SE, n=24) of the upper 15 cm of the soil at the two sites. Different 482 482 lowercase letters indicate significant differences among the altitudes for the same index (p < 0.05). 483 Altitude Organic matter (g/kg) N (g/kg) Hydrolyzed N (mg/kg) P (g/kg) Available P (mg/kg) Available K (mg/kg) pH Bulk density (g/cm3) Soil type 215m 38.62±3.91 c 1.74±0.12 b 192.94±18.88 c 0.26±0.05 a 3.66±0.29 b 41.78±3.21 b 4.7±0.5 a 0.98±0.14 a Ferric alisols 1400m 51.36±4.56 a 2.01±0.22 a 215.17±21.02 a 0.29±0.05 a 4.09±0.32 a 47.35±4.86 a 4.8±0.4 a 0.93±0.13 a Haplic alisols lowercase letters indicate significant differences among the altitudes for the same index (p < 0.05). 483 484 499 Appendix 3. Biomarkers of selected phospholipid fatty acids (PLFAs) used in this study. 500 Appendix 3. Biomarkers of selected phospholipid fatty acids (PLFAs) used in this study. 500 Groups Biomarker PLFAs Bacteria 15:0, i15:0, a15:0, i16:0, 16:1ω7c, i17:0, a17:0, cy17:0, cy19:0, 18:1ω7c Frostegård & Bååth (1996) and Zak et al. (1996) G+ bacteria 15:0, i15:0, a15:0, i16:0, a17:0, i17:0 Zelles (1999) G- bacteria 16:1ω7c, cy17:0, cy19:0, 18:1ω7c Zak et al. (1996) Fungi 18:2ω6,9c, 18:1ω9c Frostegård et al. (2011) Actinomycetes 10Me 16:0, 10Me 17:0, 10Me 18:0 Zak et al. (1996) AMF 16:1ω5c Olsson et al. (1999) Total biomass 15:0, i15:0, a15:0, i16:0, 16:1ω7c, i17:0, a17:0, cy17:0, cy19:0, 18:1ω7c, 16:1ω5c, 10Me 16:0, 10Me 17:0, 10me 18:0, 18:2ω6,9c, 18:1ω9c AMF, Arbuscular mycorrhizal fungi. 501 502 503 504 505 506 507 508 509 510 511 512 513 Groups Biomarker PLFAs Bacteria 15:0, i15:0, a15:0, i16:0, 16:1ω7c, i17:0, a17:0, cy17:0, cy19:0, 18:1ω7c Frostegård & Bååth (1996) and Zak et al. (1996) G+ bacteria 15:0, i15:0, a15:0, i16:0, a17:0, i17:0 Zelles (1999) G- bacteria 16:1ω7c, cy17:0, cy19:0, 18:1ω7c Zak et al. (1996) Fungi 18:2ω6,9c, 18:1ω9c Frostegård et al. (2011) Actinomycetes 10Me 16:0, 10Me 17:0, 10Me 18:0 Zak et al. (1996) AMF 16:1ω5c Olsson et al. (1999) Total biomass 15:0, i15:0, a15:0, i16:0, 16:1ω7c, i17:0, a17:0, cy17:0, cy19:0, 18:1ω7c, 16:1ω5c, 10Me 16:0, 10Me 17:0, 10me 18:0, 18:2ω6,9c, 18:1ω9c Frostegård & Bååth (1996) and Zak et al. (1996) Frostegård & Bååth (1996) and Zak et al. (1996) Frostegård & Bååth (1996) and Zak et al. (1996) Zelles (1999) Zak et al. (1996) Frostegård et al. (2011) Zak et al. (1996) Zak et al. (1996) a15:0, i16:0, 16:1ω7c, i17:0, a17:0, cy17:0, cy19:0, 18:1ω7c, 16:1ω5c, 10Me 16:0, 10Me 17:0, 10me 18:0, 18:2ω6,9c, 18:1ω9c 513 Figure 1. Patterns of mass loss of two tree species of CWD (fresh wood) at each altitude (215m and 1400m) and forest-edge distance (edge and 60m from the forest edge) during 24 months of decomposition in Lushan Mountain 515 of subtropical China. 516 Cinnamomum camphora 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 20 40 60 80 100 % intial mass remaining Pinus taiwanensis 215m vs. 1400m: F=11.37, p＜0.01 edge vs. 60m: F=18.56, p＜0.05 Interaction: F=4.06, p＞0.05 215m edge 215m 60m 1400m edge 1400m 60m 20 40 60 80 100 0 3 6 9 12 15 18 21 24 % intial mass remaining Months Cinnamomum camphora 215m vs. 1400m: F=13.12, p＜0.01 edge vs. 60m: F=23.65, p＜0.01 Interaction: F=3.89, p＞0.05 forest-edge distance (edge and 60m from the forest edge) during 24 months 515 of subtropical China. 516 Cinnamomum camphora 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 20 40 60 80 100 % intial mass remaining Pinus taiwanensis 215m vs. 1400m: F=11.37, p＜0.01 edge vs. 60m: F=18.56, p＜0.05 Interaction: F=4.06, p＞0.05 215m edge 215m 60m 1400m edge 1400m 60m 20 40 60 80 100 0 3 6 9 12 15 18 21 24 % intial mass remaining Months Cinnamomum camphora 215m vs. 1400m: F=13.12, p＜0.01 edge vs. 60m: F=23.65, p＜0.01 Interaction: F=3.89, p＞0.05 forest-edge distance (edge and 60m from the forest edge) during 24 months of decomposition in Lushan Mountain 515 of subtropical China. 516 Cinnamomum camphora 517 518 519 520 521 522 523 524 525 526 527 20 40 60 80 100 % intial mass remaining Pinus taiwanensis 215m vs. 1400m: F=11.37, p＜0.01 edge vs. 60m: F=18.56, p＜0.05 Interaction: F=4.06, p＞0.05 215m edge 215m 60m 1400m edge 1400m 60m 20 40 60 80 100 0 3 6 9 12 15 18 21 24 % intial mass remaining Months Cinnamomum camphora 215m vs. 1400m: F=13.12, p＜0.01 edge vs. 60m: F=23.65, p＜0.01 Interaction: F=3.89, p＞0.05 forest-edge distance (edge and 60m from the forest edge) during 24 mo 515 of subtropical China. Frostegård & Bååth (1996) and Zak et al. (1996) 516 Cinnamomum camphora 517 518 519 520 521 522 523 524 525 526 20 40 60 80 100 % intial mass remaining Pinus taiwanensis 215m vs. 1400m: F=11.37, p＜0.01 edge vs. 60m: F=18.56, p＜0.05 Interaction: F=4.06, p＞0.05 215m edge 215m 60m 1400m edge 1400m 60m 20 40 60 80 100 0 3 6 9 12 15 18 21 24 % intial mass remaining Months Cinnamomum camphora 215m vs. 1400m: F=13.12, p＜0.01 edge vs. 60m: F=23.65, p＜0.01 Interaction: F=3.89, p＞0.05 forest-edge distance (edge and 60m from the forest edge) during 24 months of decomposition in Lushan Mountain 515 Figure 2. Temperature and moisture content of CWD of the two tree species at each altitude (215m and 1400m) and 536 forest-edge distance (edge and 60m from the forest edge) during the 24-month incubation. 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 40 60 80 100 Moisture content (%) Cinnamomum camphora 215m vs. 1400m: F=8.14, p＜0.05 edge vs. 60m: F=6.98, p＜0.05 Interaction: F=3.88, p＞0.05 40 60 80 100 Moisture content (%) Pinus taiwanensis 215m vs. 1400m: F=7.89, p＜0.05 edge vs. 60m: F=6.51, p＜0.05 Interaction: F=3.67, p＞0.05 215m edge 215m 60m 1400m edge 1400m 60m 0.0 10.0 20.0 30.0 40.0 3 6 9 12 15 18 21 24 Temperature (℃) Months Pinus taiwanensis 215m vs. 1400m: F=9.19, p＜0.05 edge vs. 60m: F=7.12, p＞0.05 Interaction: F=4.03, p＞0.05 0.0 10.0 20.0 30.0 40.0 3 6 9 12 15 18 21 24 Temperature (℃) Months Cinnamomum camphora 215m vs. 1400m: F=9.94, p＜0.05 edge vs. 60m: F=7.54, p＞0.05 Interaction: F=4.34, p＞0.05 Figure 2. Temperature and moisture content of CWD of the two tree species at each altitude (215m and 1400m) and 536 forest-edge distance (edge and 60m from the forest edge) during the 24-month incubation. 537 538 539 540 541 542 543 544 545 546 547 40 60 80 100 Moisture content (%) Cinnamomum camphora 215m vs. 1400m: F=8.14, p＜0.05 edge vs. 60m: F=6.98, p＜0.05 Interaction: F=3.88, p＞0.05 40 60 80 100 Moisture content (%) Pinus taiwanensis 215m vs. 1400m: F=7.89, p＜0.05 edge vs. 60m: F=6.51, p＜0.05 Interaction: F=3.67, p＞0.05 215m edge 215m 60m 1400m edge 1400m 60m 0.0 10.0 20.0 30.0 40.0 3 6 9 12 15 18 21 24 Temperature (℃) Months Pinus taiwanensis 215m vs. 1400m: F=9.19, p＜0.05 edge vs. Frostegård & Bååth (1996) and Zak et al. (1996) 60m: F=7.12, p＞0.05 Interaction: F=4.03, p＞0.05 0.0 10.0 20.0 30.0 40.0 3 6 9 12 15 18 21 24 Temperature (℃) Months Cinnamomum camphora 215m vs. 1400m: F=9.94, p＜0.05 edge vs. 60m: F=7.54, p＞0.05 Interaction: F=4.34, p＞0.05 forest-edge distance (edge and 60m from the forest edge) during the 24-month incubation. 537 40 60 80 100 Moisture content (%) Cinnamomum camphora 215m vs. 1400m: F=8.14, p＜0.05 edge vs. 60m: F=6.98, p＜0.05 Interaction: F=3.88, p＞0.05 5 Moisture content (%) Moisture content (% 543 544 545 546 547 0.0 10.0 20.0 30.0 40.0 3 6 9 12 15 18 21 24 Temperature (℃) Months Pinus taiwanensis 215m vs. 1400m: F=9.19, p＜0.05 edge vs. 60m: F=7.12, p＞0.05 Interaction: F=4.03, p＞0.05 0.0 10.0 20.0 30.0 40.0 3 6 9 12 15 18 21 24 Temperature (℃) Months Cinnamomum camphora 215m vs. 1400m: F=9.94, p＜0.05 edge vs. 60m: F=7.54, p＞0.05 Interaction: F=4.34, p＞0.05 548 549 550 551 552 553 554 555 556 557 Figure 3. Soil phospholipid fatty acid (PLFA) (mean ± SE; ng g-1 dry soil) signatures (0-5cm) under CWD of the 558 two tree species at each forest-edge distance (edge and 60m from the forest edge) and altitude (215m and 1400m) in 559 Lushan Mountain of subtropical China. Total, total PLFA concentrations; B, bacterial PLFAs; F, fungal PLFAs; F/B, 560 fungal to bacterial ratio; G+, Gram-positive bacteria; G-, Gram-negative bacteria; G+/G-, ratio of Gram-positive to 561 Gram-negative bacteria; AMF, arbuscular mycorrhizal fungi. 562 563 564 565 566 567 568 569 570 571 572 573 0 200 400 600 PLFA (ng g-1 dry soil) Pinus taiwanensis 215m vs. 1400m: F=18.47, p＜0.001 edge vs. 60m: F=15.32, p＜0.05 Interaction: F=4.22, p＞0.05 215m edge 215m 60m 1400m edge 1400m 60m 0 200 400 600 800 AMF Fungi Total Fungi G+ G- Total B Total PLFA (ng g-1 dry soil) Microbial community Cinnamomum camphora 215m vs. 1400m: F=21.43, p＜0.001 edge vs. 60m: F=17.84, p＜0.01 Interaction: F=3.98, p＞0.05 Gram-negative bacteria; AMF, arbuscular mycorrhizal fungi. 562 563 564 565 566 567 568 569 570 571 572 573 0 200 400 600 PLFA (ng g-1 dry soil) Pinus taiwanensis 215m vs. 1400m: F=18.47, p＜0.001 edge vs. 60m: F=15.32, p＜0.05 Interaction: F=4.22, p＞0.05 215m edge 215m 60m 1400m edge 1400m 60m 0 200 400 600 800 AMF Fungi Total Fungi G+ G- Total B Total PLFA (ng g-1 dry soil) Microbial community Cinnamomum camphora 215m vs. 1400m: F=21.43, p＜0.001 edge vs. Frostegård & Bååth (1996) and Zak et al. (1996) 60m: F=17.84, p＜0.01 Interaction: F=3.98, p＞0.05 to Gram-negative bacteria; AMF, arbuscular mycorrhizal fungi. 583 584 585 586 587 588 589 590 591 592 593 594 0 50 100 150 PLFA (ng g-1 dry wood material) Pinus taiwanensis 215m vs. 1400m: F=23.76, p＜0.001 edge vs. 60m: F=20.23, p＜0.001 Interaction: F=4.59, p＞0.05 215m edge 215m 60m 1400m edge 1400m 60m 0 60 120 180 AMF Fungi Total Fungi G+ G- Total B Total PLFA (ng g-1 dry wood material) Microbial community Cinnamomum camphora 215m vs. 1400m: F=26.74, p＜0.001 edge vs. 60m: F=23.61, p＜0.001 Interaction: F=4.08, p＞0.05 to Gram-negative bacteria; AMF, arbuscular mycorrhizal fungi. 583 584 585 586 587 588 589 590 591 592 593 594 0 50 100 150 PLFA (ng g-1 dry wood material) Pinus taiwanensis 215m vs. 1400m: F=23.76, p＜0.001 edge vs. 60m: F=20.23, p＜0.001 Interaction: F=4.59, p＞0.05 215m edge 215m 60m 1400m edge 1400m 60m 0 60 120 180 AMF Fungi Total Fungi G+ G- Total B Total PLFA (ng g-1 dry wood material) Microbial community Cinnamomum camphora 215m vs. 1400m: F=26.74, p＜0.001 edge vs. 60m: F=23.61, p＜0.001 Interaction: F=4.08, p＞0.05 Figures Figure 1 Figure 1 Patterns of mass loss of two tree species of CWD (fresh wood) at each altitude (215m and 1400m) and forest-edge distance (edge and 60m from the forest edge) during 24 months of decomposition in Lushan Mountain of subtropical China. Patterns of mass loss of two tree species of CWD (fresh wood) at each altitude (215m and 1400m) and forest-edge distance (edge and 60m from the forest edge) during 24 months of decomposition in Lushan Mountain of subtropical China. Figure 2 Temperature and moisture content of CWD of the two tree species at each altitude (215m and 1400m) and forest-edge distance (edge and 60m from the forest edge) during the 24-month incubation. Figure 2 Temperature and moisture content of CWD of the two tree species at each altitude (215m and 1400m) and forest-edge distance (edge and 60m from the forest edge) during the 24-month incubation. Temperature and moisture content of CWD of the two tree species at each altitude (215m and 1400m) and forest-edge distance (edge and 60m from the forest edge) during the 24-month incubation. Temperature and moisture content of CWD of the two tree species at each altitude (215m and 1400m) and forest-edge distance (edge and 60m from the forest edge) during the 24-month incubation. Figure 3 Figure 4 Figure 4 Figure 4 Phospholipid fatty acid (PLFA) (mean ± SE; ng g-1 dry wood material) signatures of the CWD of the two tree species at each forest-edge distance (edge and 60m from the forest edge) and altitude (215m and 1400m) in Lushan Mountain of subtropical China. Total, total PLFA concentrations; B, bacterial PLFAs; fungal PLFAs; F/B, the fungal to bacterial ratio; G+, Gram-positive bacteria; G-, Gram-negative bacteria; G+/G-, ratio of Gram-positive to Gram-negative bacteria; AMF, arbuscular mycorrhizal fungi. Figure 3 Soil phospholipid fatty acid (PLFA) (mean ± SE; ng g-1 dry soil) signatures (0-5cm) under CWD of the two tree species at each forest-edge distance (edge and 60m from the forest edge) and altitude (215m and 1400m) in Lushan Mountain of subtropical China. Total, total PLFA concentrations; B, bacterial PLFAs; F, fungal PLFAs; F/B, fungal to bacterial ratio; G+, Gram-positive bacteria; G-, Gram-negative bacteria; G+/G-, ratio of Gram-positive to Gram-negative bacteria; AMF, arbuscular mycorrhizal fungi. Figure 4 Phospholipid fatty acid (PLFA) (mean ± SE; ng g-1 dry wood material) signatures of the CWD of the two tree species at each forest-edge distance (edge and 60m from the forest edge) and altitude (215m and 1400m) in Lushan Mountain of subtropical China. Total, total PLFA concentrations; B, bacterial PLFAs; F, fungal PLFAs; F/B, the fungal to bacterial ratio; G+, Gram-positive bacteria; G-, Gram-negative bacteria; G+/G-, ratio of Gram-positive to Gram-negative bacteria; AMF, arbuscular mycorrhizal fungi.
https://openalex.org/W2171262411	https://research-information.bris.ac.uk/ws/files/48620822/Harm_is_all_you_need.pdf	English	null	Harm is all you need? Best interests and disputes about parental decision-making	Journal of medical ethics	2,015	cc-by	6,110	Birchley, G. (2016). Harm is all you need? Best interests and disputes about parental decision-making. Journal of Medical Ethics, 42(2), 111- 115. https://doi.org/10.1136/medethics-2015-102893 Birchley, G. (2016). Harm is all you need? Best interests and disputes about parental decision-making. Journal of Medical Ethics, 42(2), 111- 115. https://doi.org/10.1136/medethics-2015-102893 Birchley, G. (2016). Harm is all you need? Best interests and disputes about parental decision-making. Journal of Medical Ethics, 42(2), 111- 115. https://doi.org/10.1136/medethics-2015-102893 Publisher's PDF, also known as Version of record License (if available): CC BY Link to published version (if available): 10.1136/medethics-2015-102893 Link to publication record on the Bristol Research Portal PDF-document This is the final published version of the article (version of record). It first appeared online via BMJ at http://jme.bmj.com/content/42/2/111. Please refer to any applicable terms of use of the publisher. Law ethics and medic JME Online First, published on September 23, 2015 as 10.1136/medethics-2015-102893 Law ethics and medi First, published on September 23, 2015 as 10.1136/medethics-2015-102893 PAPER Giles Birchley more understandable and strike the correct balance between parental liberty and children’s interests? English law already contains a harm threshold, perhaps overlooked by bioethicists because it is rarely used in determinations of medical best inter- ests. The experiences of the English courts in applying the harm threshold suggest that it is con- sidered signiﬁcantly more evaluative than, and suffers from similar levels of indeterminacy to, the best interests test. This paper therefore argues that mediating best interests with a harm threshold is ill judged, and efforts should instead be spent on reducing indeterminacy by specifying the values that should guide best interests decisions. Correspondence to Giles Birchley, Centre for Ethics in Medicine, University of Bristol, Canygne Hall, 39 Whatley Road, Clifton, Bristol BS8 2PS, UK; giles.birchley@bristol.ac.uk Received 12 May 2015 Revised 28 July 2015 Accepted 31 August 2015 ABSTRACT Correspondence to Giles Birchley, Centre for Ethics in Medicine, University of Bristol, Canygne Hall, 39 Whatley Road, Clifton, Bristol BS8 2PS, UK; giles.birchley@bristol.ac.uk ABSTRACT A growing number of bioethics papers endorse the harm threshold when judging whether to override parental decisions. Among other claims, these papers argue that the harm threshold is easily understood by lay and professional audiences and correctly conforms to societal expectations of parents in regard to their children. English law contains a harm threshold which mediates the use of the best interests test in cases where a child may be removed from her parents. Using Diekema’s seminal paper as an example, this paper explores the proposed workings of the harm threshold. I use examples from the practical use of the harm threshold in English law to argue that the harm threshold is an inadequate answer to the indeterminacy of the best interests test. I detail two criticisms: First, the harm standard has evaluative overtones and judges are loath to employ it where parental behaviour is misguided but they wish to treat parents sympathetically. Thus, by focusing only on ‘substandard’ parenting, harm is problematic where the parental attempts to beneﬁt their child are misguided or wrong, such as in disputes about withdrawal of medical treatment. Second, when harm is used in genuine dilemmas, court judgments offer different answers to similar cases. This level of indeterminacy suggests that, in practice, the operation of the harm threshold would be indistinguishable from best interests. Since indeterminacy appears to be the greatest problem in elucidating what is best, bioethicists should concentrate on discovering the values that inform best interests. Received 12 May 2015 Revised 28 July 2015 Accepted 31 August 2015 THE HARM CONSENSUS M To cite: Birchley G. J Med Ethics Published Online First: [please include Day Month Year] doi:10.1136/ medethics-2015-102893 Many commentators suggest that best interests is an opaque test1 4 6 7 that, taken literally, places unfair demands upon parents who will face the paradox of simultaneously trying to maximise beneﬁts to all of their children while somehow maintaining their own well-being.2 6–8 In the face of these apparent problems with the best interests test, these com- mentators argue that a harm threshold should mediate (or perhaps replace) best interests.3–7 9 Calls to replace the best interests test5 are ambigu- ous because it is not clear if it is meant to apply only to speciﬁc uses of the test. To keep things simple, I will concentrate on claims that the harm standard should mediate best interests, in other words a harm threshold should govern which cases we consider appropriate to be decided by the best interests test. Many commentators suggest that best interests is an opaque test1 4 6 7 that, taken literally, places unfair demands upon parents who will face the paradox of simultaneously trying to maximise beneﬁts to all of their children while somehow maintaining their own well-being.2 6–8 In the face of these apparent problems with the best interests test, these com- mentators argue that a harm threshold should mediate (or perhaps replace) best interests.3–7 9 5 To cite: Birchley G. J Med Ethics Published Online First: [please include Day Month Year] doi:10.1136/ medethics-2015-102893 University of Bristol – Bristol Research Portal General rights This document is made available in accordance with publisher policies. Please cite only the published version using the reference above. Full terms of use are available: http://www.bristol.ac.uk/red/research-policy/pure/user-guides/brp-terms/ Copyright Article author (or their employer) 2015. Produced by BMJ Publishing Group Ltd under licence. PAPER THE HARM THRESHOLD EXPLAINED Diekema considers that parenting children is a basic liberty right. He identiﬁes the harm threshold ﬁrst with Mill’s13 asser- tion that the only circumstance in which personal liberty may be infringed by a community is to prevent harm to others, and second with Feinberg’s14 argument that such infringement must be effective and the option of last resort. In order to further deﬁne the concept, Diekema links harm to a second concept, basic needs.i While Diekema does not deﬁne these, basic needs are predicated upon Rawls’ natural primary goods, the things ‘a rational man wants whatever else he wants’ (at 79),15 and for children might form a notional list containing the minimum that will ‘enable children to embark … on the process of self- discovery, self-determination and self-fulﬁlment’ (at 42).16 Using Feinberg’s contention that harm is the thwarting of an interest, Diekema argues that the existence of harms in children can be determined by the degree to which children’s basic needs are provided for. Where a parent persistently fails to provide for a child’s basic needs, harm is caused. Should these failures be sig- niﬁcant and serious, intervention is warranted. A second argument advanced by a number of authors4 6 7 9 is that the harm threshold is more readily understood than best interests by legislators, doctors and parents. This claim is prob- lematic for two reasons. First, although harm may appear a readily understandable concept, the ready understandability hides the fact that judgements of harm, especially in complex ethical dilemmas, contain complex value judgments. For example, fatal withdrawal of treatment is a harm of indeﬁnable proportions—perhaps catastrophic, or perhaps not even harm at all. While, for the sake of brevity, I accept without argument (here) that fatal withdrawal of treatment is sometimes justiﬁably in a patient’s interests,20 such situations do not always sit well with the concept of harm as understood in the vernacular. INTRODUCTION Although accepting that best interests is the best guide for decision-making on behalf of a child, Diekema argues that the harm threshold is superior to best interests when judging whether intervention is justiﬁed against parental refusals of medical treat- ment.4 9 Elliston6 argues that intervention against parental decisions should only be made on the basis of harm, and only then if the choice that leads to those harms is unreasonable. Others suggest more speciﬁc uses for the harm threshold. Shah7 proposes a ‘secure child standard’ should govern parental decisions to enter children into research. Under this standard the courts should ‘defer to parental decision-making unless the child is exposed to some unjustiﬁed risk of signiﬁcant harm’ (at 179). de Vos et al3 claim that parental requests for withdrawal of life-sustaining treatment should be allowed if they do not increase the risk of preventable harm. Gillam10 argues that harm indicates the scope of parental decision-making in situations of genuine dilemma. Indeed, a recent Where medical decisions must be made for infants, English law directs decision-makers—primarily parents and doctors—to follow the best interests test. Yet judgments of best interests have long been criticised for their indeterminacy since they can be informed by a variety of values.1 Many bioethicists have also argued that parents, who must juggle their own needs, and potentially those of siblings as well, with those of the child, are poorly directed by the literal demand that they do what is best for the child.2 Of the many critiques that have arisen from such analysis is the claim that the best interests test should be mediated3 4 (or perhaps replaced)5 with a harm threshold for overriding parental decisions. These critics maintain that a harm threshold more adequately reﬂects the wide range of discretion parents are customarily allowed in their behaviours towards their child, and guides professionals by indicating that only harmful parental actions justify intervention. But would a harm threshold really be Birchley G. J Med Ethics 2015;0:1–5. doi:10.1136/medethics-2015-102893 Birchley G. J Med Ethics 2015;0:1–5. doi:10.1136/medethics-2015-102893 Birchley G. J Med Ethics 2015;0:1–5. doi:10.1136/medethics-2015-102893 Law, ethics and medicine serious harm. Discussing the child’s ‘best interest’ fails to focus on the relevant standard for determining when state action is jus- tiﬁed. The harm standard focuses discussion in the proper place.4 review11 suggests that there is widespread consensus among bioethicists that the harm threshold should determine the per- missibility of overriding parental wishes. THE HARM THRESHOLD CRITICISED AND DEFENDED THE HARM THRESHOLD CRITICISED AND DEFENDED Diekema’s contention that parental authority is a liberty right akin to any other exercise of autonomy is vulnerable to critique. The notion that personal liberty extends to parental autonomy —quite possibly a misuse of Mill’s doctrineiii—conﬂicts with claims that children have rights on their own account. Such rights are often argued to extend beyond simple claims to phys- ical integrity to at least leave some space for presumptive self- determination,16 17 most cogently seen in Feinberg’s18 argument that children have a right to an open future.iv On such a basis Hester19 argues bioethicists should go further than simply pre- venting harm and specify some positive interests of children which we are obliged to protect. While he does not develop the argument, Diekema’s speciﬁcation of harm as damage to basic needs does not prevent us from making some positive speciﬁca- tion of harms, which might deﬂect this critique somewhat. INTRODUCTION I dispute the usefulness of these conclusions. While avoiding harm has been convin- cingly claimed to be the single moral principle likely to be common to all cultures and philosophies,12 this is only likely because harm can be understood in numerous ways, and these will not always be consistent with one another. The use of the harm threshold in English law exempliﬁes this, and I shall discuss this in due course. Yet I suggest that Diekema, and others who have adopted his arguments, make a fundamental mistake. The challenge of inde- terminacy is the central problem of the best interests test, and merely switching terminologies from best interests to harm does nothing to address this in any of the circumstances it has been proposed. Indeed, for reasons I explain below, the change in ter- minology is also unhelpful. No critique of the harm threshold can ignore Diekema’s4 seminal paper, which remains the most considered and widely referenced discussion of the issue. While I concentrate upon Diekema’s analysis, my focus here is on developing a general cri- tique of insufﬁciency of the harm threshold for overriding par- ental decisions. iLists of basic needs are rarely speciﬁed, and Rawls speciﬁed only an incomplete list which included health and vigour, intelligence and imagination. Basic needs (or their inverse, basic interests) may be criticised separately, although this is not the focus of this paper. iiThereby anticipating criticisms from those who feel that harm is too anaemic or plastic a concept. iiiSince Mill argued the state might place very great restrictions upon parenting and complains: “it is in the case of children, that misapplied notions of liberty are a real obstacle to the fulﬁlment by the State of its duties. One would think that a man’s children were supposed to be literally, rather than metaphorically, a part of himself, so jealous is opinion of the smallest interference of law with his absolute and exclusive control over them; more jealous than of almost any interference with his own freedom of action: so much less do the generality of mankind value liberty than power” (at 88–89).13 ivEven those who dispute claims that children have rights tend to accept that children have interests. viAlthough my focus is on English law, my understanding is that other common law jurisdictions such as the USA and Australia use the harm threshold similarly in cases of child abuse or neglect. viiThe Children Act 1989 section 1 (3–4) speciﬁes a harm threshold is to be used residence and contact orders, and care and supervision orders, respectively known also as section 8 orders and part 4 orders. The circumstances of which these orders may be sought is elaborated at section 31(2). viiiIndeed Elliston explicitly favours the extension of this model.6 ixA number of commentators argue that they do so implicitly, however I do not think this negates my argument, since I acknowledge the indeterminacy of best interests test is a problem.22 xWhether the care is indeed substandard depends upon one’s metaethical (and jurisprudential) stance. THE HARM THRESHOLD IN ENGLISH LAW English law uses a harm threshold in cases where a child may be removed from its parents.vii Only if a child is being harmed or is at risk of harm will the court decide what home serves the child’s best interests, removing the implication that the law will seek better-than-adequate parents for a child.21 Thus the law has similarities to the harm threshold envisaged by some commentators for medical decision-making,viii and examining how the threshold operates in the courts highlights two potential problems. First, as I shall detail in a moment it seems arguable that judges see evaluative overtones in a con- clusion that a parent is harming their child, which they do not (explicitly)ix read into best interests. This indicates more general difﬁculties with a harm threshold when a decision to override parental consent appears necessary on grounds other than substandard care. This non-pejorative approach is gener- ally taken because there is sympathy towards the parents’ motivation—for instance in disputes over the withdrawal or withholding of treatment or disputes motivated by the parents’ religious beliefs—and the label of substandard care appears insensitive to pluralism.x Second, as we shall see shortly, when the harm threshold is used to address genuine dilemmas, judgments of harm become as indeterminate as judgments of best interests. xiConsider refusal of consent to blood in, for example, Birmingham Children’s NHS Trust v B & C (2014) EWHC 531 (Fam); M Children’s Hospital NHS Foundation Trust v Mr & Mrs Y (2014) EWHC 2651 (Fam) NHS v Child B, Mr and Mrs B (2014) EWHC 3486 (Fam); Refusal of consent for HIV treatment represents a rare case that attracts the opposite approach e.g. Re JA (A Minor) (2014) EWHC 1135 (Fam). It could be convincingly argued that the approach here was due to the facts of the case. xiiConsider, for example, Re H-B (2015) EWCA Civ 389 xiiiThe persistent ambiguity of corporal punishment in English law is another ﬁne example, although since it is usually outside the ambit of the harm threshold (being instead governed by the Children Act 2004, s58(3) and case law, most notably R v H (2001) 2 FLR 431) it would be digressive to include it in the main discussion. xivCompare Re D (Care: Threshold Criteria) (1998) Fam Law 656 and A Local Authority v N (2005) EWHC 2956 (Fam). xiConsider refusal of consent to blood in, for example, Birmingham Children’s NHS Trust v B & C (2014) EWHC 531 (Fam); M Children’s Hospital NHS Foundation Trust v Mr & Mrs Y (2014) EWHC 2651 (Fam) NHS v Child B, Mr and Mrs B (2014) EWHC 3486 (Fam); Refusal of consent for HIV treatment represents a rare case that attracts the opposite approach e.g. Re JA (A Minor) (2014) EWHC 1135 (Fam). It could be convincingly argued that the approach here was due to the facts of the case. THE HARM THRESHOLD EXPLAINED This muddles the claim that harm is the correct threshold because it is simple enough to be generally understood and suggests harm is at least as problematic as best interests in this regard.v Diekema acknowledges that the non-self-evident nature of harm, even when coupled with terms like ‘signiﬁcant’ and ‘serious’, means that the harm principle suffers from problems of indeterminacy.ii Diekema counterargues that a threshold based upon harm better ﬁts the point where intervention against parental wishes is justiﬁed in practice and is therefore less likely to cause confusion in clinicians than the obfuscatory language of best interests. Dresser,5 arguing along similar lines, makes this point succinctly when she argues that it is difﬁcult to conceive that male circumcision for religious purposes serves the child’s best interests, yet the purportedly harmless nature of male cir- cumcision leads to its widespread acceptance in many cultures. While harm is nevertheless indeterminate, it is only fair to say that Diekema does not appear overly concerned with indeter- minacy, for he states (at 253): This indicates a second, related, problem: harm may not capture all the considerations we need to capture to make a decision. This is problematic because if harm is the only concept on which attention can justiﬁably be focused, this invites any otherwise justiﬁable claims against interests which are not readily labelled as harms to nevertheless be labelled as harms. Such labelling inevitably transforms the harm threshold The biggest problem with a best interest standard is not its sub- jectivity, but that it represents the wrong standard. State interven- tion is not justiﬁed because a decision is contrary to the child’s best interest, but because it places the child at signiﬁcant risk of iLists of basic needs are rarely speciﬁed, and Rawls speciﬁed only an incomplete list which included health and vigour, intelligence and imagination. Basic needs (or their inverse, basic interests) may be criticised separately, although this is not the focus of this paper. iiThereby anticipating criticisms from those who feel that harm is too anaemic or plastic a concept. vAlbeit we have little evidence of how best interests are understood in this context. Birchley G. J Med Ethics 2015;0:1–5. THE HARM THRESHOLD EXPLAINED doi:10.1136/medethics-2015-102893 2 Law, ethics and medicine as harm in contemporary cases.xi While I acknowledge a variety of explanations for this approach—for instance application of the harm threshold is demanded by statute only in cases where the courts consider applications for care and supervision or resi- dence and contact orders—an approach favouring the use of best interests under the inherent jurisdiction of the court has arguably arisen because of reservations about using the harm standard where the courts perceive a legitimate plurality of views. into a complex test when it is used in the wide variety of situa- tions wherein the best interest test is currently employed. While I concede that, unembellished, claims of self-evident physical harm might provide one plausible standard to judge overriding a parental consent and refusal in some contexts (such as vaccin- ation, intrasibling tissue donation and paediatric research)— albeit one that remains contestable on the basis of the non-self-evidence of parental rights—any appeal to clarity is lost once harm is the only gateway to the best interests test. Indeed, because English law contains a harm threshold, we have exam- ples of these problems at ﬁrst hand.vi as harm in contemporary cases.xi While I acknowledge a variety of explanations for this approach—for instance application of the harm threshold is demanded by statute only in cases where the courts consider applications for care and supervision or resi- dence and contact orders—an approach favouring the use of best interests under the inherent jurisdiction of the court has arguably arisen because of reservations about using the harm standard where the courts perceive a legitimate plurality of views. If the aim is to respect parental privacy in parenting, it is of course sustainable to say that the pejorative connotations of the language used are moot since interference on any basis is prob- lematic for the parents concerned. Yet the family courts regu- larly perceive that, as well as the parents concerned, they address standards of parenting in general,xii and in this context concerns about language may gain more traction. It might nevertheless be argued that the lack of transparency in the best interests test make it a more problematic way to offer this more general guidance to parents. Yet this assumes that guidance on the basis of harms is more transparent, a claim I shall now dispute. , p , ( ) xiiiThe persistent ambiguity of corporal punishment in English law is another ﬁne example, although since it is usually outside the ambit of the harm threshold (being instead governed by the Children Act 2004, s58(3) and case law, most notably R v H (2001) 2 FLR 431) it would be digressive to include it in the main discussion. i xivCompare Re D (Care: Threshold Criteria) (1998) Fam Law 656 and A Local Authority v N (2005) EWHC 2956 (Fam). xiiConsider, for example, Re H-B (2015) EWCA Civ 389 THE INDETERMINACY OF HARMS h d f h doi:10.1136/medethics-2015-102893 3 Law, ethics and medicine to the UK. None of the women were DNA matches for their child, although all were nevertheless convinced the child was theirs. In every case their physical and emotional care of the child was thought to be exemplary. Despite these strong similar- ities, the courts reached differing conclusions about whether the harm threshold had been reached. Thus in Haringey Local Authority v C,23 while Ryder J believed that the ‘mother’ had been the victim of a cruel deception, he ruled the child was at risk of harms consisting of growing up with false beliefs that he was a miraculous birth, potentially experiencing devastating consequences if he discovered his true origin and of facing pos- sible difﬁculties due to the lack of family medical history. The child was permanently removed from the ‘mother’ on this basis. While there is no public report on the outcome of Re E,24 the judge was unimpressed with the ‘mother’ as a witness, and her comments suggest a similar conclusion regarding harms was drawn.xv Conversely, in Re D,25 the judge considered both parents to have been the victims of an elaborate scam. The case attracted signiﬁcant attention and the media reported the couple had been allowed to keep the baby.27 28 Most recently, a media report following Re A26 states the putative parents were allowed to keep the child,29 the judge in this case concluding that, although the child had been harmed, the harm was not inﬂicted by the couple. complex ethical judgements. Once harm is invested with com- plexity, unless the complex rules it follows are explained, it becomes open to charges of indeterminacy. All we have done is rename the best interests test while dealing with none of its fail- ings. Moreover, because harm is implicitly evaluative, it is prob- lematic when there is genuine cause to intervene in parental behaviour, but no cause for public condemnation. In fact, I suggest that in these situations the technical language of best interests is actually helpful, precisely because it can house complex concepts without attaching condemnation. The trigger and scope for intervention against parental wishes does of course vary depending upon the situation encountered. But it would be a mistake to view harm as the only consider- ation we should pay heed to in such cases. THE INDETERMINACY OF HARMS h d f h The indeterminate nature of harms raises a second problem that challenges claims that the harm threshold is more readily under- standable than best interests. The deﬁnition of harm at section 31(9) of the Children Act 1989 as ‘ill-treatment or the impair- ment of health or development’ might appear clear and unam- biguous. Yet, given that such a deﬁnition will include a wide range of circumstances, juridical use of the harm threshold has suffered from indeterminacy:xiii for example, the question of whether a child’s cultural background should affect an assess- ment of the seriousness of harm has been answered negatively and positively in different cases.xiv A stark demonstration of the indeterminacy of harms is in a series of reports of baby trafﬁcking cases Haringey Local Authority v C,23 Re E,24 Re D25 and Re A.26 These cases share a series of key features: all concern childless couples of African origin living in the UK who attend charismatic Christian churches. Conventional fertility treatments had failed these couples and all had sought unconventional treatments at clinics in Africa. Most admitted paying large sums of money for these treatments, after which many of the women had experienced phantom pregnancy (pseudocyesis). All had contacted their family doctors in the UK in the belief they were pregnant, and, if examined (one was not), were found not to be. All had under- gone labour in African clinics, and were presented with babies who were removed by the state authorities when they returned My ﬁrst claim is that the courts view ‘harm’ as implicitly a more evaluative term than ‘best interests’. This is not to claim that the best interests test does not involve evaluation, but instead to argue that, as a legal term of art, a judgement that an action is against a child’s best interests may appear less pejora- tive than a judgement that an action is harmful. The way the courts use harm to narrowly focus upon (defective) parental care, and reserve best interests to consider a much wider range of issues than the harm threshold gives reasons for thinking this is the case. An indication of this is the general avoidance of the term ‘harm’ in English legal cases where the courts do not wish to indicate that parental care is substandard. For example, religious refusal to consent to life-saving blood products is not described Birchley G. J Med Ethics 2015;0:1–5. Almost 30 years ago Moreno30 observed: in the case of non-competent patients—those who never have been in a position to develop relevant preferences such as young children […] some best interests test, founded on the ethical prin- ciple of beneﬁcence, seems required. But, in itself, the notion of best interests is vacuous, and so further principles must be invoked in its interpretation. xvParker J (at paragraph 3) states the harm threshold is ‘amply made out’ despite not forming part of the (fact ﬁnding) ruling. THE INDETERMINACY OF HARMS h d f h Issues such as refusal of vaccination, or the place of parental consent for children’s entry into clinical trials raise issues of communal as well as child-centred responsibilities. Issues which balance one child’s interests against another require a broad consideration of bene- ﬁts to one party as well as harms to the other. Harm can be a valuable intuition pump that can lead us to consider important factors pertaining to a child’s best interests. However, absent some list of other values, judging best interests and harms is inevitably subjective. The best response to this indeterminacy is not to attempt to rename the best interests test, but to identify the values informing best interests. It is likely that rather than a single monolithic value, there are many such values that apply more or less to different situations. Although identifying such values is therefore beyond the scope of this paper, examples abound. For instance, it is clear that many bioethicists, including many advocates of the harm threshold, consider that the child’s interests can be subordinate to the interests of their parents.4 6 7 9 Other theorists favour a broadly collectivist approach that requires a restriction of parental capacity to beneﬁt as well as to disbeneﬁt.17 32 To publicise such positions in a decision about best interests allows transparency and offers grounds for challenge. To rehearse and debate the validity of such positions is genuinely informative of assessments of best interests in a way that adopting a harm threshold is not. Common law is open to interpretation, and I shall not argue that the judges in one case or another erred in their application of the harm threshold. Instead I claim that, wherever it is employed, the harm threshold can readily be used to reach opposing conclusions in very similar circumstances. This indi- cates the harm threshold suffers problems of indeterminacy of similar magnitude to the best interests test. For this reason, replacing or mediating the best interests test with a harm thresh- old is misguided; the real issue that bioethicists should focus upon is reducing indeterminacy. REDUCING INDETERMINACY 30 Almost 30 years ago Moreno30 observed: Almost 30 years ago Moreno30 observed: REFERENCES 22 Huxtable R. Autonomy, best interests and the public interest: treatment, non-treatment and the values of medical law. Med Law Rev 2014;22:459 1 Mnookin RH. Child-Custody Adjudication: Judicial Functions in the Face of Indeterminacy. Law Contemp Probl 1975;39:226–93. 1 Mnookin RH. Child-Custody Adjudication: Judicial Functions in the Face of Indeterminacy. Law Contemp Probl 1975;39:226–93. 23 Haringey Local Authority v C [2004] EWHC 2580 (Fam). Indeterminacy. Law Contemp Probl 1975;39:226–93. 24 Re E (a child) (fact-ﬁding hearing: assessment of biological parents) [2011] EWHC 3453 (Fam). 2 Ross LF. Children, families, and health care decision making. Oxford: Oxford University Press, 1998. 24 Re E (a child) (fact-ﬁding hearing: assessment of biological parents) [2011] EWHC 3453 (Fam). 2 Ross LF. Children, families, and health care decision making. Oxford: Oxford University Press, 1998. 25 Re D (A Child) [2012] EWHC 4231. 3 de Vos MA, Seeber AA, Gevers SK, et al. Parents who wish no further treatment for their child. J Med Ethics 2015;41:195–200. 26 Re A (a Child)(fact ﬁnding hearing: biological parents) [2014] EWHC 75 (Fam. ; 4 Diekema DS. Parental refusals of medical treatment: the harm principle as threshold for state intervention. Theor Med Bioeth 2004;25:243–64. 27 Crawford C. Couple can keep Nigerian baby after ‘scam’ ruling. BBC News 18 October 2012. http://www.bbc.co.uk/news/uk-19996987 (accessed 1 May 2015). 5 Dresser R. Standards for family decisions: replacing best interests with harm prevention. Am J Bioeth 2003;3:54–5. 28 Press Association. Nigeria Baby-Selling Scams Attracting ‘Desperate’ Childless British Couples. Hufﬁngton Post 25th October 2012. http://www.hufﬁngtonpost.co.uk/ 2012/10/25/nigeria-baby-selling-scams_n_2016024.html (accessed 15 Apr 2015). 6 Elliston S. The best interests of the child in healthcare. London: Routledge Cavendish, 2007. 29 Press Association. Woman fooled into believing she had given birth allowed to keep child. Daily Telegraph 1st September 2014. http://www.telegraph.co.uk/news/ uknews/law-and-order/11068833/Woman-fooled-into-believing-she-had-given-birth- allowed-to-keep-child.html. (accessed 15 Apr 2015). 7 Shah SK. Does research with children violate the best interests standard? An empirical and conceptual analysis. Northwest J Law Soc Policy 2013;8:121–73. p p y y 8 Buchanan A, Brock D. Deciding for others. Cambridge: Cambridge University Press, 1990. 30 Moreno JD. Ethical and legal issues in the care of the impaired newborn. Clin Perinatol 1987;14:345–60 at 356. 9 Diekema DS. Revisiting the best interest standard: uses and misuses. J Clin Ethics 2011;22:128–33. 31 DeGrazia D. Value theory and the best interests standard. Bioethics 1995;9:50–61. 31 DeGrazia D. Value theory and the best interests standard. Bioethics 1995;9:50–61. 10 Gillam L. Provenance and peer review Not commissioned; externally peer reviewed. 18 Feinberg J. The child’s right to an open future. In: Aitken W, LaFollette H, eds. Whose child? Children’s rights, parental authority, and state power. Totowa, NJ: Rowman and Littleﬁeld, 1980:124–53. venance and peer review Not commissioned; externally peer revie Provenance and peer review Not commissioned; externally peer reviewed. Open Access This is an Open Access article distributed in accordance with the terms of the Creative Commons Attribution (CC BY 4.0) license, which permits others to distribute, remix, adapt and build upon this work, for commercial use, provided the original work is properly cited. See: http://creativecommons.org/ licenses/by/4.0/ 19 Hester DM. Interests and neonates: there is more to the story than we explicitly acknowledge. Theor Med Bioeth 2007;28:357–72. 19 Hester DM. Interests and neonates: there is more to the story than we explicitly acknowledge. Theor Med Bioeth 2007;28:357–72. g 20 Larcher V, Craig F, Bhogal K, et al. Making decisions to limit treatment in life-limiting and life-threatening conditions in children: a framework for practice. Arch Dis Child 2015;100(Suppl 2):s1–s23. 20 Larcher V, Craig F, Bhogal K, et al. Making decisions to limit treatment in life-limiting and life-threatening conditions in children: a framework for practice. Arch Dis Child 2015;100(Suppl 2):s1–s23. pp 21 Bainham A. Children: the modern law. 3rd edn. Bristol: Jordan Publishing, 2005. pp 21 Bainham A. Children: the modern law. 3rd edn. Bristol: Jordan Publishing, 2 Competing interests None declared. y 17 Archard D. Children: rights and childhood. 2nd edn. London: Routledge, 2004. CONCLUSION M Many commentators advocate a harm threshold to mediate the best interests test because they claim it is more determinate than the best interests test, and more clearly reﬂects the freedoms of parents to decide how and when to beneﬁt their children. Diekema,4 the harm threshold’s most convincing advocate, acknowledges that harm has similar problems with indetermin- acy to best interests, but also argues that a harm threshold is better understood by clinicians than best interests, an argument that others have extended to the judiciary and to parents themselves. Rather than specify some of these principles, advocates of the harm threshold seek to simplify the best interests test. Diekema’s4 tying of the threshold to the concept of basic needs is alert to this problem, since an ‘objective’ list of such welfare needs would answer this criticism—although reaching agree- ment on the speciﬁcs of such a list is problematic.31 However, harm does not describe all the parental behaviours that merit state intervention, and Diekema errs in suggesting that it does. ‘Harm’ is a catch-all term with which few could disagree. It cap- tures the wrongness of hitting a child around the head with a saucepan or abusing a child for pleasure. Yet these situations do not represent genuine dilemmas when we would need the guid- ance of a harm threshold, since almost nobody would argue that such actions were permissible. It is in situations of genuine ethical complexity that a focus on harm becomes inadequate, and, if used in such situations, harm merely frames more While recognising the potential for a harm threshold to have uses as an intuition pump or a cross-cultural standard, this paper argues that where a harm threshold is used to determine intervention, it is inferior to the best interests test. I have used the way the harm threshold operates in English law to demon- strate it is insufﬁciently broad in its scope to be helpful in genu- inely troubling cases without carrying with it signiﬁcant elements of indeterminacy. Such complexity invalidates claims that the harm threshold is readily understandable in all cases. Furthermore, the language of harm is strongly evaluative, ren- dering it less suitable than the technicolegal language of best interests when intervening against well-intentioned parental behaviours without wishing to condemn such behaviours. Birchley G. J Med Ethics 2015;0:1–5. doi:10.1136/medethics-2015-102893 4 Law, ethics and medicine Law, ethics and medicine None of this mitigates the indeterminacy of the best interests test, and this paper has contended that this is the greatest chal- lenge to deciding children’s interests. Recourse to a harm threshold offers nothing to this project, and the focus of bioethicists should be upon identifying and mapping the values that (should) inform best interests decisions. 11 McDougall RJ, Notini L. Overriding parents’ medical decisions for their children: a systematic review of normative literature. J Med Ethics 2014;40:448–52. y 12 DeGrazia D. Common morality, coherence, and the principles of biomedical ethics. Kennedy Inst Ethics J 2003;13:219–30. y 13 Mill JS. On liberty. New York: Dover Publications, 2002 [1859]. 14 Feinberg J. The moral limits of the criminal law volume 1: harm to others. Oxford: Oxford University Press, 1987. y 15 Rawls J. A theory of Justice. Revised edn. Cambridge: Harvard University Press, 1999. Funding Wellcome Trust (WT097725MF). 16 Miller RB. Children, ethics and modern medicine. Bloomington, Indiana: Indiana University Press, 2003. Competing interests None declared. REFERENCES Childrens bioethics and the zone of parental discretion. Monash Bioeth Rev 2010;29:09.1–3. 32 Blustein J. Doing the best for one’s child: satisﬁcing versus optimizing parentalism. Theor Med Bioeth 2012;33:199–205. 32 Blustein J. Doing the best for one’s child Theor Med Bioeth 2012;33:199–205. 32 Blustein J. Doing the best for one’s child Theor Med Bioeth 2012;33:199–205. Birchley G. J Med Ethics 2015;0:1–5. doi:10.1136/medethics-2015-102893 5
https://openalex.org/W3215855715	https://eprints.whiterose.ac.uk/181687/12/keab862.pdf	English	null	Ultrasound subclinical synovitis in anti-CCP-positive at-risk individuals with musculoskeletal symptoms: an important and predictable stage in the rheumatoid arthritis continuum	Rheumatology	2,021	cc-by	6,354	Original article Rheumatology Rheumatology Rheumatology Original article Rheumatology Abstract Objectives. To investigate whether anti-CCP2-positive at-risk individuals with musculoskeletal (MSK) symptoms but without clinical synovitis (CCP2þ at-risk) develop US subclinical synovitis before inﬂammatory arthritis and if US subclinical synovitis can be predicted. Methods. First, US scans of CCP2þ at-risk individuals who developed inﬂammatory arthritis (‘progressors’) were reviewed for subclinical synovitis prior to inﬂammatory arthritis development. Patients in whom the pre-progression US scan was negative but the scan was conducted >6 months before progression were excluded. Subsequently, regression analyses were performed to identify predictors of US synovitis in CCP2þ at-risk individuals without base- line US abnormalities who had one or more longitudinal US scan and a complete dataset. g p Results. US subclinical synovitis was detected in one or more scan in 75 of 97 progressors (77.3%) fmedian time to inﬂammatory arthritis development from ﬁrst evidence of US synovitis 26.5 weeks [interquartile range (IQR) 7–60]g, in whom one or more scan was available, excluding those with a negative scan >6 months from inﬂammatory arthritis development (n ¼ 38). In 220 CCP2þ at-risk individuals with normal baseline US scans, who had one or more longitu- dinal US scan and a complete dataset, US synovitis was detected in 69/220 (31.4%) [median time to ﬁrst developing US synovitis 56.4weeks (IQR 33.0–112.0)]. In the multivariable analysis, only anti-CCP3 antibodies were predictive for the development of US synovitis [odds ratio 4.75 (95% CI 1.97, 11.46); P < 0.01]. CLINICAL SCIENCE Conclusions. In anti-CCP2þ at-risk individuals, a stage of subclinical synovitis usually precedes the development of inﬂammatory arthritis. Anti-CCP2þ/CCP3þ individuals without clinical or US subclinical synovitis may represent the optimal window of opportunity for intervention to prevent joint disease. Key words: ACPA, ultrasound, at-risk, third-generation anti-CCP antibodies, anti-CCP3, subclinical synovitis, prediction, rheumatoid arthritis, inﬂammatory arthritis Ultrasound subclinical synovitis in anti-CCP-positive at-risk individuals with musculoskeletal symptoms: an important and predictable stage in the rheumatoid arthritis continuum Downloaded from https://academic.oup.com/rheumatology/article/61/8/3192/6438028 by guest on 20 December 2022 Andrea Di Matteo 1,2,3, Laurence Duquenne1,2, Edoardo Cipolletta 3, Jacqueline L. Nam1,2, Leticia Garcia-Montoya 1,2, Richard J. Wakefield1,2, Michael Mahler 4, Kulveer Mankia 1,2,* and Paul Emery 1,2,* Andrea Di Matteo 1,2,3, Laurence Duquenne1,2, Edoardo Cipolletta 3, Jacqueline L. Nam1,2, Leticia Garcia-Montoya 1,2, Richard J. Wakefield1,2, Michael Mahler 4, Kulveer Mankia 1,2,* and Paul Emery 1,2,* Rheumatology 2022;61:3192–3200 https://doi.org/10.1093/rheumatology/keab862 Advance Access publication 24 November 2021 Rheumatology 2022;61:3192–3200 https://doi.org/10.1093/rheumatology/keab862 Advance Access publication 24 November 2021 Introduction The identification of those individuals who are at immi- nent risk, ideally in the window before the occurrence of any joint involvement (i.e. before the ‘second hit’ of RA occurs), is of utmost importance for risk stratification and consequently for disease prevention. However, this is challenging to achieve and therefore biomarkers that can identify those individuals who will develop joint dis- ease would be of great value. The concept of early RA has recently evolved. It is now considered a ‘disease continuum’ rather than a fixed phenotype, in which individuals with risk factors pro- gress through different stages before the development of clinical arthritis [1–3]. In 2012, the EULAR Standing Committee on Investigative Rheumatology defined six categories along the preclinical ‘RA continuum’: genetic (phase A) and envir- onmental (phase B) risk factors for RA, RA-related system- ic autoimmunity (phase C), musculoskeletal (MSK) symptoms without clinical arthritis (phase D), undifferenti- ated arthritis (phase E) and RA (phase F) [4] (Fig. 1). The aims of this study were 2-fold: to investigate whether US subclinical synovitis represents a distinct stage of the ‘continuum’ in second-generation IgG anti-CCP antibody–positive (CCP2þ) at-risk individu- als prior to the development of clinical inflammatory arthritis and to determine in anti-CCP2þ at-risk individuals with MSK symptoms but before joint in- volvement (neither clinical nor subclinical synovitis), clinical and/or serological predictors of US subclinical synovitis. Downloaded from https://academic.oup.com/rheumatology/article/61/8/3192/6438028 by guest on 20 December 2022 Recently, several studies have highlighted the pres- ence of another important population along this ‘con- tinuum’, which sits between phases D and E. This is represented by at-risk individuals (i.e. those with sys- temic autoimmunity and MSK symptoms but without clinical arthritis) who have subclinical joint inflammation on US or MRI [5–7]. These studies have demonstrated that high-resolution imaging is able to detect subclinical joint inflammation in at-risk individuals before clinical synovitis occurs. Indeed, the detection of baseline sub- clinical joint inflammation and/or joint damage on US has been demonstrated to greatly increase the risk of progression to inflammatory arthritis in at-risk individuals [8–11]. When US power Doppler (PD) is detectable in four or more joints, the future development of RA is al- most certain, suggesting this occurs at a late stage in the ‘continuum’ [12]. Rheumatology key messages Rheumatology key messages . The majority of anti-CCP2-positive at-risk individuals go through a stage of subclinical synovitis before the development of inﬂammatory arthritis. . CCP2-positive at-risk individuals can be identiﬁed prior to any joint involvement by the presence of anti-CCP3 antibodies. . This may represent the optimal ‘window of opportunity’ for intervention to prevent joint disease. 1Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, 2National Institute for Health Research, Leeds Biomedical Research Centre, Leeds Teaching Hospitals NHS Trust, Leeds, UK, 3Rheumatology Unit, Department of Clinical and Molecular Sciences, ‘Carlo Urbani’ Hospital, Polytechnic University of Marche,Jesi, Ancona, Italy and 4Research and Development, Werfen Autoimmunity, San Diego, CA, USA Submitted 28 July 2021; accepted 11 November 2021 Correspondence to: Paul Emery, Leeds Institute of Rheumatic and Musculoskeletal Medicine, Chapel Allerton Hospital, Leeds LS7 4SA, UK. E-mail: p.emery@leeds.ac.uk K.M. and P.E. jointly supervised this work. . This may represent the optimal ‘window of opportunity’ for intervention to prevent joint disease. 1Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, 2National Institute for Health Research, Leeds Biomedical Research Centre, Leeds Teaching Hospitals NHS Trust, Leeds, UK, 3Rheumatology Unit, Department of Clinical and Molecular Sciences, ‘Carlo Urbani’ Hospital, Polytechnic University of Marche,Jesi, Ancona, Italy and 4Research and Development, Werfen Autoimmunity, San Diego, CA, USA Correspondence to: Paul Emery, Leeds Institute of Rheumatic and Musculoskeletal Medicine, Chapel Allerton Hospital, Leeds LS7 4SA, UK. E-mail: p.emery@leeds.ac.uk K.M. and P.E. jointly supervised this work. Submitted 28 July 2021; accepted 11 November 2021 V C The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Rheumatology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https: reproduction in any medium, provided the original work is properly cited. US subclinical synovitis in anti-CCPþ at-risk individuals Introduction An additional and important aspect that has emerged is that the occurrence of MSK symp- toms seems to precede, in most at-risk individuals, the development of subclinical joint inflammation and/or joint damage on US [13, 14]. Symptoms in the absence of clinical or subclinical inflammation may represent the critical time point for preventive treatments, particularly for joint disease. om https://academic.oup.com/rheumatology/article/61/8/3192/6438028 by guest on 20 December 2022 Statistical analysis Descriptive statistics were used to describe the main characteristics of the study population and reported as absolute frequencies with the corresponding percentage for categorical variables, mean (S.D.) for continuous vari- ables with a normal distribution and median with inter- quartile range (IQR) for non-normally distributed continuous variables. The chi-squared test was used for comparing categorical variables. The Mann–Whitney U test was used to compare continuous variables. A uni- variable analysis was performed to define the predictive value of age, gender, anti-CCP2 antibodies (high/low), anti-CCP3 antibodies (positive/negative), RF (positive/ negative), EMS >30 min and tenderness in the small joints of the hands on physical examination for the de- velopment of US subclinical synovitis at follow-up. The multivariable regression analysis was adjusted for those parameters that were significant in the univariable ana- lysis. In addition, we performed three different multivari- able models excluding either anti-CCP2 (high level), anti-CCP3 or RF, to rule out the potential influence of the collinearity between these variables on the multivari- able analysis results. The collinearity between anti- CCP2, anti-CCP3 and RF was also explored using Crame´ r’s V. A coefficient >0.60 was considered to be indicative of collinearity. Kaplan–Meier analysis and log- rank tests were performed to evaluate the US subclinical synovitis-free survival time for anti-CCP3 antibodies and RF. Statistical analyses were performed using SPSS ver- sion 25.0 for Windows (IBM, Armonk, NY, USA). The level of significance was set at 5%. Downloaded from https://academic.oup.com/rheumatology/article/61/8/3192/6438028 by guest on 20 December 2022 A full US protocol was performed as part of the Leeds CCP study. The following joints were scanned: elbows, wrists, first–fifth MCP joints, first–fifth PIP joints, knees, ankles and first–fifth MTP joints. The first MTP joint was not included in the analyses, as US abnormalities have been frequently detected at this level in other non- inflammatory joint diseases, such as OA, as well as in asymptomatic healthy subjects [20, 21]. US synovitis (synovial hypertrophy 1 and PD signal 1) and bone erosions were identified according to the EULAR/ OMERACT and OMERACT definitions, respectively [22, 23]. Three different US machines were used during the study: an ATL HDI 5000 (Philips US, Cambridge, MA, USA), employing 5–12 MHz and 8–15 MHz transducers, and an S7 and Logiq E9 (GE Healthcare, Chicago, IL, USA), both employing a 6–15 MHz transducer. Methods CCP2þ (BioPlex 2200, Bio-Rad Laboratories, Hercules, CA, USA) at-risk individuals with MSK symptoms, but without clinical synovitis, taking part in the Leeds CCP study from June 2008 to March 2020 were included. Full details of ‘The CCP Study: Coordinated Programme to Prevent Arthritis – Can We Identify Arthritis at a Pre- clinical Stage?’ have been previously reported [15, 16]. Briefly, in this national study, individuals 18 years of age with new MSK symptoms, who test positive for anti-CCP antibodies, are invited to a dedicated research clinic at Chapel Allerton Hospital (Leeds, UK) as part of an observational study. Anti-CCP2 antibody is the most used test to detect ACPA in many places, including the UK. Different from some other at-risk cohorts currently being followed internationally [17, 18], all subjects in the Leeds CCP study are anti-CCP2þ. FIG. 1 Categories along the RA continuum deﬁned by the EULAR Standing Committee on Investigative Rheumatology UA: undifferentiated arthritis. UA: undifferentiated arthritis. 3193 https://academic.oup.com/rheumatology Andrea Di Matteo et al. In anti-CCP2þ at-risk individuals, the following data were collected at baseline: age, gender, early morning stiffness (EMS) duration, tenderness in the small joints of the hands on physical examination, anti-CCP2 level, third-generation IgG anti-CCP antibody (anti-CCP3) level (QUANTA Lite CCP3, Inova Diagnostic, San Diego, CA, USA) and IgM RF status (BNII nephelometry before February 2010, AdviaXPT turbidometry after February 2010; Siemens, Munich, Germany). neither US synovitis nor bone erosions) who had one or more longitudinal US scan and in whom a complete dataset was available. In these patients, we investigated predictors of subclinical synovitis on longitudinal scans. This study was approved by the NHS Health Research Authority National Research Ethics Service Committee Yorkshire & the Humber–Leeds West. All individuals participating in the study provided full written informed consent. neither US synovitis nor bone erosions) who had one or more longitudinal US scan and in whom a complete dataset was available. In these patients, we investigated predictors of subclinical synovitis on longitudinal scans. This study was approved by the NHS Health Research Authority National Research Ethics Service Committee Yorkshire & the Humber–Leeds West. All individuals participating in the study provided full written informed consent. The anti-CCP2 and anti-CCP3 test positivity threshold was set according to the manufacturer’s cut-offs (>2.99 IU/ml and 20 units, respectively). Methods The anti-CCP2 level was considered low or high when it was <3 or 3 times the positivity threshold, respectively, according to the ACR/EULAR 2010 criteria [19]. Statistical analysis PD was set as follows: pulse repetition frequency (PRF) 700– 1000 Hz, Doppler frequency 6 MHz for the ATL HDI 5000 and 10 MHz for the S7 and Logic E9. Sensitivity analy- ses between the first two US machines (ATL HDI 5000 and S7) were performed due to the change in the US machine during the study [8]. Given the positive results of this analyses and the fact that the S7 was replaced with another GE machine (Logiq E9), the same analyses were not repeated for the third US machine used. This study consisted of two parts. In the first part (US subclinical synovitis prior to the development of inflam- matory arthritis), among at-risk individuals who pro- gressed to inflammatory arthritis, the prevalence and distribution of subclinical synovitis was evaluated in the US scans conducted prior to inflammatory arthritis de- velopment. Patients in whom the pre-progression US scan was negative, but the scan was conducted >6 months before progression, were excluded. The rea- son for this choice was based on the fact that the ma- jority of anti-CCP2þ individuals with US synovitis who progress to inflammatory arthritis do so within a short- term follow-up (i.e. median 7.9–9.9 months) [15, 16]. Therefore, individuals with a negative US scan several months before progression may well have subsequently developed an undetected subclinical synovitis. In the se- cond part of the study (predicting the development of US subclinical synovitis), we selected only anti-CCP2þ individuals without US abnormalities at baseline (i.e. US subclinical synovitis prior to the development of inflammatory arthritis In 220 CCP2þ at-risk individuals with normal baseline US scans who had one or more longitudinal US scan Downloaded from https://academic.oup.com/rheumatology/article/61/8/3192/6438028 b TABLE 1 Baseline demographic and clinical characteristics of the anti-CCP2þ at-risk individuals who developed inﬂam- matory arthritis TABLE 1 Baseline demographic and clinical characteristics of the anti-CCP2þ at-risk individuals who developed inﬂam- matory arthritis Characteristics Total population (N 5 135) US subclinical synovitis preceding clinical inflam- matory arthritis (n 5 75) No US subclinical synovitis preceding clinical inflammatory arthritis (n 5 22) Individuals not included in the analysis* (n 5 38) Age, years, mean (S.D.) 53.9 (12.8) 56.3 (12.8) 50.0 (10.5) 51.5 (12.4) Female, n (%) 102 (75.5) 54 (72.0) 15 (68.2) 33 (86.8) Tenderness in the hands, n (%) 62 (45.9) 38 (50.7) 8 (36.4) 16 (42.1) EMS, minutes, median (IQR) 15 (0–60) 15 (0–42.5) 37.5 (15–60) 10 (0–30) Anti-CCP2 antibodies, n (%) Low 16 (11.9) 11 (14.7) 1 (4.5) 4 (10.5) High 119 (88.1) 64 (85.3) 21 (95.5) 34 (89.5) Anti-CCP3 antibodies, n (%) Not available 11 (8.1) 7 (9.3) 0 (0.0) 4 (10.5) Negative 11 (8.1) 6 (8.0) 3 (13.6) 2 (5.3) Positive 113 (83.8) 62 (82.7) 19 (86.4) 32 (84.2) RF, n (%) Negative 49 (36.3) 24 (32.0) 8 (36.4) 17 (44.7) Positive 86 (63.7) 51 (68.0) 14 (63.6) 21 (55.3) Only anti-CCP2þ at-risk individuals who progressed to inﬂammatory arthritis and had one or more US scan prior to inﬂam- matory arthritis development are included. Individuals in whom the most recent scan before progression was negative and where this scan was >6 months before progression. Only anti-CCP2þ at-risk individuals who progressed to inﬂammatory arthritis and had one or more US scan prior to inﬂam- matory arthritis development are included. Individuals in whom the most recent scan before progression was negative and where this scan was >6 months before progression. US subclinical synovitis prior to the development of inflammatory arthritis A total of 155/620 (25.0%) anti-CCP2þ at-risk individuals progressed to inflammatory arthritis [median time to de- velop inflammatory arthritis from the baseline visit: 51weeks (IQR 24–107.2)]. At least one US scan per- formed prior to the development of clinical arthritis was available in 135/155 progressors (87.1%). Thirty-eight individuals in whom the most recent scan before progres- sion was negative and where this scan was >6 months before progression were excluded. US subclinical syno- vitis was detected in one or more scan in 75 of the remaining 97 individuals (77.3%) [median time to inflam- matory arthritis development from first developing US 3194 https://academic.oup.com/rheumatology US subclinical synovitis in anti-CCPþ at-risk individuals Predicting the development of US subclinical synovitis synovitis: 26.5 weeks (IQR 7.0–60.0); median number of joints with US synovitis: 2.0 (IQR 1.0–3.0)]. The demographic and clinical characteristics of pro- gressors with one or more US scan available is reported in Table 1. A total of 220 CCP2þ individuals with a normal baseline US scan (i.e. no US synovitis or bone erosions) who had one or more longitudinal US scan and in whom a com- plete dataset was available were included in this ana- lysis. The clinical and demographic characteristics of individuals included are reported in Table 2. US subclinical synovitis was detected in the follow- ing anatomical areas: wrists in 45/75 (60.0%) CCP2þ individuals, MCP joints in 30/75 (40.0%), MTP joints in 22/75 (29.3%), PIP joints in 17/75 (22.7%), knees in 6/ 75 (8.0%), elbows in 1/75 (1.3%) and ankles in 1/75 (1.3%). Only anti-CCP2þ at-risk individuals with normal baseline US scan who had one or more longitudinal US scan and in whom a complete dataset was available are included. US subclinical synovitis prior to the development of inflammatory arthritis TABLE 2 Baseline demographic, clinical and imaging characteristics of the anti-CCP2þ at-risk individuals TABLE 2 Baseline demographic, clinical and imaging characteristics of the anti-CCP2þ at-risk individuals Characteristics Baseline (N 5 220) Longitudinal analyses Developed US subclinical syno- vitis (ever) (n 5 69) Developed US subclinical syno- vitis (12 months) (n 5 28) Developed US subclinical synovitis (24 months) (n 5 49) Did not develop subclinical US synovitis (ever) (n 5 151) Age, years, mean (S.D.) 48.9 (12.4) 53.0 (10.8) 53.2 (9.6) 52.3 (10.9) 24.9 (12.6) Female, n (%) 164 (74.5) 49 (71.0) 20 (71.4) 33 (67.3) 115 (76.2) Tenderness in the hands, n (%) 71 (32.3) 20 (28.2) 8 (28.6) 12 (24.5) 51 (33.8) EMS, minutes, median (IQR) 5 (0–30) 10 (0–30) 30 (2.5–60) 20 (0–60) 0 (0–30) Anti-CCP2 antibodies, n (%) Low 83 (37.7) 19 (27.5) 8 (28.6) 13 (26.5) 64 (42.4) High 137 (62.3) 50 (72.5) 20 (71.4) 36 (73.5) 87 (57.6) Anti-CCP3 antibodies, n (%) Negative 109 (49.5) 17 (24.6) 7 (25.0) 11 (22.4) 59 (39.1) Positive 111 (50.5) 52 (75.4) 21 (75.0) 38 (77.6) 92 (60.9) RF, n (%) Negative 161 (73.2) 39 (56.5) 16 (57.1) 28 (57.1) 122 (80.8) Positive 59 (26.8) 30 (43.5) 12 (42.9) 21 (42.9) 29 (19.2) Only anti-CCP2þ at-risk individuals with normal baseline US scan who had one or more longitudinal US scan and in whom a complete dataset was available are included. BLE 2 Baseline demographic, clinical and imaging characteristics of the anti-CCP2þ at-risk individuals Only anti-CCP2þ at-risk individuals with normal baseline US scan who had one or more longitudinal US scan and in whom a complete dataset was available are included. 3195 https://academic.oup.com/rheumatology Andrea Di Matteo et al. (P< 0.01 for both) (Fig. 2a). Similar results were observed in the subgroup of high-level CCP2þ individuals at the 1 and 2year follow-up; respectively, 19.4% and 32.7% of high-level CCP2þ/anti-CCP3þ, but only 2.6% and 10.3% of high-level CCP2þ individuals with negative anti-CCP3 antibodies developed subclinical synovitis on longitudinal scans (P¼ 0.01 and P< 0.01, respectively) (Fig. 2b). The impact of RF on subclinical synovitis-free survival rate was not significant in individuals with positive anti-CCP3 anti- bodies (Fig. 2c). Conversely, this was notable in individuals with anti-CCP2þ/RFþ [i.e. 21/59 (35.6%) developed US subclinical synovitis within 2years of follow-up] compared with individuals with positive anti-CCP2þ and negative RF [i.e. 28/161 (17.4%), P¼ 0.01] (Fig. 2d). US subclinical synovitis prior to the development of inflammatory arthritis and a complete dataset, US synovitis was detected in 69/220 (31.4%) [median time to first developing US synovitis: 56.4 weeks (IQR 33.0–112.0); median number of US scans: 2.0 (IQR 1.0–3.0); median number of joints with US synovitis: 2.0 (IQR 1.0–2.0)]. US subclinical synovitis was detected on longitudinal scans in the following anatomical areas: wrists in 44/69 (63.8%) CCP2þ individuals, MCP joints in 30/69 (43.5%), MTP joints in 14/69 (20.3%), knees in 11/69 (15.9%), PIP joints in 4/69 (5.8%) and elbows in 1/69 (1.4%). j ( ) ( ) In the univariable analysis, age [OR 1.04 (95% CI 1.02, 1.07), P < 0.01], high level anti-CCP2þ [OR 1.94 (95% CI 1.04, 3.60), P ¼ 0.04], anti-CCP3 antibodies [OR 4.77 (95% CI 2.52, 9.03), P < 0.01] and RF [OR 3.24 (95% CI 1.73, 6.05), P < 0.01] were predictive for the de- velopment of US subclinical synovitis on subsequent US scans. In the multivariable analysis, only anti-CCP3 anti- bodies remained significantly predictive [OR 4.75 (95% CI 1.97, 11.46), P < 0.01] while borderline results were observed with age [OR 1.04 (95% CI 1.01, 1.07), P ¼ 0.01] (Table 3). The predictive value of anti-CCP3 for US subclinical synovitis development was also observed when the multivariable analysis was carried out exclud- ing high-level anti-CCP2 [anti-CCP3 OR 3.60 (95% CI 1.74, 7.44), P < 0.01] or RF [anti-CCP3 OR 5.64 (95% CI 2.50, 12.76), P < 0.01] (Supplementary Table S1, avail- able at Rheumatology online). Interestingly, RF was pre- dictive for the development of US subclinical synovitis when anti-CCP3 antibodies were not included in the multivariable analysis [RF OR 2.61 (95% CI 1.34, 5.08), P ¼ 0.01]. No significant collinearity between anti-CCP2, anti-CCP3 and RF was found (V ¼ 0.54, 0.28 and 0.52 for anti-CCP2/anti-CCP3, anti-CCP2/RF and anti-CCP3/ RF, respectively). Downloaded from https://academic.oup.com/rheumatology/article/61/8/3192/6438028 by guest on 20 December 2022 Discussion rom https://academic.oup.com/rheumatology/article/61/8/3192/6438028 by guest on 20 December 2022 The results of the current study show that in CCP2þ at- risk individuals with MSK symptoms, but without clinical synovitis, the majority of progressors go through a stage of US subclinical joint inflammation prior to developing inflammatory arthritis; this represents a distinct stage of the ‘RA continuum’. Moreover, this is an important group to recognize, as it also represents the first transi- tion of systemic autoimmunity into articular inflamma- tion, the so-called ‘second hit’ in the pathogenesis of RA [24] (Fig. 3). There is increasing evidence from patient-reported outcomes and the very high predictive value of subclin- ical synovitis on imaging that the presence of subclinical synovitis in multiple joints may represent a distinct dis- ease in its own right [25, 26]. In support of this, serial US assessments in a cohort of anti-CCP2þ at-risk indi- viduals suggest that subclinical synovitis occurs directly before the development of clinical synovitis [27] and the fact that the majority of anti-CCP2þ individuals with US synovitis who develop inflammatory arthritis do so within 12 months of follow-up [15, 16]. Of note, in our study, >60% of progressors had evidence of subclinical syno- vitis on US scans performed within 6 months prior to in- flammatory arthritis development. CCP2þ individuals with positive anti-CCP3 antibodies show a significantly reduced US subclinical synovitis-free survival rate compared with individuals with negative anti- CCP3 antibodies (Fig. 2). At the 1 and 2year follow-up, 18.9% and 34.2%, respectively, of individuals with dual CCP2/CCP3 positivity developed subclinical synovitis on longitudinal scans compared with 6.4% and 10.1% of CCP2þ individuals with negative anti-CCP3 antibodies TABLE 3 Univariable and multivariable regression analyses for the development of US synovitis TABLE 3 Univariable and multivariable regression analyses for the development of US synovitis Variables Univariable analysis Multivariable analysis OR (95% CI) P-value OR (95% CI) P-value Gender (male) 1.30 (0.69, 2.48) 0.42 – – Age 1.04 (1.02, 1.07) <0.01 1.04 (1.01, 1.07) 0.01 Tenderness in the hands 1.25 (0.67, 2.32) 0.48 – – EMS 1.00 (0.99, 1.01) 0.11 – – Anti-CCP2þ (high level) 1.94 (1.04, 3.60) 0.04 0.60 (0.26, 1.41) 0.24 Anti-CCP3þ 4.77 (2.52, 9.03) <0.01 4.75 (1.97, 11.46) <0.01 RFþ 3.24 (1.73, 6.05) <0.01 1.46 (0.70, 3.05) 0.31 Signiﬁcant results are in bold. Signiﬁcant results are in bold. 3196 US subclinical synovitis in anti-CCPþ at-risk individuals FIG. https://academic.oup.com/rheumatology Discussion 2 Kaplan–Meier analysis shows US subclinical synovitis-free survival time in CCP2þ at-risk individuals G. 2 Kaplan–Meier analysis shows US subclinical synovitis-free survival time in CCP2þ at-risk individuals FIG. 2 Kaplan–Meier analysis shows US subclinical synovitis-free survival time in CCP2þ at-risk individuals Percentages refer to the individuals who developed US subclinical synovitis at 12 and 24 months follow-up (black lines). HL: high level. Downloaded from https://academic.oup.com/rheumatology/article/61/8/3192/6438028 b ademic.oup.com/rheumatology/article/61/8/3192/6438028 by guest on 20 December 2022 Percentages refer to the individuals who developed US subclinical synovitis at 12 and 24 months follow-up (black lines). HL: high level. FIG. 3 The ‘disease continuum’ of CCP2þ at-risk individuals Anti-CCPþ at-risk individuals with MSK symptoms but without any joint involvement (clinical or subclinical) may repre- sent the critical time point for timing interventions to prevent the onset of joint disease. UA: undifferentiated arthritis. FIG. 3 The ‘disease continuum’ of CCP2þ at-risk individuals Anti-CCPþ at-risk individuals with MSK symptoms but without any joint involvement (clinical or subclinical) may repre- sent the critical time point for timing interventions to prevent the onset of joint disease. UA: undifferentiated arthritis. therapy may only delay eventual disease progression rather than truly preventing arthritis [28]. Instead, anti- CCPþ individuals with MSK symptoms in whom subclin- ical joint inflammation is not present but imminent may represent ‘ideal targets’ for therapeutic trials for arthritis prevention. Halting disease progression in this population Certainly one might argue that US subclinical inflamma- tion represents a late feature in the development of in- flammatory arthritis and, when present, may represent an inevitable progression to clinical synovitis, especially when multiple joints are affected; patients who have progressed this far already have joint inflammation and 3197 https://academic.oup.com/rheumatology Andrea Di Matteo et al. would prevent the development of any joint inflammation, the cardinal feature of RA, and would likely produce bet- ter outcomes. This is also a valuable population to use for investigating the immunopathology of RA develop- ment, as the immunological events that drive the initial joint involvement in RA (i.e. the ‘second hit’) are not clear. The position of this population along the early ‘RA con- tinuum’ is well suited to address this. Indeed, further research is needed to establish which joints, and indeed how many joints, need to be imaged for optimum predictive accuracy. Most of the US studies in at-risk cohorts have adopted comprehensive US pro- tocols that evaluate multiple pathological findings (i.e. Discussion PD signal, grey-scale synovitis and/or bone erosions), including most or all relevant small joints. In addition, the interpretation of US-detected synovitis should be performed in the context of other joint findings. Indeed, it is well known that inflammation accompanies struc- tural changes of OA in small and large joints [33]. The results of the present study show that anti-CCP3 antibodies have a predictive role for the imminent devel- opment of subclinical synovitis in CCP2þ at-risk individ- uals. Such predictive value was not replicated with either high-level anti-CCP2 antibodies or RF, neither of which were predictive for the development of US syno- vitis in the multivariable analysis (Table 3). However, it should be noted that RF was predictive for the develop- ment of US subclinical synovitis when anti-CCP3 anti- bodies were not included in the multivariable analysis. Of note, around a third of high-level anti-CCP2þ/CCP3þ individuals developed US subclinical synovitis within 2 years of follow-up. On the other hand, only 4/39 (10.3%) high-level anti-CCP2þ/CCP3 individuals devel- oped US subclinical synovitis on one or more longitudin- al scan in this time frame (Fig. 2b). Thus the CCP3 result had a 3-fold effect on the prognostic value of high-level anti-CCP2 antibodies. As shown in Fig. 2d, a similar trend was also observed in individuals with double posi- tive anti-CCP2 and RF in comparison with individuals with positive anti-CCP2 but negative RF. Our results suggest that anti-CCP2þ/CCP3þ individu- als without subclinical joint disease are at a critical tran- sition point in the evolution of RA (i.e. the transition from autoimmunity to joint inflammation). This transition to the first detectable phase of joint involvement may represent the so-called ‘second hit’ in RA pathogenesis and may be viewed as an additional distinct stage in the ‘RA con- tinuum’. Moreover, this point may represent a unique time point for preventing the onset of joint disease. However, caution is warranted not to dismiss the possi- bility of participation in prevention trials of those who al- ready have subclinical joint inflammation. Downloaded from https://academic.oup.com/rheumatology/article/61/8/3192/6438028 by guest on 20 December 2022 To our knowledge, this is the first study aimed at identifying at-risk individuals just before the develop- ment of subclinical joint involvement. Indeed, this nov- elty is the main strength of the current study. Moreover, data are presented from one of the largest prospective cohorts with the longest follow-up of CCPþ at-risk indi- viduals. However, all individuals were anti-CCP2þ. Conclusions In CCP2þ at-risk individuals with MSK symptoms but without clinical synovitis, the majority of progressors go through a stage of US subclinical joint inflammation prior to the development of inflammatory arthritis, thus representing a distinct and important stage of the ‘RA continuum’. However, progression to inflammatory arthritis in at- risk individuals with RA-related antibodies and MSK symptoms is not always inevitable, even in those with US subclinical joint inflammation. In a recent study, baseline US synovitis was not associated with the devel- opment of clinical arthritis at 12 months follow-up in 54% of ACPAþ individuals with arthralgia [31]. The rate of progression to inflammatory arthritis in at-risk individ- uals with subclinical synovitis is variable and depends on several factors, such as the target population (i.e. asymptomatic first-degree relatives vs individuals with MSK symptoms and RA-related antibodies), distribution and number of joints or tendons involved and the type of US pathological findings detected (e.g. grey-scale synovitis, PD signal) [32]. In the current study, progres- sors were found to have a median of 2 joints with US subclinical synovitis (IQR 1.0–3.0) prior to inflammatory arthritis development. US subclinical synovitis was main- ly detected in the wrists, MCP joints and MTP joints (60.0%, 40.0% and 29.3% of progressors, respectively). Anti-CCP3 antibodies have a potential role in the iden- tification of CCP2þ individuals who are about to develop clinical or subclinical RA-related joint inflammation (i.e. before the ‘second hit’ in RA pathogenesis occurs). This may be the ideal population for interventions to prevent joint disease. This is also a unique population for investi- gating the drivers of joint involvement in the develop- ment of RA. Discussion Therefore the results of our study can be interpreted only in the context of anti-CCP2þ at-risk individuals. Our group has previously demonstrated the predict- ive role of anti-CCP3 for the development of inflam- matory arthritis [29]. This is likely to be related to dual anti-CCP2/CCP3 positivity reflecting an expanded ACPA repertoire (i.e. different antigenic targets/ACPA fine specificity being detected) and suggests that in anti-CCP2þ at-risk individuals, anti-CCP3 antibodies may be identifying a more advanced stage of auto- immunity driving the onset of subclinical and later clinical inflammation [29, 30]. Acknowledgements This study was conducted while A.D.M. was an ARTICULUM Fellow. P.E. is National Institute for Health Research (NIHR) Biomedical Research Centre (BRC) dir- ector and BRC funds supported this work. L.G.-M. and L.D. are NIHR BRC fellows. A.D.M. was one of the clini- cians of the study, collected and analysed data and wrote the manuscript. K.M. was one of the clinicians of https://academic.oup.com/rheumatology 3198 US subclinical synovitis in anti-CCPþ at-risk individuals Study Group for Risk Factors for Rheumatoid Arthritis. Ann Rheum Dis 2012;71:638–41. the study, designed the study and contributed to the analysis of data and writing the manuscript. L.D. was one of the clinicians of the study, collected data and performed statistical analysis. J.L.N. was one of the clinicians of the study, contributed to the design of the study and collected data. L.G.-M. was one of the clini- cians of the study and contributed to the collection and analysis of data. E.C. helped with the statistical analysis and contributed to the analysis of data. R.J.W. and M.M. contributed to the analysis of data and writing the manuscript. P.E. established the cohort, designed the study, analysed data and contributed to writing the manuscript. All co-authors contributed to revising the manuscript critically and approved the ﬁnal version to be published. 5 5 Zabotti A, Finzel S, Baraliakos X et al. Imaging in the preclinical phases of rheumatoid arthritis. Clin Exp Rheumatol 2020;38:536–42. 6 Di Matteo A, Mankia K, Azukizawa M et al. The role of musculoskeletal ultrasound in the rheumatoid arthritis continuum. Curr Rheumatol Rep 2020;22:41. 7 Hunt L, Euge´ nio G, Grainger AJ. Magnetic resonance imaging in individuals at risk of rheumatoid arthritis. Best Pract Res Clin Rheumatol 2017;31:80–9. Downloaded from https://academic.oup.com/rheumatology/article/61/8/3192/6438028 by guest on 20 December 2022 8 Nam JL, Hensor EMA, Hunt L et al. Ultrasound ﬁndings predict progression to inﬂammatory arthritis in anti-CCP antibody-positive patients without clinical synovitis. Ann Rheum Dis 2016;75:2060–7. 9 Zufferey P, Rebell C, Benaim C et al. Ultrasound can be useful to predict an evolution towards rheumatoid arthritis in patients with inﬂammatory polyarthralgia without anticitrullinated antibodies. Joint Bone Spine 2017;84:299–303. Funding: The study was supported by the NIHR Leeds BRC (grant IS-BRC-1215-20015). Funding: The study was supported by the NIHR Leeds BRC (grant IS-BRC-1215-20015). Disclosure statement: R.J.W. has received honoraria from AbbVie, Novartis and GE for ultrasound-related educational activities (all <$10 000). M.M. is an em- ployee of Inova Diagnostics, commercializing CCP3. K.M. Data availability statement 13 van de Stadt LA, Bos WH, Meursinge Reynders M et al. The value of ultrasonography in predicting arthritis in auto-antibody positive arthralgia patients: a prospective cohort study. 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https://openalex.org/W2067290870	https://europepmc.org/articles/pmc4384120?pdf=render	English	null	Transcriptional Regulation of Chemokine Expression in Ovarian Cancer	Biomolecules	2,015	cc-by	8,975	Bipradeb Singha, Himavanth R. Gatla and Ivana Vancurova * Department of Biological Sciences, St. John’s University, 8000 Utopia Parkway, New York, NY 11439, USA; E-Mails: bipradeb.singha10@my.stjohns.edu (B.S.); himavanthreddy.gatla12@my.stjohns.edu (H.R.G.) * Author to whom correspondence should be addressed; E-Mail: vancuroi@stjohns.edu; Tel.: +1-718-990-6409. Academic Editor: Jürg Bähler Academic Editor: Jürg Bähler Received: 8 December 2014 / Accepted: 9 March 2015 / Published: 17 March 2015 Received: 8 December 2014 / Accepted: 9 March 2015 / Published: 17 March 2015 Abstract: The increased expression of pro-inflammatory and pro-angiogenic chemokines contributes to ovarian cancer progression through the induction of tumor cell proliferation, survival, angiogenesis, and metastasis. The substantial potential of these chemokines to facilitate the progression and metastasis of ovarian cancer underscores the need for their stringent transcriptional regulation. In this Review, we highlight the key mechanisms that regulate the transcription of pro-inflammatory chemokines in ovarian cancer cells, and that have important roles in controlling ovarian cancer progression. We further discuss the potential mechanisms underlying the increased chemokine expression in drug resistance, along with our perspective for future studies. Keywords: chemokines; interleukin-8; NFB; ovarian cancer; transcriptional regulation Biomolecules 2015, 5, 223-243; doi:10.3390/biom5010223 Biomolecules 2015, 5, 223-243; doi:10.3390/biom5010223 1. Introduction Chemokines are a family of cytokines that induce chemotaxis of target cells. Though they were originally discovered for their ability to induce leukocyte migration into the infected or injured sites, more recently, it became clear that they could also promote cancer progression [1–9]. In addition to inducing tumor cell proliferation, angiogenesis and metastasis, chemokines and their receptors regulate tumor cell differentiation and survival. Currently, the human chemokine network includes more than Biomolecules 2015, 5 Biomolecules 2015, 5 224 45 known chemokines and 20 chemokine receptors. Based on the number and spacing of conserved N-terminal cysteine residues that form disulfide bonds, chemokines are divided into four groups: (X)C, CC, CXC, and CX3C [10–12]. Epithelial ovarian cancer (EOC) is among the leading causes of cancer death in women. Since most ovarian cancers relapse and become drug-resistant, the survival rates remain low. Progression of ovarian cancer (OC) has been associated with the increased expression and release of pro-inflammatory chemokines, which contribute to ovarian cancer development through their induction of tumor cell proliferation, survival, migration, and angiogenesis [13–15]. The chemokine expression by ovarian cancer cells is controlled at several levels that include transcriptional regulation, post-transcriptional regulation and regulation of mRNA stability, translation, and mechanisms regulating the cytokine intracellular storage, transport, and release. Table 1 summarizes chemokines produced by ovarian cancer cells. Several excellent reviews have addressed the physiological and cellular functions of these chemokines in ovarian cancer [9,16,17]. Thus, in this review, we focus instead on the main mechanisms that regulate transcription of these chemokines in ovarian cancer cells. Table 1. Chemokines released by ovarian cancer cells. 2.1. Chemokine Regulation by NFB and Epigenetic Acetylation 2.1. Chemokine Regulation by NFB and Epigenetic Acetylation 2.1. Chemokine Regulation by NFB and Epigenetic Acetylation Chemokines are regulated at the transcriptional level by binding of transcription factors and repressors to gene promoter and enhancer regions. The transcription factors that control the expression of most inflammatory chemokines include the nuclear factor-B (NFB), activator protein-1 (AP-1) and the signal transducers and activators of transcription (STAT) family. The NFB activity is constitutively increased in aggressive ovarian cancers, and inhibition of NFB signaling suppresses angiogenesis and tumorigenicity of ovarian cancer cells and increases their sensitivity to chemotherapy and apoptosis [37–40]. The underlying mechanisms likely involve the NFB-regulated chemokine expression, since several studies have demonstrated that the expression of CCL2, CXCL1, CXCL2, and IL-8/CXCL8 is mediated by NFB in ovarian cancer cells [28–30,41]. The increased activity of NFB in ovarian cancer cells is mediated by enzymes of the IB kinase (IKK) complex, which phosphorylate the NFB inhibitory protein, IB, resulting in IB proteasomal degradation and nuclear translocation of NFB subunits [42–45]. In addition to phosphorylating IB, IKKs can also phosphorylate the NFB subunits, particularly p65 [46]. While the cytoplasmic degradation of IB, resulting in the nuclear translocation of NFB subunits, represents a general step in NFB activation, the specificity of NFB-regulated responses is mediated by the subunit composition of NFB complexes and their post-translational modifications [47,48]. In addition to transcription factor binding to promoter sequences, chemokine expression is regulated by epigenetic modifications that include histone modifications as well as post-translational modifications of transcription factors, particularly the p65 subunit of NFB. It is believed that while histone acetylation and acetylation of transcription factors induced by histone acetyl transferases (HATs) generally promotes transcriptional activation, hypoacetylation induced by histone deacetylase (HDAC) activity is associated with transcriptional repression. Since hypoacetylation of tumor suppressor genes by HDACs has been linked to tumor development, HDACs inhibitors are now being evaluated for their therapeutic effects in cancer, including ovarian cancer [49–51]. Clinical studies using HDAC inhibitors in the treatment of ovarian cancer are summarized in the recent elegant review by Khabele [52]. Numerous studies have shown that HDACs regulate chemokine expression in different cell types [53–58]; however, their role in the regulation of chemokine expression in ovarian cancer has yet to be documented. 1. Introduction Systematic Name Alternate Human Names Tissue/Cells Reference CCL2 Monocyte chemotactic protein 1 (MCP-1) Tumor biopsies, serum and ascites Negus et al., 1995 [18] Milliken et al., 2002 [19] CCL5 RANTES Tumor ascites, plasma and peritoneal fluid Milliken et al., 2002 [19] Negus et al., 1997 [20] CCL11 Eotaxin Primary ovarian cancer cells obtained from ascites Levina et al., 2009 [21] Nolen et al., 2010 [22] CCL25 Thymus expressed chemokine (TECK) Tumor tissue Singh et al., 2011 [23] CCL28 Mucosae-associated epithelial chemokine (MEC) Tumor tissue Facciabene et al., 2011 [24] CXCL1 Growth-regulated protein (GRO-) Plasma and tumor ascites Lee et al., 2006 [25] Yang et al., 2006 [26] CXCL2 Growth-regulated protein (GRO-) Ovarian cancer cell lines Son et al., 2007 [27] Kavandi et al., 2012 [28] CXCL8 Interleukin 8 (IL-8) Tumor tissue, ascites, serum and cyst fluid Lee et al., 1996 [29] Xu et al., 1999 [30] CXCL12 Stromal cell-derived factor (SDF-1) Tumor biopsies, tissues and ascites Zou et al., 2001 [31] Scotton et al., 2002 [32] CXCL16 Transmembrane chemokine CXCL16 Epithelial ovarian carcinoma tissue Guo et al., 2011 [33] Gooden et al., 2014 [34] CX3CL1 Fractalkine Epithelial ovarian carcinoma tissue Gaudin et al., 2011 [35] XCL1/2 Lymphotactin Tumor ascites and ovarian cancer cell lines Kim et al., 2012 [36] Table 1. Chemokines released by ovarian cancer cells. Biomolecules 2015, 5 225 2. Mechanisms Regulating Chemokine Transcription in Ovarian Cancer Cells 2. Mechanisms Regulating Chemokine Transcription in Ovarian Cancer Cells 2.1. Chemokine Regulation by NFB and Epigenetic Acetylation 2. Mechanisms Regulating Chemokine Transcription in Ovarian Cancer Cells 2.1. Chemokine Regulation by NFB and Epigenetic Acetylation 2.2. Chemokine Modulation by Hypoxia and Metabolism Ovarian cancer tissues and ascites are characterized by decreased oxygen content, which stabilizes the -subunit of the transcription factor hypoxia-inducible factor-1 (Hif-1) [59]. Hif-1 responds to hypoxia by increasing the transcription of genes that promote survival in low-oxygen conditions, thus promoting angiogenesis and oncogenesis. Indeed, the increased expression of Hif-1 has been detected in epithelial ovarian cancer, and correlates with poor prognosis [60–62]. Hypoxia induces IL-8 [30], CXCL12 [63], and CCL28 [24] expression in ovarian cancer cells. The seminal study by Xu et al. [30] demonstrated that hypoxic conditions increase the IL-8 expression in ovarian cancer cells by increasing NFB and AP-1 binding to IL-8 promoter. The mechanisms of how hypoxia increases the NFB-dependent IL-8 transcription involve activation of the transforming growth factor beta-activated Biomolecules 2015, 5 Biomolecules 2015, 5 226 kinase 1 (TAK1), resulting in increased IKK activation, and p65 NFB recruitment to the IL-8 promoter [64,65]. In addition, hypoxia induces a direct binding of Hif-1 to the hypoxia-response element (HRE) located next to the NFB binding site in human IL-8 promoter, resulting in the increased IL-8 expression [66]. One of the consequences of Hif-1 activation is the increased expression of glycolytic genes, resulting in increased aerobic glycolysis, glucose consumption, and lactic acid production (Warburg effect) [67–69]. The high rate of glucose consumption and lactic acid production contributes to the acidification of the tumor environment and cancer progression. Xu et al. showed that acidic pH increases the IL-8 transcription by enhancing the binding of AP-1 and NFB to IL-8 promoter in ovarian cancer cells [70]. In addition, in endothelial cells, lactate was shown to activate the NFB-dependent IL-8 transcription by inducing degradation of IB [71]. The role of lactate and other metabolites of the glycolytic pathway in the regulation of pro-angiogenic chemokine expression in ovarian cancer cells is yet to be investigated, especially since recent studies have indicated high levels of aerobic glycolysis and lactate production in ovarian tumors [72,73]. While hyperglycemia and obesity are thought to be contributing factors to cancer development and progression, caloric restriction has been associated with reduced cancer incidence [74–77]. During reduced calorie intake or exercise, the body switches to obtaining energy from fatty acid oxidation, which results in ketone bodies production. Intriguingly, the recent study by Shimazu et al. 2.2. Chemokine Modulation by Hypoxia and Metabolism [78] has demonstrated that the ketone body -hydroxybutyrate (OHB) is an endogenous and specific inhibitor of HDACs, and that administration of exogenous OHB increases histone acetylation, correlating with changes in transcription. Since HDACs regulate chemokine transcription by both deacetylating histones and p65 NFB [53–58], it will be important to analyze whether OHB and other HDAC inhibitors regulate chemokine expression in ovarian cancer cells, and whether this is modulated by the metabolic state. 2.3. Chemokine Modulation by Chemotherapeutic Interventions There is growing evidence that the increased chemokine expression by tumor cells modulates not only cancer development but also cancer responsiveness and resistance to chemotherapy [79]. A major contributor to the acquired chemoresistance of ovarian cancer cells is the increased expression of NFB-dependent chemokines that is induced by the platinum-based drugs carboplatin and cisplatin, and by the mitotic inhibitors docetaxel and paclitaxel [29,80–83]. The mechanisms responsible for the increased IL-8 expression induced by paclitaxel in ovarian cancer cells involve increased expression of toll-like receptors (TLRs) and increased p65 NFB binding to IL-8 promoter [80,83]. Bortezomib (BZ) is the first FDA approved proteasome inhibitor, which has shown a limited effectiveness in ovarian cancer treatment as a single agent [84–87]. However, BZ has been considered in combination with cisplatin, since BZ prevents the cisplatin-induced degradation of cisplatin influx transporter, resulting in enhanced cisplatin uptake and tumor cell killing [88,89]. We have recently shown that BZ increases expression of IL-8 and CCL2 in ovarian cancer cells, while it does not affect expression of other NFB-dependent genes. The responsible mechanisms involve a gene specific and IKK-dependent recruitment of S536 phosphorylated p65 NFB to IL-8 and CCL2 promoters, suggesting that anti-inflammatory therapy targeting IKK might increase the BZ effectiveness in ovarian cancer treatment [41]. Since approximately 50% of women diagnosed with ovarian cancer die from Biomolecules 2015, 5 227 chemoresistant metastatic disease, understanding the molecular mechanisms by which chemotherapeutic interventions increase the chemokine expression in ovarian cancer cells should lead to the development of more effective combination strategies. STAT1-3; Signal transducer and activator of transcription 1-3, NFB; Nuclear factor kappa B. * Experimentally confirmed binding sites, Hif-1; Hypoxia inducible factor-1, AP-1; Activator protei 3. Chemokine Transcriptional Regulation in Ovarian Cancer Cells Chemokines listed in Table 1 have all been identified in ovarian cancer cells and tissues. Various online databases can be used to assess putative transcription factor binding sites. For this review, we have obtained chemokine promoter sequences from the NCBI database and used the Alggen promoter-mapping program to search for the transcription factor binding sites [90,91]. All found putative binding sites are listed in Tables 2–5; the binding sites that have been experimentally confirmed are highlighted in bold and labeled with an asterisk. Below, we limit discussion of the transcriptional mechanisms only to the chemokines that have been experimentally confirmed in ovarian cancer cells. While the first insights into the chemokine transcriptional regulation were obtained by using in vitro electrophoretic mobility shift assays (EMSA) or overexpression experiments, chromatin immunoprecipitations (ChIP) generally provides a more realistic picture about the transcription factor binding to endogenous promoter sequences in living cells. Table 2. List of putative transcription factor binding sites in human CCL2 promoter. Factor Site Sequence Factor Site Sequence SP-1 -54/-44 ACTCCGCCCT c-Fos -1465/-1457 CTGACTCC Nkx-1 -65/-58 CCTCCTG p53 -1541/-1534 GGGCAGG Elk-1 -76/-71 GGAAG HOX-11 -1571/-1564 CCTAACG GATA -88/-82 CTTATC PEA3 -1644/-1636 AAACATCC C/EBP -112/-106 TTGCTC GR -1790/-1782 TTGTTCTC ELF -143/-130 CTACTTCCTGGAA AR -1789/-1781 TGTTCTCT Hif-1 * -127/-122 CACAG FOXP3 -1959/-1950 AAACATTTT AP-1 * -139/-131 TTCCTGGAA C/EBP -1980/-1973 TTGCACA STAT1-3 * -139/-131 TTCCTGGAA Pbx-1 -2132/-2120 AGCATGACTGGA C-Ets1 -140/-133 CTTCCTG FOXO-3 -2184/-2176 CTTATTTA NF-AT -181/-172 GGAAAAAGT CUTL-1 -2309/-2303 ATTGGT E47 -239/-232 GTCTGGG PR -2358/-2351 GAACACT RP58 -256/-245 GTTCACATCTG Smad3 -2521/-2511 GAGGCAGACA HNF-1 -654/-646 TAATATTT ER -2570/-2562 CTGACCTC TMF -708/-701 TATAACA c-Jun -2580/-2574 CATGGG HNF-3 -742/-735 CTATTTA NFB * -2600/-2591 GGAATTTCC AP-2 -747/-741 GCAGGC ZDX/BCL6 -2632/-2621 GGGAACTTCC c-Jun -942/-935 TGACTTA E47 -2678/-2671 ATCTGGA HMG1 -1042/-1035 GGAAATT ETF -2717/-2708 CACAGCCCC IRF-3 -1089/-1082 GCTTTCC GATA -2902/-2893 CTTTATCT BTEB3 -1287/-1278 AGGAGGAGG PU-1 -3041/-3031 TTACTTCCTC NF-Y -1315/-1307 ATTGGGCA YY1 -3264/-3257 AAAATGG USF-2b -1447/-1439 GTCATTTG RAR -3429/-3421 ATCTCACC * Experimentally confirmed binding sites, Hif-1; Hypoxia inducible factor-1, AP-1; Activator protein-1, STAT1-3; Signal transducer and activator of transcription 1-3, NFB; Nuclear factor kappa B. Table 2. List of putative transcription factor binding sites in human CCL2 promoter. 228 Biomolecules 2015, 5 Table 3. List of putative transcription factor binding sites in human CXCL1 promoter. * Experimentally confirmed binding sites, c-Jun; Jun proto-oncogene. 3. Chemokine Transcriptional Regulation in Ovarian Cancer Cells Factor Site Sequence Factor Site Sequence IRF-3 -50/-43 GCTTTCC Elk-1 -771/-766 GGAAG HMG I -75/-68 AATTTCC FOXP3 -791/-782 CAACATTTT MBP-1 -78/-68 GGGAATTTCC MZF-1 -810/-803 CAGGGGA NFB * -79/-68 CGGGAATTTCC TGIF -870/-862 TGACAACC CDP * -97/-87 GGGATCGATC C/EBP -980/-974 TTGCAC E47 -90/-83 ATCTGGA YY-1 -1061/-1054 TAAATGG E2F-1 -126/-119 GGCGGGG c-Ets -1076/-1069 CAGGAAG SP3 -128/-119 GGGGCGGGG AR -1394/-1386 TGTTCTCT SP-1 * -130/-121 GGGGGCGGG c-Jun -1491/-1483 TGACTCAT R2 -137/-131 TCCACC Pax -1909/-1902 CCTTGAC LF-A1 -247/-240 TGGGGCA ER -2057/-2050 TGGGTCAA AP-2 * -279/-273 GCAGGC NF-Y -2060/-2052 ATTGGGTC AREB6 -296/-288 CAGGTGGT LEF-1 -2807/-2799 CTTTGTTG Smad3 -563/-553 TTCACAGACA HNF-1 -2966/-2958 TAATATTT PR -602/-595 GAACATT RAR -3102/-3094 ATGCCTTAG GR -605/-596 GCAGAACAT NHP-1 -3103/-3096 TGACCTT TMF -739/-732 TGTTATA PEA3 -3110/-3102 GGATGTAT GATA -767/-761 GATAAG ATF -3452/-3443 TGACGTAAA * Experimentally confirmed binding sites, CDP; CAATT displacement protein, SP-1; Specificity protein 1, AP-2; Activator protein 2. * Experimentally confirmed binding sites, CDP; CAATT displacement protein, SP-1; Specificity protein 1, AP-2; Activator protein 2. Table 4. List of putative transcription factor binding sites in human CXCL2 promoter. Factor Site Sequence Factor Site Sequence NFB * -76/-67 GGGAATTTCC BTEB3 -862/-853 AAGCGGAGT CREB -83/-74 CGGACGTCA NF-Y -970/-962 GAACCAAT ATF-2 -83/-74 CGGACGTCA HMG I -999/-992 AATTTCC HLF -104/-95 GTTACGCAA IRF -999/-992 AATTTCC E2F-1 -111/-104 GGCGGGA NF-AT -1001/-992 AAAATTTCC NF-1 -113/-108 TTGGC CUTL1 -1085/-1079 ATTGAT LF-A1 -139/-132 CGGGGCA FOXP3 -1115/-1106 CTTAATTTT GATA -192/-184 GGTTATCT PR A -1257/-1250 GAACACT AP2 -198/-192 GCAGGC C/EBP -1367/-1360 TGAGCAA STAT3 * -218/-210 TTGGGGAA MZF1 -1380/-1373 CAGGGGA ER -241/-233 CTGACCCA HNF-1 -1440/-1432 ATATTAAC PEA3 -276/-268 GGATGTAG TMF -1880/-1873 TATAACA Elk-1 -296/-292 GAAG E47 -1830/-1823 TTCTGGA STAT3 * -318/-310 GGGATCGATC Nkx2 -1827/-1820 CTGGAGG p53 -339/-332 CTTGCCC HNF -2153/-2146 TAAATGG AhR -418/-410 GCGTGCGT YY1 -2153/-2146 TAAATGG c-Jun * -437/-430 TGACACA HSF1 -2409/-2401 ATTCTAGG c-Fos -451/-443 TGCGTCAT ETF -2505/-2496 GGGGCTGTC c-Ets -473/-467 CAGGAAG AP3 -2636/-2629 GAGTTAG USF-1 -508/-499 ACACGTGAT Smad3 -3112/-3102 CAGTCAGACA AREB6 -574/-566 AACACCTG LEF-1 -3101/-3093 CAACAAAG FOXJ2 -621/-611 AAAATAAACA TCF-1 -3102/-3093 ACAACAAAG AR -673/-665 TGTTCCAA GR -3256/-3247 ACAGAACAT * Experimentally confirmed binding sites, c-Jun; Jun proto-oncogene. Table 4. List of putative transcription factor binding sites in human CXCL2 promoter. 229 Biomolecules 2015, 5 Table 5. List of putative transcription factor binding sites in human CXCL8 promoter. 3. Chemokine Transcriptional Regulation in Ovarian Cancer Cells Factor Site Sequence Factor Site Sequence NFB * -80/-70 GGAATTTCC E47 -859/-852 ATCTGGA PU-1 -83/-73 GGAATTTCCTC PR -868/-861 ACTCTTC NRF * -88/-77 ATTCCTCTGA HSF1 -867/868 CCTTGAAT C/EBP * -94/-87 TTGCAAA IRF -973/-964 TTTCCATTA MZF-1 -112/-105 GAGGGA RAR -1068/-1061 AGAGGTC EBF -118/-107 TGCCCTGAGGG ER -1067/-1060 GAGGTCA C/EBP * -119/-112 TTGCACA p53 -1258/-1251 CTTGCCC AP-1 * -129/-121 TGACTCAG FOXP3 -1304/-1295 AAAATGAAG c-Ets -141/-132 TAGGAAGTC RelA -1367/-1357 GGCATTCCCC Elk-1 -139/-134 GGAAG YY1 -1372/-1365 AAAATGG LEF-1 -187/-179 GATCAAAG Smad3 -1403/-1393 GAAACAGACA Hif-1 * -234/-229 GTGCG Nkx1 -1457/-1450 CCTCAAG GR -335/-327 TTGTTCTA AP2 -1473/-1467 CCAGGC AREB6 -328/-320 AACACCTG TCF1 -1663/-1654 ACAACAAAG AR -334/-326 TGTTCTAA NF-AT -1687/-1677 CTAATTTTCC NF -424/-416 ATTGGCTC HMGI -1685/-1677 AATTTTCC AP3 -535/-528 TAAATC HLF -1695/-1686 TTGTGTAAC HNF-3 -606/-599 TAAATGT CUTL1 -1858/1852 TTGGT FOXO3 -651/-641 CTTATCTA PEA3 -2174/-2166 GCACATCC GATA -651/-644 CTTTATCT HOX11 -2200/-2193 CGTTAGG c-Myb -792/-784 CAACTGCC RAR -2225/-2217 GGCTCACC C/EBP -798/-792 TTGCTC AIRE -2555/-2545 ATGGTTATCT GR -847/-838 CTGTTCTCT Oct1 -2744/-2733 TCACTTTGCAT * Experimentally confirmed binding sites, C/EBP; CCAAT enhancer binding protein, NRF; NFB repressing factor. 3.1. CCL2 CCL2 (MCP-1) is an important determinant of macrophage infiltration in ovarian tumors [92,93]. Although CCL2 has been originally thought to have an inhibitory effect on ovarian cancer progression [94–96], recent studies have indicated that CCL2 increases invasion of ovarian cancer cells and resistance to chemotherapy [97,98]. The putative transcription factor binding sites identified in human CCL2 promoter are listed in Table 2. Experimental studies demonstrated binding of NFB, STAT1, STAT3, AP-1, and Hif-1 to the CCL2 promoter in OC cells (Figure 1). Even though the NFB binding site is located in the distal regulatory region of human CCL2 promoter (Figure 1), several studies have demonstrated p65 NFB involvement in the regulation of CCL2 expression in OC cells [27,41,99]. In addition, CCL2 expression is regulated by IKK-dependent recruitment of the transcription factor EGR-1, and inhibition of IKK activity decreases p65 and EGR-1 promoter recruitment and CCL2 expression [41]. Interestingly, the NFB binding site in human CCL2 promoter has the same nucleotide sequence as the NFB site in human IL-8/CXCL8 promoter. Curiously, both CCL2 and IL-8 are increased by paclitaxel [83] and bortezomib [41], indicating that the paclitaxel and BZ-induced CCL2 (and IL-8) increase is promoter specific. Biomolecules 2015, 5 230 Biomolecules 2015, 5 230 Figure 1. Schematic illustration of human CCL2 promoter. Figure 1. Schematic illustration of human CCL2 promoter. Figure 1. Schematic illustration of human CCL2 promoter. Activity of the transcription factors STAT-1 and STAT-3 is also constitutively increased in OC cells, where it promotes cell motility and invasiveness [100]. Phosphorylation of STAT3 at tyrosine residues 705 and 727 increases its transcriptional activity [101]. In OC cells, IL-6 [102] and M-CSF [103] induce phosphorylation and activation of STAT3, and increase the CCL2 expression. In addition to NFB and STAT transcription factors, studies in other cell types indicated that the CCL2 expression is positively regulated by AP-1 and Hif-1 [104–107]. Though no transcription factors have been reported to be involved in the negative regulation of CCL2 in OC cells, studies involving other cell types have reported negative regulators of CCL2. Specifically, NFB p50/p50 homodimers, HDAC1, and the transcription factors Nrf2 and SMRT have been suggested to suppress the CCL2 expression in hepatic cells and adipocytes [108–110]. 3.2. CXCL1 CXCL1 (GRO-) contributes to ovarian cancer progression by inducing endothelial and epithelial cell proliferation and migration [25,26]. The putative transcription factor binding sites identified in human CXCL1 promoter are listed in Table 3. Experimental studies have demonstrated binding of the transcription factors p65 NFB, AP-2, CCAAT displacement protein (CDP), and the stimulating protein-1 (SP-1) to the CXCL1 promoter in human cells (Figure 2). In ovarian cancer cells, though, the CXCL1 gene expression was found to be regulated mainly by NFB pathway, specifically by the p65 DNA binding [25,27,28,111,112]. In addition to the positive regulation by p65 NFB, AP-2 and SP-1, studies using human melanocytes have indicated that the CXCL1 expression is negatively controlled by the transcriptional repressors CDP and the poly(ADPribose) polymerase-1 (PARP-1) [113,114]. The exact mechanisms of how CDP and PARP-1 inhibit the CXCL1 expression are not fully understood; however, they likely involve displacement of trans-activating factors that bind to CXCL1 promoter, resulting in transcriptional repression. 231 Biomolecules 2015, 5 omolecules 2015, 5 23 Figure 2. Schematic illustration of human CXCL1 promoter. Figure 2. Schematic illustration of human CXCL1 promoter. 3.3. CXCL2 The putative transcription factor binding sites identified in human CXCL2 (GRO-) promoter are listed in Table 4. However, experimental studies have demonstrated only binding of NFB, AP-1, and STAT3 to human CXCL2 promoter (Figure 3). In ovarian cancer cells, the CXCL2 expression is dependent on IB [28] and IKK [44]. In addition, the CXCL2 expression in OC cells is induced by TNF, and is inhibited by overexpression of the tumor suppressor p53 [115]. Figure 3. Schematic illustration of human CXCL2 promoter. Figure 3. Schematic illustration of human CXCL2 promoter. 3.4. CXCL8 4. Conclusions and Perspectives As we continue to improve our understanding of the mechanisms regulating chemokine expression in ovarian cancer cells, our knowledge will contribute to the development of new therapeutic strategies targeting the increased chemokine expression in chemoresistant metastatic ovarian cancer. Several important questions remain to be answered: What are the specific molecular targets and mechanisms responsible for the chemokine expression induced by chemotherapeutic drugs and hypoxia? What is the role of HDACs and other transcriptional repressors in regulating the chemokine expression in ovarian cancer cells? What is the role of the metabolic state of ovarian cancer cells in regulating the chemokine expression? Answers to these questions may open new avenues for therapeutic approaches for treating ovarian cancer. Author Contributions All authors have contributed to the drafting, writing and critical revision of the manuscript, and have approved the final version of the manuscript. Acknowledgments We apologize to any scientists whose work could not be cited in this review due to space limitations; Work in the Vancurova lab is supported by grant CA173452 from the National Institutes of Health. Biomolecules 2015, 5 233 Biomolecules 2015, 5 Transcription of IL-8 is also regulated by the transcription factor AP-1 that consists of Fos, FosB, Jun, and Jun-B subunits. Activation of AP-1 mediates the increased IL-8 expression in hypoxia, paclitaxel, and lysophosphatidic acid (LPA) treated OC cells [30,80,135]. Interestingly, a recent study has shown that the stress hormones norepinephrine and epinephrine enhance the IL-8 expression by a FosB-dependent mechanism [136]. Table 5 lists all putative transcription factor binding sites identified in the human CXCL8/IL-8 promoter. Although studies from other cell types have shown that the IL-8 expression is negatively regulated by the NFB repressing factor NRF, nuclear receptor corepressor (NCoR), the silencing mediator for retinoic acid and thyroid hormone receptor SMRT, and HDACs [54,137–139], the potential involvement of these corepressors in OC cells has yet to be demonstrated. Considering the important role these corepressors play in the IL-8 regulation, it will be important to elucidate their function in ovarian cancer setting. Conflicts of Interest The authors declare no conflicts of interest. 3.4. CXCL8 CXCL8 (IL-8), an inflammatory chemokine originally discovered as the neutrophil chemoattractant and inducer of leukocyte-mediated inflammation [1–3], contributes to cancer progression through its induction of tumor cell proliferation, migration and angiogenesis [4–9]. The expression levels of IL-8 Biomolecules 2015, 5 232 directly correlate with ovarian cancer progression, and suppression of IL-8 expression inhibits angiogenesis and tumorigenicity of ovarian cancer cells [13,116–118]. A number of studies have identified a minimal region in human IL-8 promoter that spans nucleotides -1 to -140, is necessary for IL-8 transcription, and contains binding sites for NFB, AP-1, CCAAT enhancer-binding protein beta (C/EBP or NF-IL6), Hif-1, and NFB-repressing factor (NRF) [119–127]. In addition, the IL-8 transcription in ovarian cancer cells is positively regulated by the transcription factor early growth response-1 (EGR-1) binding to IL-8 promoter, and by enzymes of IKK complex that phosphorylate both IB, leading to its cytoplasmic degradation, and p65 NFB, resulting in its increased transcriptional activity (Figure 4) [41–45]. Figure 4. Human CXCL8 promoter with the identified transcription factor binding sites. Figure 4. Human CXCL8 promoter with the identified transcription factor binding sites. NFB is crucial for the IL-8 expression, and regulates IL-8 in all cell types [128]. The NFB binding sequence (GGAATTTCC) is located between -80 and -70 of the IL-8 gene [120]. In most cell types, the IL-8 transcription is regulated predominantly by p65 homodimers [37,121,129–131]. Phosphorylation of p65 NFB on serines 276 and 536 increases its transcriptional activity and interaction with other transcription factor and regulators, and decreases its affinity for nuclear IB [129–133]. We have recently shown that in ovarian cancer cells, the IL-8 transcription is regulated by S536-p65 NFB, IKK, and EGR-1, and that proteasome inhibition developed as a strategy to inhibit NFB-dependent transcription, paradoxically increases the IL-8 expression in ovarian cancer cells by increasing the S536-p65, IKK and EGR-1 recruitment to IL-8 promoter [41]. Adjacent to the NFB site in the IL-8 promoter are C/EBP and Hif-1 binding sites (Figure 4). 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https://openalex.org/W1961974861	https://fixedpointtheoryandalgorithms.springeropen.com/counter/pdf/10.1186/s13663-015-0447-6	Latin	null	Viscosity approximation method for generalized asymptotically quasi-nonexpansive mappings in a convex metric space	Fixed point theory and applications	2,015	cc-by	6,909	© 2015 Khan et al. 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. R ES EA RCH Open Access Viscosity approximation method for generalized asymptotically quasi-nonexpansive mappings in a convex metric space Abdul Rahim Khan1, Nusrat Yasmin2, Haﬁz Fukhar-ud-din1,3 and Sami Atif Shukri1 Correspondence: nusyasmin@yahoo.com 2Centre for Advanced Studies in Pure and Applied Mathematics, Bahauddin Zakariya University, Multan, 60800, Pakistan Full list of author information is available at the end of the article Abstract A general viscosity iterative method for a ﬁnite family of generalized asymptotically quasi-nonexpansive mappings in a convex metric space is introduced. Special cases of the new iterative method are the viscosity iterative method of Chang et al. (Appl. Math. Comput. 212:51-59, 2009), an analogue of the viscosity iterative method of Fukhar-ud-din et al. (J. Nonlinear Convex Anal. 16:47-58, 2015) and an extension of the multistep iterative method of Yildirim and Özdemir (Arab. J. Sci. Eng. 36:393-403, 2011). Our results generalize and extend the corresponding known results in uniformly convex Banach spaces and CAT(0) spaces simultaneously. MSC: 47H09; 47H10; 47J25 Keywords: convex metric space; viscosity iterative method; generalized asymptotically quasi-nonexpansive mapping; uniformly Hölder continuous function; common ﬁxed point; strong convergence; △-convergence Khan et al. Fixed Point Theory and Applications ( 2015) 2015:196 DOI 10.1186/s13663-015-0447-6 Khan et al. Fixed Point Theory and Applications ( 2015) 2015:196 DOI 10.1186/s13663-015-0447-6 1 Introduction and preliminaries Let C be a nonempty subset of a metric space X and T : C →C be a mapping. We as- sume that F(T), the set of ﬁxed points of T, is nonempty and I = {,,,...,r}. The map- ping T is (i) quasi-nonexpansive if d(Tx,Ty) ≤d(x,y) for x ∈C, y ∈F(T); (ii) asymptot- ically quasi-nonexpansive if there exists a sequence of real numbers {un} in [,∞) with limn→∞un = such that d(Tnx,p) ≤(+ un)d(x,p) for all x ∈C, p ∈F(T) and n ≥; (iii) generalized asymptotically quasi-nonexpansive [] if there exist two sequences of real numbers {un} and {cn} in [,∞) with limn→∞un = = limn→∞cn such that d(Tnx,p) ≤ d(x,p) + und(x,p) + cn for all x ∈C, p ∈F(T) and n ≥; (iv) uniformly L-Lipschitzian if there exists a constant L > such that d(Tnx,Tny) ≤Ld(x,y) for all x,y ∈C and n ≥; (v) uniformly Hölder continuous if there are constants L > , γ > such that d(Tnx,Tny) ≤ Ld(x,y)γ for all x,y ∈C and n ≥; and (vi) semi-compact if for a sequence {xn} in C with limn→∞d(xn,Txn) = , there exists a subsequence {xni} of {xn} such that xni converges to a point in C. Clearly, the class of generalized asymptotically quasi-nonexpansive mappings includes the class of asymptotically quasi-nonexpansive mappings. Page 2 of 13 Khan et al. Fixed Point Theory and Applications ( 2015) 2015:196 The following example improves and extends Example .in [] to a ﬁnite family of generalized asymptotically quasi-nonexpansive mappings. Example .Let E = R and C = [–  π ,  π ] and deﬁne Tix = x i+sin(  x) if x ̸= and Tix = if x = for all x ∈C and i ∈I. Then Tn i x →uniformly (see []). For each ﬁxed n, deﬁne fin(x) = ∥Tn i x∥– ∥x∥for all x in C and i ∈I. Set cin = supx∈C{fin(x),}. Then limn→∞cin =  and Tn i x ≤∥x∥+ cin. This shows that {Ti : i ∈I} is a ﬁnite family of generalized asymptotically quasi-nonex- pansive mappings with i∈I F(Ti) ̸= ∅. Convergence theorems for various mappings through diﬀerent iterative methods have been obtained by a number of authors (e.g., [, , ] and the references therein). For more on the study of ﬁxed point iteration process, the interested reader is referred to Berinde [] and Ciric [, ]. 1 Introduction and preliminaries A nonempty subset C of a convex metric space X is convex if W(x,y,α) ∈C for all x,y ∈C and α ∈J. All normed linear spaces are convex met- ric spaces, but there are convex metric spaces which are not linear; for example, a CAT() space [, ]. for all x,y,u ∈X and α ∈J = [,]. The metric space X together with a convex structure W is known as a convex metric space. A nonempty subset C of a convex metric space X is convex if W(x,y,α) ∈C for all x,y ∈C and α ∈J. All normed linear spaces are convex met- ric spaces, but there are convex metric spaces which are not linear; for example, a CAT() space [, ]. A convex metric space X is uniformly convex if for any ε > , there exists δ = δ(ε) >  such that for all r > and x,y,z ∈X with d(z,x) ≤r, d(z,y) ≤r and d(x,y) ≥rε imply that d(z,W(x,y,  )) ≤(– δ)r. A mapping η : (,∞) × (,] →(,] which provides such δ = η(r,ϵ) for given r > and ε ∈(,] is called modulus of uniform convexity. We call η monotone if it decreases with r (for a ﬁxed ϵ). Obviously, uniformly convex Banach spaces are uniformly convex metric spaces. In general, a convex structure W is not continuous []. Throughout this paper, we as- sume that W is continuous. We now devise a general iterative method which extends the methods in (.) and (.) simultaneously in a convex metric space. We now devise a general iterative method which extends the methods in (.) and (.) simultaneously in a convex metric space. We deﬁne an Sn-mapping generated by a family {Ti : i ∈I} of generalized asymptotically quasi-nonexpansive mappings on C as Snx = Urnx, (.) (.) Snx = Urnx, where Un = I (the identity mapping), Unx = W(Tn r Unx,Unx,arn),Unx = W(Tn r–Unx, Unx,a(r–)n),...,Urnx = W(Tn U(r–)nx,U(r–)nx,an). where Un = I (the identity mapping), Unx = W(Tn r Unx,Unx,arn),Unx = W(Tn r–Unx, Unx,a(r–)n),...,Urnx = W(Tn U(r–)nx,U(r–)nx,an). where Un = I (the identity mapping), Unx = W(Tn r Unx,Unx,arn),Unx = W(Tn r–Unx, Unx,a(r–)n),...,Urnx = W(Tn U(r–)nx,U(r–)nx,an). where Un = I (the identity mapping), Unx = W(Tn r Unx,Unx,arn),Unx = W(Tn r–Unx, Unx,a(r–)n),...,Urnx = W(Tn U(r–)nx,U(r–)nx,an). 1 Introduction and preliminaries Let C be a convex subset of a normed space. Yildirim and Özdemir [] introduced the following multistep iterative method: x∈C, xn+= (– an)yn+r–+ anTn yn+r–, yn+r–= (– an)yn+r–+ anTn yn+r–, ( ) x∈C, xn+= (– an)yn+r–+ anTn yn+r–, yn+r–= (– an)yn+r–+ anTn yn+r–, (.) xn+= (– an)yn+r–+ anTn yn+r–, yn+r–= (– an)yn+r–+ anTn yn+r–, xn+= (– an)yn+r–+ anTn yn+r–, (.) yn+= (– a(r–)n)yn + a(r–)nTn (r–)yn, yn = (– arn)xn + arnTn r xn, r ≥,n ≥, yn = (– arn)xn + arnTn r xn, r ≥,n ≥, where {Ti : i ∈I} is a family of self-mappings of C, ain ∈[ϵ,– ϵ], for some ϵ ∈(,  ), for all n ≥. where {Ti : i ∈I} is a family of self-mappings of C, ain ∈[ϵ,– ϵ], for some ϵ ∈(,  ), for all n ≥. If T= T= ··· = Tr and αjn = for j = ,...,r and r ≥, then the iterative method (.) reduces to the Mann iterative method []. Let us note that the scheme (.) and multistep scheme (.) in [] are independent of each other. Moudaﬁ[] proposed a viscosity iterative method by selecting a particular ﬁxed point of a given nonexpansive mapping. The so-called viscosity iterative method has been studied by many authors (see, for example, [, ]). These methods are very important because of their applicability to convex optimization, linear programming, monotone inclusions and elliptic diﬀerential equations []. Recently, Chang et al. [] introduced and studied the following viscosity iterative method: xn+= (– αn)f (xn) + αnTnyn, yn = (– βn)xn + βnTnxn, n ≥, (.) (.) where T is an asymptotically nonexpansive mapping [] and f is a ﬁxed contraction. where T is an asymptotically nonexpansive mapping [] and f is a ﬁxed contraction. Page 3 of 13 Khan et al. Fixed Point Theory and Applications ( 2015) 2015:196 Page 3 of 13 Page 3 of 13 The iterative methods in (.) and (.) involve convex combinations, and so a convex structure is needed to deﬁne them on a nonlinear domain. A mapping W : X× J →X is a convex structure [] on a metric space X if d u,W(x,y,α) ≤αd(u,x) + (– α)d(u,y) for all x,y,u ∈X and α ∈J = [,]. The metric space X together with a convex structure W is known as a convex metric space. 1 Introduction and preliminaries For {αn} ⊂J, a ﬁxed contractive mapping f on C and Sn given in (.), we deﬁne {xn} as follows: x∈C, xn+= W f (xn),Snxn,αn (.) x∈C, xn+= W f (xn),Snxn,αn (.) and call it a general viscosity iterative method in a convex metric space. and call it a general viscosity iterative method in a convex metric space. and call it a general viscosity iterative method in a convex metric space. The purpose of this paper is to: The purpose of this paper is to: The purpose of this paper is to: (i) establish a necessary and suﬃcient condition for convergence of iterative method (i) establish a necessary and suﬃcient condition for convergence of iterative method (.) to a common ﬁxed point of a ﬁnite family of generalized asymptotically quasi-nonexpansive mappings on a convex metric space; (ii) prove strong convergence and △-convergence results for the iterative me to a common ﬁxed point of a ﬁnite family of generalized asymptotically quasi-nonexpansive mappings on a uniformly convex metric space. We now assume that F = i∈I F(Ti) ̸= ∅. We need the following known results for our convergence analysis. Khan et al. Fixed Point Theory and Applications ( 2015) 2015:196 Page 4 of 13 Lemma .(cf. []) Let the sequences {an} and {un} of real numbers satisfy an+≤(+ un)an, an ≥,un ≥, ∞ n= un < +∞. Then (i) limn→∞an exists; (ii) if liminfn→∞an = , then limn→∞an = . Then (i) limn→∞an exists; (ii) if liminfn→∞an = , then limn→∞an = . Lemma .([]) Let X be a uniformly convex metric space. Let x ∈X and {an} be a sequence in [b,c] for some b,c ∈(,). If {un} and {vn} are sequences in X such that limsupn→∞d(un,x) ≤r, limsupn→∞d(vn,x) ≤r and limn→∞d(W(un,vn,an),x) = r for some r ≥, then limn→∞d(un,vn) = . 2 Convergence in convex metric spaces In this section, we prove some results for the viscosity iterative method (.) to converge to a common ﬁxed point of a ﬁnite family of generalized asymptotically quasi-nonexpansive mappings in a convex metric space. Lemma .Let C be a nonempty, closed and convex subset of a convex metric space X and {Ti : i ∈I} be a family of generalized asymptotically quasi-nonexpansive self-mappings of C, i.e., d(Tn i x,pi) ≤(+ uin)d(x,pi) + cin for all x ∈C and pi ∈F(Ti), i ∈I, where {uin} and {cin} are sequences in [,∞) with ∞ n=uin < ∞, ∞ n=cin < ∞for each i. Then, for the sequence {xn} in (.) with ∞ n=αn < ∞, there are sequences {νn} and {ξn} in [,∞) satisfying ∞ n=νn < ∞, ∞ n=ξn < ∞such that (a) d(xn+,p) ≤(+ νn)rd(xn,p) + ξn for all p ∈F and all n ≥; (b) d(xn+m,p) ≤M(d(xn,p) + ∞ n=ξn) for all p ∈F and n ≥, m ≥, M> . Proof (a) Let p ∈F and νn = maxi∈I uin for all n ≥. Since ∞ n=uin < ∞for each i, therefore ∞ n=νn < ∞. Now we have d(Unxn,p) = d W Tn r Unxn,Unxn,arn ,p ≤(– arn)d(xn,p) + arnd Tn r xn,p ≤(– arn)d(xn,p) + arn (+ urn)d(xn,p) + crn ≤(+ urn)d(xn,p) + crn ≤(+ νn)d(xn,p) + crn. Assume that d(Uknxn,p) ≤(+ νn)kd(xn,p) + (+ νn)k–k i=c(r–i+)n holds for some < k. Consider Assume that d(Uknxn,p) ≤(+ νn)kd(xn,p) + (+ νn)k–k i=c(r–i+)n holds for some < k. Consider Assume that d(Uknxn,p) ≤(+ νn)kd(xn,p) + (+ νn)k–k i=c(r–i+)n holds for some < k. Consider Consider d(U(k+)nxn,p) = d W Tn r–kUknxn,Uknxn,a(r–k)n ,p ≤(– a(r–k)n)d(Uknxn,p) + a(r–k)nd Tn r–kUknxn,p ≤(– a(r–k)n)d(Uknxn,p) + a(r–k)n (+ u(r–k)n)d(Uknxn,p) + c(r–k)n ≤(+ νn)d(Uknxn,p) + c(r–k)n d(U(k+)nxn,p) = d W Tn r–kUknxn,Uknxn,a(r–k)n ,p ≤(– a(r–k)n)d(Uknxn,p) + a(r–k)nd Tn r–kUknxn,p ≤(– a(r–k)n)d(Uknxn,p) + a(r–k)n (+ u(r–k)n)d(Uknxn,p) + c(r–k)n ≤(+ νn)d(Uknxn,p) + c(r–k)n heory and Applications ( 2015) 2015:196 Page 5 of 13 Khan et al. Fixed Point Theory and Applications ( 2015) 2015:196 Page 5 of 13 Khan et al. Fixed Point Theory and Applications ( 2015) 2015:196 Page 5 of 13 Khan et al. 2 Convergence in convex metric spaces Fixed Point Theory and Applications ( 2015) 2015:196 Page 5 of 13 ≤(+ νn) (+ νn)kd(xn,p) + (+ νn)k– k i= c(r–i+)n + c(r–k)n ≤(+ νn)k+d(xn,p) + (+ νn)k k+ i= c(r–i+)n. ≤(+ νn) (+ νn)kd(xn,p) + (+ νn)k– k i= c(r–i+)n + c(r–k)n ≤(+ νn)k+d(xn,p) + (+ νn)k k+ i= c(r–i+)n. By mathematical induction, we have By mathematical induction, we have By mathematical induction, we have By mathematical induction, we have d(Ujnxn,p) ≤(+ νn)jd(xn,p) + (+ νn)j– j i= c(r–i+)n, ≤j ≤r. (.) (.) Hence d(Snxn,p) = d(Urnxn,p) ≤(+ νn)rd(xn,p) + (+ νn)r– r i= c(r–i+)n. (.) (.) Now, by (.) and (.), we obtain Now, by (.) and (.), we obtain d(xn+,p) = d W f (xn),Snxn,αn ,p ≤αnd f (xn),p + (– αn)d(Snxn,p) ≤αnd(xn,p) + αnd f (p),p + (– αn) (+ νn)rd(xn,p) + (+ νn)r– r i= c(r–i+)n ≤(+ νn)rd(xn,p) + (– αn)(+ νn)r– r i= c(r–i+)n + αnd f (p),p ≤(+ νn)rd(xn,p) + αnd f (p),p + (+ νn)r– r i= c(r–i+)n. i= ( ) ≤(+ νn)rd(xn,p) + (– αn)(+ νn)r– r i= c(r–i+)n + αnd f (p),p ≤(+ νn)rd(xn,p) + αnd f (p),p + (+ νn)r– r i= c(r–i+)n. Setting max{d(f (p),p),sup(+ νn)r–} = M, we get that Setting max{d(f (p),p),sup(+ νn)r–} = M, we get that d(xn+,p) ≤(+ νn)rd(xn,p) + M αn + r i= c(r–i+)n . d(xn+,p) ≤(+ νn)rd(xn,p) + M αn + r i= c(r–i+)n . d(xn+,p) ≤(+ νn)rd(xn,p) + M αn + r i= c(r–i+)n . That is, That is, d(xn+,p) ≤(+ νn)rd(xn,p) + ξn, d(xn+,p) ≤(+ νn)rd(xn,p) + ξn, where ξn = M(αn + r i=c(r–i+)n) and ∞ n=ξn < ∞. (b) We know that + t ≤et for t ≥. Thus, by part (a), we have where ξn = M(αn + r i=c(r–i+)n) and ∞ n=ξn < ∞. where ξn = M(αn + r i=c(r–i+)n) and ∞ n=ξn < ∞. (b) We know that + t ≤et for t ≥. Thus, by part (a), we have d(xn+m,p) ≤(+ νn+m–)rd(xn+m–,p) + ξn+m– d(xn+m,p) ≤(+ νn+m–)rd(xn+m–,p) + ξn+m– ≤erνn+m–d(xn+m–,p) + ξn+m– ≤erνn+m–d(xn+m–,p) + ξn+m– ≤erνn+m–d(xn+m–,p) + ξn+m– ≤er(νn+m–+νn+m–)d(xn+m–,p) + ξn+m–+ ξn+m– ≤er(νn+m–+νn+m–)d(xn+m–,p) + ξn+m–+ ξn+m– heory and Applications ( 2015) 2015:196 Page 6 of 13 Khan et al. 2 Convergence in convex metric spaces Fixed Point Theory and Applications ( 2015) 2015:196 Page 6 of 13 Page 6 of 13 ≤er n+m– i=n vid(xn,p) + n+m– i=n+ vi n+m– i=n ξi ≤er ∞ i=vi d(xn,p) + ∞ i= ξi = M d(xn,p) + ∞ i= ξi , where M= er ∞ i=vi. □ ≤er n+m– i=n vid(xn,p) + n+m– i=n+ vi n+m– i=n ξi ≤er ∞ i=vi d(xn,p) + ∞ i= ξi = M d(xn,p) + ∞ i= ξi , where M= er ∞ i=vi. □ □ The next result deals with a necessary and suﬃcient condition for the convergence of {xn} in (.) to a point of F. Theorem .Let C, {Ti : i ∈I}, F, {uin} and {cin} be as in Lemma .. Let X be complete. The sequence {xn} in (.) with ∞ n=αn < ∞converges strongly to a point in F if and only if liminfn→∞d(xn,F) = , where d(x,F) = infp∈F(x,p). Proof The necessity is obvious; we only prove the suﬃciency. By Lemma .(a), we have d(xn+,p) ≤(+ νn)rd(xn,p) + ξn for all p ∈F and n ≥. d(xn+,p) ≤(+ νn)rd(xn,p) + ξn for all p ∈F and n ≥. d(xn+,p) ≤(+ νn)rd(xn,p) + ξn for all p ∈F and n ≥. herefore, Therefore, d(xn+,F) ≤(+ νn)rd(xn,F) + ξn = + r k= r(r – )···(r – k + ) k! νk n d(xn,F) + ξn. d(xn+,F) ≤(+ νn)rd(xn,F) + ξn = + r k= r(r – )···(r – k + ) k! νk n d(xn,F) + ξn. As ∞ n=νn < +∞, so ∞ n= r k= r(r–)···(r–k+) k! νk n < ∞. Now ∞ n=ξn < ∞in Lemma .(a), so by Lemma .and liminfn→∞d(xn,F) = , we get that limn→∞d(xn,F) = . Next, we prove that {xn} is a Cauchy sequence in X. Let ε > . From the proof of Lemma .(b), we have d(xn+m,xn) ≤d(xn+m,F) + d(xn,F) ≤(+ M)d(xn,F) + M ∞ i=n ξi. (.) (.) As limn→∞d(xn,F) = and ∞ i=ξi < ∞, so there exists a natural number nsuch that As limn→∞d(xn,F) = and ∞ i=ξi < ∞, so there exists a natural number nsuch that d(xn,F) ≤ ε (+ M) and ∞ i=n ξi < ε M for all n ≥n. 2 Convergence in convex metric spaces So, for all integers n ≥n, m ≥, we obtain from (.) that So, for all integers n ≥n, m ≥, we obtain from (.) that So, for all integers n ≥n, m ≥, we obtain from (.) that d(xn+m,xn) < (M+ ) ε (+ M) + M ε M = ε. Thus, {xn} is a Cauchy sequence in X and so it converges to q ∈X. Finally, we show that q ∈F. For any ε > , there exists a natural number nsuch that Thus, {xn} is a Cauchy sequence in X and so it converges to q ∈X. Finally, we show that q ∈F. For any ε > , there exists a natural number nsuch that d(xn,F) = inf p∈F d(xn,p) < ε  and d(xn,q) < ε  for all n ≥n. Khan et al. Fixed Point Theory and Applications ( 2015) 2015:196 Page 7 of 13 d p∗,q ≤d xn,p∗ + d(xn,q) < ε. d p∗,q ≤d xn,p∗ + d(xn,q) < ε. □ Remark .A generalized asymptotically nonexpansive mapping is a generalized asymp- totically quasi-nonexpansive mapping. So Theorem .holds good for the class of gener- alized asymptotically nonexpansive mappings. 3 Results in a uniformly convex metric space The aim of this section is to establish some convergence results for the iterative method (.) of generalized asymptotically quasi-nonexpansive mappings on a uniformly convex metric space. Lemma .Let C be a nonempty, closed and convex subset of a uniformly convex metric space X and {Ti : i ∈I} be a family of uniformly Hölder continuous and generalized asymp- totically quasi-nonexpansive self-mappings of C, i.e., d(Tn i x,pi) ≤(+ uin)d(x,pi) + cin for all x ∈C and pi ∈F(Ti), where {uin} and {cin} are sequences in [,∞) with ∞ n=uin < ∞ and ∞ n=cin < ∞, respectively, for each i ∈I. Then, for the sequence {xn} in (.) with ain ∈[δ,– δ] for some δ ∈(,  ) and ∞ n=αn < ∞, we have limn→∞d(xn,Tjxn) = for each j ∈I. Proof Let p ∈F and νn = maxi∈I uin for all n ≥. By Lemma .(i) and Lemma .(a), it follows that limn→∞d(xn,p) exists for all p ∈F. Assume that lim n→∞d(xn,p) = c. (.) (.) lim n→∞d(xn,p) = c. lim n→∞d(xn,p) = c. Inequality (.) together with (.) gives that Inequality (.) together with (.) gives that Inequality (.) together with (.) gives that limsup n→∞ d(Ujnxn,p) ≤c, ≤j ≤r. (.) (.) limsup n→∞ d(Ujnxn,p) ≤c, ≤j ≤r. By (.), we have By (.), we have d(xn+,p) = d W f (xn),Snxn,αn ,p ≤αnd f (xn),p + (– αn)d(Snxn,p) ≤αnd f (xn),p + αnd f (p),p + (– αn)d(Urnxn,p), and hence c ≤liminf n→∞d(Urnxn,p). (.) Combining (.) and (.), we get lim n→∞d(Urnxn,p) = c. and hence and hence (.) lim n→∞d(Urnxn,p) = c. Page 8 of 13 Khan et al. Fixed Point Theory and Applications ( 2015) 2015:196 Note that d(Urnxn,p) = d W Tn U(r–)nxn,U(r–)nxn,an ,p ≤and Tn U(r–)nxn,p + (– an)d(U(r–)nxn,p) ≤an (+ un)d(U(r–)nxn,p) + cn + (– an)d(U(r–)nxn,p) ≤an(+ νn)d(U(r–)nxn,p) + ancn ≤an(+ νn) an(+ νn)d(U(r–)nxn,p) + ancn + an(+ νn)cn ≤anan(+ νn)d(U(r–)nxn,p) + anan(+ νn)cn + ancn ... ≤anan ···a(j–)n(+ νn)j–d(U(r–(j–))nxn,p) + anan ···a(j–)n(+ νn)(j–)–c(j–)n + anan ···a((j–)–)n(+ νn)(j–)–c((j–)–)n + ··· + anan(+ νn)cn + ancn. Hence c ≤liminf n→∞d(U(r–(j–))nxn,p), ≤j ≤r. (.) Using (.) and (.), we have lim n→∞d(U(r–(j–))nxn,p) = c. That is, lim n→∞d W Tn j U(r–j)nxn,U(r–j)nxn,ajn ,p = c for ≤j ≤r. 3 Results in a uniformly convex metric space This together with (.), (.) and Lemma .gives that lim n→∞d Tn j U(r–j)nxn,U(r–j)nxn, =  for ≤j ≤r. (.) If j = r,we have by (.) lim d Tnx x =  Note that d(Urnxn,p) = d W Tn U(r–)nxn,U(r–)nxn,an ,p ≤and Tn U(r–)nxn,p + (– an)d(U(r–)nxn,p) ≤an (+ un)d(U(r–)nxn,p) + cn + (– an)d(U(r–)nxn,p) ≤an(+ νn)d(U(r–)nxn,p) + ancn ≤an(+ νn) an(+ νn)d(U(r–)nxn,p) + ancn + an(+ νn)cn ≤anan(+ νn)d(U(r–)nxn,p) + anan(+ νn)cn + ancn ... ≤anan ···a(j–)n(+ νn)j–d(U(r–(j–))nxn,p) + anan ···a(j–)n(+ νn)(j–)–c(j–)n + anan ···a((j–)–)n(+ νn)(j–)–c((j–)–)n + ··· + anan(+ νn)cn + ancn. Note that Note that Note that ≤an(+ νn) an(+ νn)d(U(r–)nxn,p) + ancn + an(+ νn)cn ≤anan(+ νn)d(U(r–)nxn,p) + anan(+ νn)cn + ancn ≤anan ···a(j–)n(+ νn)j–d(U(r–(j–))nxn,p) + anan ···a(j–)n(+ νn)(j–)–c(j–)n + anan ···a((j–)–)n(+ νn)(j–)–c((j–)–)n + ··· + anan(+ νn)cn + ancn. ≤anan ···a(j–)n(+ νn)j–d(U(r–(j–))nxn,p) + anan ···a(j–)n(+ νn)(j–)–c(j–)n + anan ···a((j–)–)n(+ νn)(j–)–c((j–)–)n + ··· + anan(+ νn)cn + ancn. Hence (.) c ≤liminf n→∞d(U(r–(j–))nxn,p), ≤j ≤r. Using (.) and (.), we have lim n→∞d(U(r–(j–))nxn,p) = c. That is, lim n→∞d W Tn j U(r–j)nxn,U(r–j)nxn,ajn ,p = c for ≤j ≤r. This together with (.), (.) and Lemma .gives that lim n→∞d Tn j U(r–j)nxn,U(r–j)nxn, =  for ≤j ≤r. (.) (.) lim n→∞d Tn j U(r–j)nxn,U(r–j)nxn, =  for ≤j ≤r. (.) If j = r,we have by (.) lim n→∞d Tn r xn,xn = . In case j ∈{,,,...,r – }, we observe that In case j ∈{,,,...,r – }, we observe that In case j ∈{,,,...,r – }, we observe that d(xn,U(r–j)nxn) = d xn,W Tn j+U(r–(j+))nxn,U(r–(j+))nxn,a(j+)n ≤a(j+)nd Tn j+U(r–(j+))nxn,xn + (– a(j+)n)d(U(r–(j+))nxn,xn) ≤(+ νn)d(U(r–(j+))nxn,xn) + c(j+)n Page 9 of 13 Khan et al. Fixed Point Theory and Applications ( 2015) 2015:196 ≤(+ νn)r–jd(Unxn,xn) + (+ νn)r–j–crn + (+ νn)r–j–c(r–)n + ··· + (+ νn)c(j+)n + c(j+)n. Hence, lim n→∞d(xn,U(r–j)nxn) = . (.) (.) lim n→∞d(xn,U(r–j)nxn) = . Since Tj is uniformly Hölder continuous, therefore the inequality d Tn j xn,xn ≤d Tn j xn,Tn j U(r–j)nxn + d Tn j U(r–j)nxn,U(r–j)nxn + d(U(r–j)nxn,xn) ≤Ld(xn,U(r–j)nxn)γ + d(xn,U(r–j)nxn) + d Tn j U(r–j)nxn,U(r–j)nxn d Tn j xn,xn ≤d Tn j xn,Tn j U(r–j)nxn + d Tn j U(r–j)nxn,U(r–j)nxn + d(U(r–j)nxn,xn) ≤Ld(xn,U(r–j)nxn)γ + d(xn,U(r–j)nxn) + d Tn j U(r–j)nxn,U(r–j)nxn ≤Ld(xn,U(r–j)nxn)γ + d(xn,U(r–j)nxn) + d Tn j U(r–j)nxn,U(r–j)nxn together with (.) and (.) gives that together with (.) and (.) gives that together with (.) and (.) gives that lim n→∞d Tn j xn,xn = . Hence, Hence, d Tn j xn,xn → as n →∞for ≤j ≤r. (.) (.) As before, we can show that d(xn,xn+) = d xn,W f (xn),Snxn,αn ≤αn(+ α)d(xn,p) + αnd p,f (p) + (– αn) and U(r–)nxn,Tn U(r–)nxn + d(xn,U(r–)nxn) . Therefore, by (.) and (.), we get Therefore, by (.) and (.), we get Therefore, by (.) and (.), we get lim n→∞d(xn,xn+) = . (.) Let us observe that lim n→∞d(xn,xn+) = . (.) (.) lim n→∞d(xn,xn+) = . (.) Let us observe that d(xn,Tjxn) ≤d(xn,xn+) + d xn+,Tn+ j xn+ + d Tn+ j xn+,Tn+ j xn + d Tn+ j xn,Tjxn ≤d(xn,xn+) + d xn+,Tn+ j xn+ + Ld(xn+,xn)γ + Ld Tn j xn,xn γ . By the uniform Hölder continuity of Tj, (.) and (.), we get By the uniform Hölder continuity of Tj, (.) and (.), we get lim n→∞d(xn,Tjxn) = , ≤j ≤r. (.) □ (.) □ (.) lim n→∞d(xn,Tjxn) = , ≤j ≤r. (.) □ □ Khan et al. In case j ∈{,,,...,r – }, we observe that Fixed Point Theory and Applications ( 2015) 2015:196 Page 10 of 13 Theorem .Under the hypotheses of Lemma ., assume, for some ≤j ≤r, that Tm j is semi-compact for some positive integer m. If X is complete, then {xn} in (.) converges strongly to a point in F. Proof Fix j ∈I and suppose Tm j to be semi-compact for some m ≥. By (.), we obtain d Tm j xn,xn ≤d Tm j xn,Tm– j xn + d Tm– j xn,Tm– j xn + ··· + d T j xn,Tjxn + d(Tjxn,xn) ≤d(Tjxn,xn) + (m – )Ld(Tjxn,xn)γ →. ≤d(Tjxn,xn) + (m – )Ld(Tjxn,xn)γ →. Since {xn} is bounded and Tm j is semi-compact, {xn} has a convergent subsequence {xni} such that xni →q ∈C. Hence, by (.), we have Since {xn} is bounded and Tm j is semi-compact, {xn} has a convergent subsequence {xni} such that xni →q ∈C. Hence, by (.), we have d(q,Tiq) = lim n→∞d(xnj,Tixnj) = , i ∈I. d(q,Tiq) = lim n→∞d(xnj,Tixnj) = , i ∈I. Thus q ∈F, and so by Theorem ., {xn} converges strongly to a common ﬁxed point q of the family {Ti : i ∈I}. □ Thus q ∈F, and so by Theorem ., {xn} converges strongly to a common ﬁxed point q of the family {Ti : i ∈I}. □ An immediate consequence of Lemma .and Theorem .is the following strong con- vergence result in uniformly convex metric spaces. Theorem .Let C, {Ti : i ∈I}, F, {uin} and {cin} be as in Lemma .. If there exists a constant M such that d(xn,Tjxn) ≥Md(xn,F) for all n ≥and X is complete, then the sequence {xn} in (.) converges strongly to a point in F. The concept of △-convergence in a metric space was introduced by Lim [] and its analogue in CAT() spaces was investigated by Dhompongsa and Panyanak []. Here we study △-convergence in uniformly convex metric spaces. For this, we collect some basic concepts. Let {xn} be a bounded sequence in a uniformly convex metric space X. For x ∈X, deﬁne a continuous functional r(·,{xn}) : X →[,∞) by r x,{xn} = limsup n→∞ d(x,xn). r x,{xn} = limsup n→∞ d(x,xn). Now, we establish △-convergence of the iterative method (.). Theorem .Let C be a nonempty, closed and convex subset of a complete uniformly con- vex metric space X with monotone modulus of uniform convexity η, and let {Ti : i ∈I} be a family of uniformly L-Lipschitzian and generalized asymptotically nonexpansive self- mappings of C such that F ̸= φ, i.e., d(Tn i x,Tn i y) ≤(+ uin)d(x,y) + cin for all x,y ∈C, where {uin} and {cin} are sequences in [,∞) with ∞ n=uin < ∞and ∞ n=cin < ∞, respectively, for each i ∈I. Then the sequence {xn} in (.) with ain ∈[δ,– δ] for some δ ∈(,  ) and ∞ n=αn < ∞, △-converges to a common ﬁxed point of {Tj : j ∈I}. Proof By Lemma ., {xn} is bounded, and so by Lemma ., {xn} has a unique asymptotic center, that is, A({xn}) = {x}. Let {zn} be any subsequence of {xn} such that A({zn}) = {z}. Also by Lemma ., we have limn→∞d(zn,Tjzn) = for each j ∈I. Proof By Lemma ., {xn} is bounded, and so by Lemma ., {xn} has a unique asymptotic center, that is, A({xn}) = {x}. Let {zn} be any subsequence of {xn} such that A({zn}) = {z}. Also by Lemma ., we have limn→∞d(zn,Tjzn) = for each j ∈I. We claim that z is a common ﬁxed point of {Tj : j ∈I}. To show this, we deﬁne a sequence {wk} in C by wk = Tk j z, d(wk,zn) = d Tk j z,zn ≤d Tk j z,Tk j zn + k i= d Ti j zn,Ti– j zn ≤(+ ujn)d(z,zn) + cjn + kLd(Tjzn,zn). d(wk,zn) = d Tk j z,zn ≤d Tk j z,Tk j zn + k i= d Ti j zn,Ti– j zn ≤(+ ujn)d(z,zn) + cjn + kLd(Tjzn,zn). Taking limsup, Taking limsup, limsup n→∞ d(wk,zn) ≤limsup n→∞ d(z,zn), i.e., r(Tk j z,zn) ≤r(z,zn). It follows from Lemma .that limk→∞Tk j z = z. As Tj is uniformly continuous, we have Tjz = Tj(limk→∞Tk j z) = limk→∞Tk+ j z = z. Therefore, z is a common ﬁxed point of {Tj : j ∈I}. j Recall that limn→∞d(xn,z) exists by Lemma .. Recall that limn→∞d(xn,z) exists by Lemma .. Suppose x ̸= z. In case j ∈{,,,...,r – }, we observe that The asymptotic radius ρ = r({xn}) of {xn} is given by The asymptotic radius ρ = r({xn}) of {xn} is given by The asymptotic radius ρ = r({xn}) of {xn} is given by ρ = inf r x,{xn} : x ∈X . The asymptotic center of a bounded sequence {xn} with respect to a subset C of X is deﬁned as follows: AC {xn} = x ∈X : r x,{xn} ≤r y,{xn} for any y ∈C . If the asymptotic center is taken with respect to X, then it is simply denoted by A({xn}). A sequence {xn} in X is said to △-converge to x ∈X if x is the unique asymptotic center of {un} for every subsequence {un} of {xn}. In this case, we write △-limn xn = x and call x as △-limit of {xn}. Page 11 of 13 Khan et al. Fixed Point Theory and Applications ( 2015) 2015:196 Lemma .([]) Let (X,d) be a complete uniformly convex metric space with monotone modulus of uniform convexity. Then every bounded sequence {xn} in X has a unique asymp- totic center with respect to any nonempty closed convex subset C of X. Lemma .([]) Let C be a nonempty closed convex subset of a uniformly convex metric space and {xn} be a bounded sequence in C such that A({xn}) = {y} and r({xn}) = ρ. If {ym} is another sequence in C such that limm→∞r(ym,{xn}) = ρ, then limm→∞ym = y. Now, we establish △-convergence of the iterative method (.). Authors’ contributions Authors’ contributions The authors have contributed to this work on an equal basis. All authors read and approved the ﬁnal manuscript. Author details 1Department of Mathematics and Statistics, King Fahd University of Petroleum and Minerals, Dhahran, 31261, Saudi Arabia. 2Centre for Advanced Studies in Pure and Applied Mathematics, Bahauddin Zakariya University, Multan, 60800, Pakistan. 3Department of Mathematics, Islamia University, Bahawlpur, 63100, Pakistan. Open problem Assume that the initial point is the same in scheme (.) and multistep scheme (.) in []. Under what conditions are these schemes equivalent? Open problem Assume that the initial point is the same in scheme (.) and multistep scheme (.) in []. Under what conditions are these schemes equivalent? Now, we establish △-convergence of the iterative method (.). By the uniqueness of asymptotic centers, we obtain ≤limsup n→∞ d(xn,x) < limsup n→∞ d(xn,z) = limsup n→∞ d(zn,z), Khan et al. Fixed Point Theory and Applications ( 2015) 2015:196 Page 12 of 13 a contradiction. Hence x = z. Since {zn} is an arbitrary subsequence of {xn}, therefore A({zn}) = {z} for all subsequences {zn} of {xn}. This proves that {xn} △-converges to a com- mon ﬁxed point of {Tj : j ∈I}. □ a contradiction. Hence x = z. Since {zn} is an arbitrary subsequence of {xn}, therefore A({zn}) = {z} for all subsequences {zn} of {xn}. This proves that {xn} △-converges to a com- mon ﬁxed point of {Tj : j ∈I}. □ Competing interests Competing interests The authors declare that they have no competing interests. References 1. Shahzad, N, Zegeye, H: Strong convergence of an implicit iteration process for a ﬁnite family of generalized 1. Shahzad, N, Zegeye, H: Strong convergence of an implicit iteration process for a ﬁnite family asymptotically quasi-nonexpansive maps. Appl. Math. Comput. 189, 1058-1065 (2007) asymptotically quasi-nonexpansive maps. Appl. Math. Comput. 189, 1058-1065 (2007) 2. Kim, TH, Choi, JW: Asymptotic behavior of almost-orbits of non-Lipschitzian mappings in Banach spaces. Math. Jpn. 38, 191-197 (1993) 3. Plubtieng, S, Thammathiwat, T: A viscosity approximation method for ﬁnding a common solution of ﬁxed points and equilibrium problems in Hilbert spaces. J. Glob. Optim. 50, 313-327 (2011) 4. 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Remark . (i) Lemma ., Theorems .and .set an analogue of Theorems .-.in [] and Lemma ., Theorems .and .in [], in uniformly convex metric spaces. (ii) Lemma .and Theorem .provide an analogue of Lemma .and Theorem . in [] and Lemma .and Theorem .in [] in uniformly convex metric spaces. (iii) Theorems .and .extend Theorems ., ., and .in [], to convex metric spaces (ii) Lemma .and Theorem .provide an analogue of Lemma .and Theorem . in [] and Lemma .and Theorem .in [] in uniformly convex metric spaces. ( ) p g in [] and Lemma .and Theorem .in [] in uniformly convex metric spaces. (iii) Theorems .and .extend Theorems ., ., and .in [], to convex metric spaces. (iii) Theorems .and .extend Theorems ., ., and .in [], to convex metric spaces. (iii) Theorems .and .extend Theorems ., ., and .in [], to convex metric spaces. (iv) Our results give an analogue of the results in []. 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https://openalex.org/W1557107102	https://pure.eur.nl/files/47854718/REPUB_93695_OA.pdf	English	null	Cannabis use and support for cannabis legalization	Empirical economics	2,016	cc-by	12,916	Empir Econ DOI 10.1007/s00181-016-1172-7 Cannabis use and support for cannabis legalization Ali Palali1,2 · Jan C. van Ours3,4 Received: 27 July 2015 / Accepted: 4 August 2016 Received: 27 July 2015 / Accepted: 4 August 2016 © The Author(s) 2016. This article is published with open access at Springerlink.com y p g © The Author(s) 2016. This article is published with open access at Springerlink.com Abstract We investigate the determinants of the support for cannabis legalization ﬁnding a causal effect of personal experience with cannabis use. Current and past cannabis users are more in favor of legalization. We relate this ﬁnding to self-interest and inside information about potential dangers of cannabis. While the self-interest effect is not very surprising, the effect of inside information suggests that cannabis use is not as harmful as cannabis users originally thought it was before they started consuming. Our analysis suggests that as the share of cannabis users in the population increases, support for cannabis legalization will also increase. Keywords Cannabis use · Opinions on cannabis policy · Mixed proportional hazard model · Factor approach JEL Classiﬁcation I18 · K14 · C31 JEL Classiﬁcation I18 · K14 · C31 B Jan C. van Ours vanours@ese.eur.nl 1 Introduction In many countries the production, use and distribution of cannabis are prohibited. How- ever, the legal framework on cannabis is changing as some countries are becomingmore 1 CPB Netherlands Bureau for Economic Policy Analysis, The Hague, The Netherlands 2 Department of Economics, CentER, Tilburg University, Tilburg, The Netherlands 3 Department of Applied Economics, Erasmus University Rotterdam, Rotterdam, The Netherlands 4 Department of Economics, University of Melbourne, Parkville, Australia 123 3 A. Palali, J. C. van Ours tolerant. The Netherlands quasi-legalized cannabis use through the introduction of “coffeeshops” which are licensed cannabis sales outlets. Recently, four states in the USA—Alaska, Colorado, Oregon and Washington—legalized cannabis for personal use. A licensed retail and production system for cannabis was introduced in Uruguay in 2014. There are several alternatives to prohibition varying from decriminaliza- tion to regulation and legalization (European Monitoring Center for Drugs and Drug Addiction 2013). Whereas decriminalization refers to the removal of the criminal status for personal possession or use, regulation refers to limits on access and restric- tions on advertizing. Legalization refers to cannabis use and cannabis supply, making lawful what previously was prohibited. Although in the policy debate a distinction is made between legalization and regulation of cannabis, we consider regulation as legalization under restrictions and focus on the dichotomy between prohibition and legalization.1 In the policy debate on cannabis legalization, there are frequent references to studies which ﬁnd that cannabis use has adverse effects on physical and mental health as well as other negative effects on important life outcomes such as educational attainment and labor market position (Ellickson et al. 1999; Brook et al. 1999; French et al. 2001; Arseneault et al. 2004; Van Ours 2006, 2007; Van Ours et al. 2013). There are also frequent references to harm to society through crime and anti-social behavior of users, the impacts on drug tourism and the gateway hypothesis.2 Nevertheless, in their overview study on the effects of cannabis use, Van Ours and Williams (2015) conclude that there do not appear to be serious harmful health effects of moderate cannabis use but there is evidence of reduced mental well-being for heavy users who are susceptible to mental health problems. Furthermore, they conclude that while there is robust evidence that early cannabis use reduces educational attainment, there remains substantial uncertainty as to whether using cannabis has adverse labor market effects. 1 Introduction Negative effects of cannabis use seem to be related to a small group of users while for the majority of cannabis users these effects are absent. It is well-known that cannabis users are more in favor of legalization than non- users. A 1989 study from Norway shows that 65% of cannabis users were in favor of prohibition of cannabis while among non-users this was 95% (Skretting 1993). This study also ﬁnds that those who use cannabis do not consider drug possession as a serious crime possibly because of self-interest.3 Trevino and Richard (2002) ﬁnds that drug users in Houston have different attitudes toward drug policies than non-drug users; 68% of drug users were in favor of legalizing cannabis, while only 33% of the non-drug users were in favor. In the Netherlands, in 2008, among cannabis users only 1 What the optimal cannabis policy is from an economic point of view is not clear. Economic arguments are based on the tradeoff between legalizing cannabis which would allow taxes to be introduced on cannabis use or prohibiting cannabis use because it is easier to limit cannabis supply than to implement taxation. See for studies that analyze the pros and cons of legalization (Becker et al. 2006; Caulkins et al. 2012; Glaeser and Shleifer 2001; Miron and Zwiebel 1995). 3 The inﬂuence of self-interest is present also in the use of other (legal) drugs. Using Californian data, Green and Gerken (1989) ﬁnd that self-interest plays a decisive role in forming attitudes toward restrictions on smoking and cigarette taxes. 123 1 What the optimal cannabis policy is from an economic point of view is not clear. Economic arguments are based on the tradeoff between legalizing cannabis which would allow taxes to be introduced on cannabis use or prohibiting cannabis use because it is easier to limit cannabis supply than to implement taxation. See for studies that analyze the pros and cons of legalization (Becker et al. 2006; Caulkins et al. 2012; Glaeser and Shleifer 2001; Miron and Zwiebel 1995). 3 The inﬂuence of self-interest is present also in the use of other (legal) drugs. Using Californian data, Green and Gerken (1989) ﬁnd that self-interest plays a decisive role in forming attitudes toward restrictions on smoking and cigarette taxes. 2 The gateway hypothesis suggests cannabis use can be a gateway to hard drug use such as cocaine heroin. 123 Cannabis use and support for cannabis legalization 7% was in favor of prohibition of cannabis, while this was 50% among non-users (Van der Sar et al. 2011). In Australia, in 1998, 57% of cannabis users were in favor of legalizing cannabis while among the non-users this was only 18% (Williams et al. 2016). 7% was in favor of prohibition of cannabis, while this was 50% among non-users (Van der Sar et al. 2011). In Australia, in 1998, 57% of cannabis users were in favor of legalizing cannabis while among the non-users this was only 18% (Williams et al. 2016). All in all, several studies suggest that support for cannabis legalization is higher among cannabis users. Interesting as this may be in itself, the fact that cannabis users are more in favor of legalizing cannabis does not necessarily imply that opinions are inﬂuenced by cannabis use in a causal way. It could be that individuals who are more likely to consume cannabis are also more in favor of legalization without personal experience affecting opinions. Knowing whether or not there is a causal effect from cannabis use to opinions is interesting because if there is a causal effect, this reveals how potential dangers of cannabis use are assessed. Cannabis users may change their mind about cannabis legalization through personal experience. As Orphanides and Zervos (1995) point out there is uncertainty about whether or not individuals might get addicted if they start using drugs. Individuals may start using cannabis by way of experimenting balancing the instant pleasure it provides and probabilistic future harm. Consumption of cannabis is not equally harmful to all. Some may not be sus- ceptible to addiction while others are of the addictive type. Individuals do not know their addictive tendency and the only way to ﬁnd out is by experimentation. How- ever, if individuals of the addictive type experiment and recognize their tendency too late they are drawn into addiction. If individuals believe that their risk of addiction is high they may optimally choose not to experiment with cannabis. Once individ- uals experiment with cannabis they may conclude that they are not of the addictive type and for them there is no harm. In other words, by using cannabis individuals learn about potential harmful effects and therefore they may change their mind about legalization. 123 We argue that if cannabis use has a favorable effect on opinions about cannabis legalization, then this may reveal that cannabis use is not as harmful as what it is originallybelieved.However,suchacausaleffectmayalsohavetodowithself-interest, i.e. cannabis legalization leading to easier access to cannabis and perhaps lower prices. We argue that it is possible to make a distinction between inside information and self- interest by comparing how past cannabis use and current cannabis use affect opinions. Whereas the effect of current cannabis use may be a mixture of self-interest and inside information, the effect of past cannabis use is related to inside information only. Although previous studies clearly show that there is a signiﬁcant difference between cannabis users and non-users in the opinions about cannabis policies, they do not establish whether this has to do with a causal relationship. The only study that establishes a causal relationship between user status and opinions on cannabis policy is Williams et al. (2016). They analyze Australian data from cross-sectional surveys over the period 1993–2007 using a quasi-panel approach to account for potential endogeneity of cannabis use. The main conclusion is that preference of past users for legalization is consistent with information on net beneﬁts of cannabis use while self-interest as contributing to current users’ support for legalization cannot be ruled out. Although focusing on a similar research question, our study differs from theirs in terms of econometric speciﬁcation and identiﬁcation strategy. 12 3 A. Palali, J. C. van Ours In the empirical part of our analysis, we use a statistical model that allows us to establish the causal effects of cannabis use on opinions about cannabis policies. We focus on two policy statements in a survey conducted in the Netherlands in 2008. Individuals were asked to indicate their support for statements on prohibition and legalization of cannabis. We estimate simultaneous models that integrate cannabis use dynamics and opinions on cannabis use.4 We ﬁnd that cannabis user status is correlated with opinions on cannabis legalization. This correlation partly reﬂects a causal effect. Current cannabis users and past cannabis users seem to have learned from their experi- ences and are therefore more in favor of legalizing cannabis. Current cannabis users are more in favor of legalizing cannabis than past cannabis users. 4 In the context of a MPH model describing the dynamics in cannabis, correlated models have been used for example by Van Ours and Williams (2012) and Van Ours and Williams (2011) to study the relationship between cannabis use and health and by Van Ours (2007) to study wage effects of cannabis use. Cannabis use is not the only context in which such methods are used. In labor economics literature, Abbring et al. (2005), Arni et al. (2013) and Van Ours (2004) for example, use similar methods. Other examples are Fevang et al. (2014) who analyze Norwegian absenteeism jointly modeling the ﬂow from presence to absence and back and Richardson and van den Berg (2013) who study the effect of labor market training on the job ﬁnding rates of Swedish unemployed workers. 5 Note that the expectation about the effect of past use on opinions is not very clear. It can be also the case that past users face the negative effects of cannabis use and regret the fact that they have used cannabis. Our empirical ﬁndings suggests that there is no regret. 123 This suggests that self- interest plays a role in opinions about cannabis policies but more interestingly, it also suggests that cannabis use may not be as harmful as non-users are inclined to think.5 Ourcontributiontotheliteratureontheeconomicsofcannabisuseisthreefold.First, we present an analysis of opinions on cannabis policies in a quasi-legal environment. TheDutchrespondentshavelittleincentivetomisreportasthereisnothingillegalabout using cannabis. Because of the quasi-legal environment, they are also familiar with potential consequences of making cannabis easily available. Second, we establish a causal link from cannabis use to opinions on cannabis policy. Third, we present a novel strategy to investigate the assessment of potential harmful effects by distinguishing between self-interest and inside information. Since several aspects of cannabis use feed into opinions on cannabis policies, these harmful effects are not only related to the individuals themselves but also to the society in general. The setup of our paper is as follows. In Sect. 2 we give a brief overview of cannabis policy in the Netherlands. In Sect. 3 we discuss our data and give some stylized facts. Section 4 presents our empirical model and in Sect. 5 we discuss our baseline parameter estimates together with estimates from several sensitivity and falsiﬁcation/placebo analysis. Section 6 concludes. 2 Cannabis policy in The Netherlands As indicated in the introduction, in the Netherlands, cannabis use is quasi-legalized. Small quantities of cannabis can be bought in cannabis shops. These are retail outlets and referred to as “coffeeshops”. Cannabis policy is focused on health issues (De Graaf et al. 2010) and can be summarized as tolerant (Van Solinge 1999). The basic aim of the cannabis policy is to lessen the potential harm to users and their environment. Although 12 123 Cannabis use and support for cannabis legalization cannabis is tolerated by Dutch authorities, use of hard drugs and production and trade of soft drugs and hard drugs are classiﬁed as serious offenses (Palali and van Ours 2015). The distinction between soft and hard drugs in terms of legal measures is at the heart of drug policy. The intention is to provide an organized environment for cannabis sale, thus keeping potential customers away from dealers of more harmful illicit drugs and having a control over the quality of cannabis. Coffeeshops are regulated by law. Some of the fundamental rules are: no sale of hard drugs, no advertising, no sale to youngsters below 18years of age, no nuisance and no more than 500g of cannabis on the premises. Failures to operate within the regulations might result in shutting down of the shop. The duration of shut down depends on the seriousness of the violations committed by the owners of coffeeshops. The 1980s stand as a crucial period in the history of Dutch cannabis policies. In 1980, the policy of tolerance of coffeeshops was publicly announced by Dutch authorities and this announcement was followed by a sharp increase in the number of coffeeshops (Jansen 1991). In the mid 1990s there were around 1500 coffeeshops. However, in the 1990s the tolerant cannabis policy was increasingly criticized from inside the country as well as from other countries. In 1995, the Dutch government made changes in the rules under which coffeeshops could operate. From 1996 onwards the limit for personal possession of cannabis was decreased from 30 to 5g. Moreover, the monitoring and punishment of production and trade of cannabis were increased and more importantly, local governments were given the opportunity to decide whether or not they wanted to have a cannabis-shop in their municipality. These policies caused a substantial decrease in the number of coffeeshops (Bieleman et al. 2007). 2 Cannabis policy in The Netherlands In 1999, there were 846 coffeeshops across the Netherlands, a number that went down to 651 in 2011 and further down to 582 in 2015. 3.1 Data Our data are from the 2008 Alcohol and Drugs study, one of the assembled studies of the Dutch Longitudinal Internet Studies for the Social Sciences (LISS) panel. The LISS survey is administered by CentERdata, a research institute of Tilburg University. The data used in our study are from one of the special cross-sectional surveys conducted in November 2008. The online survey is addressed to a representative Dutch sample. Respondents answered various questions about their opinions on different types of government policies on cannabis. Furthermore, respondents were asked whether they had ever used cannabis and if they answered afﬁrmatively they were faced with the question: At what age, approximately, did you ﬁrst use cannabis? Individuals were also asked to report if they were currently using cannabis, i.e. whether they had used cannabis in the previous 30 days. Because older individuals were never confronted with cannabis supply, we perform our analysis on individuals who were born in 1960 or later. It appears that about 20% of the respondents ever used cannabis whereas less than 5% used cannabis in the previous 30days. The latter are considered to be current users whereas the others are considered to be past users. 12 3 A. Palali, J. C. van Ours 0 1 2 3 4 5 6 7 8 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 Starting rate Age 0 5 10 15 20 25 30 35 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 Cumulative starting probability Age a b Fig. 1 Starting rates and cumulative starting probabilities cannabis use. a Starting rates (% per year). 3.1 Data b Cumulative starting probabilities (%) 0 1 2 3 4 5 6 7 8 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 Starting rate Age a Age Age 0 5 10 15 20 25 30 35 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 Cumulative starting probability Age b Fig 1 Starting rates and cumulative starting probabilities cannabis use a Starting rates (% per year) Fig. 1 Starting rates and cumulative starting probabilities cannabis use. a Starting rates (% per year). b Cumulative starting probabilities (%) We use information on past cannabis use and current cannabis use, together with the retrospective question on the age of ﬁrst cannabis use to analyze cannabis use dynamics. Figure 1 shows the dynamics in cannabis use. Panel a shows that the starting rate for cannabis use has a peak at age 17. The probability to use cannabis for the ﬁrst time is at its highest point at age of 17. There are other smaller peaks at age 19 and 21. After age 25 the probability of using cannabis conditional on not using before virtually becomes zero. Panel b conﬁrms this ﬁnding by showing that the slope of the line representing the cumulative probability of using cannabis becomes very small after age 25. 3.2 Stylized facts In the main part of our empirical analysis, we focus on two statements. The respondents were asked to indicate their opinion on a scale from 1 to 5 ranging from deﬁnitely dis- agree to deﬁnitely agree with the following statement: cannabis should be prohibited. We rescaled the responses given to this statement assuming that those who indicated to agree with it would disagree with the statement that cannabis should be legalized and vice versa. The respondents were also asked to report their opinion on the state- ment coffeeshops should be allowed to sell cannabis. We assume that this statement on coffeeshops is equivalent to a statement on the current status of cannabis policy in the Netherlands. Panels a and b of Table 1 present the distribution of opinions for the full sample and subsamples distinguished by user status. On average, the responses are evenly distributed; 45% of the sample agree that cannabis should be legalized whereas another 34% state that they disagree. The remaining 21% is indifferent. Sim- ilarly, 46% of the individuals state that they agree with the idea of selling cannabis through coffeeshops whereas 35% disagree.6 Dividing the sample into three categories based on user status exhibits interest- ing results. Among current cannabis users only 1deﬁnitely disagree and 2legalized whereas 89 percent agrees. Similarly 85% of the current users of cannabis agree with the statement that coffeeshops should be allowed to sell cannabis. These percent- ages are different for past users of cannabis of whom 77% disagree with prohibition whereas 9% agree with this idea. Similarly 74% of the past cannabis users agree with the statement that coffeeshops should be allowed sell cannabis. The numbers are reversed for those who have never used cannabis in their lives. Almost half of the never users state that they disagree with the idea of legalization whereas only 31% of the never users agree. Among the never users only 32% agree with coffeeshops while 45% of these respondents disagree. The last column in Table 1 presents the p values of a Chi-square test where we test if the differences between reported percentages among current, past and never users are statistically signiﬁcant as compared to never users, never users and ever users respectively. 6 If no birth year restriction is imposed on the sample, these ﬁgures are 39 and 43%, respectively. In 1998 this distribution was very much the same: Back then, 43% of the Dutch population of 19years and older thought that coffeeshops were admissible while 46% thought they were not admissible (Sociaal en Cultureel Planbureau 1998). 123 Cannabis use and support for cannabis legalization 3.2 Stylized facts For most of the policy statements, which will be discussed in more detail below, the raw data indicate that there are signiﬁcant differences in opinions about cannabis policies depending on the user status. Table 2 provides a description and summary statistics of the variables used in our analysis. Most of the descriptions are self-explanatory. For the question on political preferences we grouped the various parties in the Netherlands as follows: Con- servatives (VVD, Trots op Nederland, PVV), Left wing (PvdA, SP, Groen-Links, D66, Partij van de Dieren), Center (CDA, Christenunie, SGP), Others (other party, would rather not say, would not vote, is not entitled to vote, blank vote, does not know). 12 3 3 A. Palali, J. C. van Ours Table 1 Opinions on various types of cannabis policy (percentages) Deﬁnitely Deﬁnitely Disagree Disagree Indifferent Agree Agree Total N P valuea a. Cannabis legalized 1 All 16 18 21 28 17 100 2016 2 Current users 0 2 8 33 56 100 96 0.00 3 Past users 4 5 14 43 34 100 529 0.00 4 Never users 21 24 24 22 9 100 1391 0.00 b. Coffeeshops permitted 1 All 19 16 19 36 10 100 2015 2 Current users 3 5 7 45 40 100 95 0.00 3 Past users 6 7 13 57 17 100 529 0.00 4 Never users 25 20 22 27 5 100 1391 0.00 c. No sale to youngsters 1 All 1 3 12 50 34 100 2015 2 Current users 3 7 15 50 26 100 95 0.01 3 Past users 1 4 11 53 31 100 529 0.04 4 Never users 1 2 12 50 36 100 1391 0.00 d. No coffeeshops near schools 1 All 2 4 11 39 44 100 2014 2 Current users 5 11 27 33 24 100 95 0.00 3 Past users 2 5 15 47 31 100 528 0.00 4 Never users 2 3 8 36 50 100 1391 0.00 e. Education campaigns 1 All 0 1 6 52 40 100 2012 2 Current users 0 1 10 52 36 100 95 0.00 3 Past users 0 1 6 57 35 100 528 0.00 4 Never users 0 1 6 50 42 100 1389 0.00 f. 3.2 Stylized facts Drugs education at schools 1 All 1 1 8 53 36 100 2005 2 Current users 2 5 14 50 29 100 95 0.49 3 Past users 0 2 9 58 30 100 528 0.03 4 Never users 1 1 7 2 39 100 1382 0.02 a The p value of a Chi-square test of independence with null hypothesis that the reported difference in opinion categories between current and never users, past and never users, and ever (current or past) and never users is not signiﬁcant. For most of the cases we reject this null hypothesis Table 1 Opinions on various types of cannabis policy (percentages) a The p value of a Chi-square test of independence with null hypothesis that the reported difference in opinion categories between current and never users, past and never users, and ever (current or past) and never users is not signiﬁcant. For most of the cases we reject this null hypothesis 4.1 Cannabis use dynamics In the analysis of cannabis use starting rates, we assume that individuals become vulnerable to the risk of cannabis use from age 13 onwards. This is because only a 123 Cannabis use and support for cannabis legalization Table 2 Description and summary statistics of the variables Variable Deﬁnition Mean Min. Max. Female 1 if the respondent is female 0.57 0 1 Religion Amount of the times that the parents of the respondent visited a church per week when the respondent was 15 1.72 0 6 Migrant 1 if the respondent is migrant 0.14 0 1 Urban 1 1 if the resident is in an extremely urban area 0.13 0 1 Urban 2 1 if the resident is in a very or moderately urban area 0.49 0 1 Urban 3 1 if the resident is in an slightly urban area 0.22 0 1 Urban 4 (Reference) 1 if the resident is in a not urban area 0.16 0 1 Primary (Reference) 1 if the respondent has primary ed. 0.05 0 1 Secondary 1 1 if the respondent has intermediate secondary ed. 0.19 0 1 Secondary 2 1 if the respondent has high secondary ed. 0.15 0 1 Vocational 1 1 if the respondent has intermediate vocational ed. 0.30 0 1 Vocational 2 1 if the respondent has high vocational ed. 0.24 0 1 University 1 if the respondent has university ed. 4.1 Cannabis use dynamics 0.08 0 1 Ever use 1 if the respondent ever used cannabis 0.31 0 1 Past use 1 if the respondent ever used cannabis but not in the last 30 days 0.26 0 1 Current use 1 if the respondent used cannabis in the last 30 days 0.05 0 1 Intensity of use: 1 if the respondent used cannabis in following intensity: 6–7days a week 0.01 0 1 2–5days a week 0.02 0 1 1day a week 0.01 0 1 <1day a week 0.01 0 1 Starting age Starting age of cannabis (conditional on ever use) 18.1 13 60 Single w/o child (Reference) 1 if the respondent is single with no children 0.13 0 1 Couple w/o child 1 if the respondent is in a couple without children 0.20 0 1 Couple w child 1 if the respondent is in a couple with children 0.60 0 1 Single w child 1 if the respondent is single with children 0.06 0 1 Other 1 if otherwise 0.01 0 1 Age 15–24 (Reference) 1 if the respondent is 15–24years old 0.16 0 1 Age 25–34 1 if the respondent is 25–34years old 0.27 0 1 Age 35–44 1 if the respondent is 35–44years old 0.40 0 1 Age 45+ 1 if the respondent is more than 45years old 0.17 0 1 Conservative (Reference) 1 if the respondent is categorized as a conservative voter 0.18 0 1 Left wing 1 if the respondent is categorized as left wing voter 0.27 0 1 Center 1 if the respondent is categorized as center voter 0.14 0 1 Others 1 if the respondent is categorized as other 0.10 0 1 I don’t know 1 if the respondent did not want to disclose his/her preference 0.28 0 1 Policy opinion 1 Cannabis should be legalized 2.91 1 5 12 3 A. Palali, J. C. van Ours Table 2 continued Variable Deﬁnition Mean Min. Max. 4.1 Cannabis use dynamics Policy opinion 2 It should be permitted to sell cannabis at coffeeshops 2.81 1 5 Policy opinion 2 Government should ensure that coffeeshops don’t sell cannabis to people below 18years old 4.29 1 5 Policy opinion 4 Coffeeshops should not be in the vicinity of schools 4.28 1 5 Policy opinion 5 Government should conduct drug education campaigns 4.30 1 5 Policy opinion 6 Government should ensure that schools provide drug education campaign 4.34 1 5 Policy opinion variables (1–6): Respondents are asked to give their opinion about the corresponding state- ment. 1 means deﬁnitely disagree while 5 means deﬁnitely agree. The number of observations is 2016 licy opinion variables (1–6): Respondents are asked to give their opinion about the corresponding state- ent. 1 means deﬁnitely disagree while 5 means deﬁnitely agree. The number of observations is 2016 few individuals start using cannabis before the age of 13. We specify the starting rate for cannabis use at time t (t = 0 at age 12) conditional on observed characteristics x and unobserved characteristics u as few individuals start using cannabis before the age of 13. We specify the starting rate for cannabis use at time t (t = 0 at age 12) conditional on observed characteristics x and unobserved characteristics u as θc(t \| x, u) = λc(t) exp(x′βc + u) (1) (1) where βc represent the effects of independent variables and λc(t) represents individual duration (age) dependence. Unobserved heterogeneity, in this case, is denoted with u which controls for differences in individuals’ unobserved susceptibility to cannabis use. We model duration (age) dependence in a ﬂexible way by using a step function λc(t) = exp(Σkλk Ik(t)), where k(= 1, . . . , 9) is a subscript for age categories and Ik(t) are time-varying dummy variables that are one in subsequent categories, 8 of which are for individual ages (age 13, . . . , 20) and the last interval is for ages above 20years. Because we also estimate a constant term, we normalize λc,1 = 0. 4.1 Cannabis use dynamics The conditional density function of the completed durations until the uptake of cannabis use can be written as fc(t \| x, u) = θc(t \| x, u) exp − t 0 θc(s \| x, u)ds (2) (2) We integrate out the unobserved heterogeneity such that density function for the dura- tion of time until cannabis uptake t conditional on x is fc(t \| x) = u fc(t \| x, u)dG(u) (3) (3) (3) where G(u) is assumed to be a discrete mixing distribution with 2 points of support ua and ub reﬂecting the presence of two types of individuals in the hazard rate for cannabis uptake. The associated probabilities are denoted as follows: Pr(u = ua) = r and Pr(u = ub + ua) = 1 −r with 0 ≤r ≤1, and r is modeled using a logit speciﬁcation, r = exp(α) 1+exp(α). 123 123 123 Cannabis use and support for cannabis legalization To account for the discrete nature of the observations of age of onset of cannabis use, the log-likelihood is speciﬁed as n i=1 dc,i log [Fc(ti −1) −Fc(ti)] + 1 −dc,i log 1 −Fc(ts,i) (4) (4) where i is an index for individual, n is the number of individuals in the sample and dc,i is a dummy variable that is equal to 1 if an individual started using cannabis and equal to 0 if an individual did not start using cannabis before being interviewed at age ts. In addition to starting rates of cannabis use, we also estimate quitting rates in order to have a complete analysis of cannabis use dynamics. The LISS panel includes questions on the last month use of cannabis. We assume that if the individual reports no use of cannabis in the last 30days, that individual stopped using cannabis in time period starting from the ﬁrst use of cannabis until 30days prior to the survey. The conditional density function for the completed durations until the last use of cannabis can be written as fq(τ \| x1, v) = θq(x1, v) exp(−θq(x1, v) · τ) (5) (5) Eventhoughwedonotobservetheexacttimeofquittingintermsofageofrespondents, we can still analyze the duration of quitting thanks to the interval censored nature of the data. However, note that due to the uncertainty about the exact time of quitting, duration dependence is not estimated here. 4.1 Cannabis use dynamics On the other hand it is certain that the duration of cannabis use, τ, will lie in the interval [0, τq] where τq is the difference between age at the time of survey and the age of the ﬁrst use. This means that we can integrate out the conditional density function over this period to account for the uncertainty of quitting time and obtain the distribution function, Fq. Individuals who report using cannabis in the last 30days are assumed to be right censored in their quitting, i.e. as yet they did not quit. Since the quitting analysis is performed only on those who ever used cannabis there are no left censored individuals. As in the analysis of the uptake of cannabis, we assume that there are 2 unobserved heterogeneity groups where the probabilities are assumed to follow a logistic distribution. The log-likelihood is speciﬁed as m i=1 dq,i log Fq(τq,i) + 1 −dq,i log 1 −Fq(τq,i) (6) (6) where m is the number of individuals that ever used cannabis and dq,i is a dummy variable which has a value of 1 if the individual stopped using cannabis and a value of 0 if the individual did not stop using cannabis. Finally, we allow for the possibility that conditional on the observed characteristics, the age of uptake and the duration of use cannabis uptake and cannabis quits are cor- related through unobserved characteristics. The joint density of completed durations until the uptake of cannabis use and completed durations of cannabis use is speciﬁed as: 12 3 A. Palali, J. C. van Ours g1(t, τ \| x1, x2) = v u fc(t \| x1, v) fq(τ \| x2, u)dG(u, v) (7) where G(u, v) is the joint distribution of unobserved heterogeneity which assume to be discrete with an unknown number of support points. where G(u, v) is the joint distribution of unobserved heterogeneity which assume to be discrete with an unknown number of support points. 7 For simplicity we write x′ 3βp = x′ 2βp + ρccc + ρpcp. 4.2 Opinions on cannabis policy G(ϵ) is assumed to be a dis- crete mixing distribution with 2 points of support ϵa and ϵb indicating that conditional on observed characteristics there are 2 types of individuals in the ordered responses given to the policy statements. The associated probabilities are denoted as follows: Pr(ϵ = ϵa) = p and Pr(ϵ = ϵb + ϵa) = 1 −p with 0 ≤p ≤1, where p is modeled using a logit speciﬁcation, p = exp(α) 1+exp(α). + p( ) There are a number of assumptions and normalizations which need to be made for model identiﬁcation. First, we assume that mean and variance of the error term e in the latent equation are 0 and 1, respectively. Since we have heterogeneity speciﬁc constants in the model, we also set the ﬁrst threshold parameter μ1 to zero. The other threshold parameters are modeled in the following way in order to ensure that probabilities are positive and thresholds are ordered: μ2 = γ 2 1 , μ3 = μ2 + γ 2 2 and μ4 = μ3 + γ 2 3 . Finally, we write the likelihood function of the ordered choice model as N Prob(y = j\|x3). N Until now, when estimating the ordered probit model for opinions, we assume that the decision to use cannabis is exogenous, i.e. independent from any factor that would affect the opinions about cannabis policies. However, it is possible that there are unobserved personal characteristics that affect both the decision to use cannabis and opinions about cannabis policies. If, for example, certain individuals have an inclination toward cannabis use due to some intrinsic factors that would also lead them to have positive attitudes toward liberal cannabis policies, then we might end up with signiﬁcant parameter estimates for ρc and ρp even though there is no causal relationship. In order to control for correlation between unobserved characteristics and establish a causal effect, we jointly estimate the ordered probit model and the mixed proportional hazard models following a discrete factor approach. 4.2 Opinions on cannabis policy We ﬁrst model the determinants of opinions on cannabis policy assuming that the dynamics in cannabis use are exogenous. Then we take possible correlation between the two processes into account. Individuals report their opinions on the relevant cannabis policy statements on a scale of 1–5, with 1 representing deﬁnitely disagree and 5 representing deﬁnitely agree. In order to exploit this ordinal character of the dependent variable, we use an ordered probit model with Heckman and Singer type discrete unobserved heterogeneity (Heckman and Singer 1984). The unobserved latent variable in the ordered response model is deﬁned as y∗= x′ 2βp + ρccc + ρpcp + ϵ + e (8) (8) where cc represent current cannabis use, cp represents past cannabis use and ϵ con- trols for discrete type of unobserved heterogeneity which is different from the error term e that represents the random error term. The parameters of interest in our study are ρc and ρp which measure the effect of current and past use of cannabis on the opinions about cannabis policy statements. Furthermore, βp measures the effect of our control variables whose descriptions and summary statistics are provided in detail in Table 2. The observed responses on the cannabis policy statements in the data are, then, assumed to be y = ⎧ ⎪⎪⎪⎪⎨ ⎪⎪⎪⎪⎩ 1 (Deﬁnitely disagree) if y∗≤μ1 2 (Disagree) if μ1 < y∗≤μ2 3 (Indifferent) if μ2 < y∗≤μ3 4 (Agree) if μ3 < y∗≤μ4 5 (Deﬁnitely agree) if μ4 < y∗ (9) (9) where μ’s are to be estimated threshold parameters in the ordered choice models. Assuming that the error term e has a standard normal distribution, we can write the following probabilities for the ordered probit model conditional on observable and unobservable individual heterogeneity7: Pr(y = 1\|x3, ϵ) = Φ(μ1 −x′ 3βp −ϵ) Pr(y = 2\|x3, ϵ) = Φ(μ2 −x′ 3βp −ϵ) −Φ(μ1 −x′ 3βp −ϵ) . Pr(y = 5\|x3, ϵ) = 1 −Φ(μ4 −x′ 3βp −ϵ) . (10) (10) 7 For simplicity we write x′ 3βp = x′ 2βp + ρccc + ρpcp. 123 Cannabis use and support for cannabis legalization where Φ(.) is standard normal cdf. Unconditional probabilities, then, can be written as Pr(y = j\|x3) = ϵ Prob(y = j\|x3, ϵ)dG(ϵ) (11) (11) where j ∈{1, 2, 3, 4, 5}, denoting ordered responses. 4.2 Opinions on cannabis policy The joint density function of the completed duration of uptake of cannabis, the duration of cannabis use and opinions on cannabis use is speciﬁed as: g2(t, τ, y = j \| x1, x2, x3) = ϵ v u fc(t \| x1, v) fq(τ \| x2, u)Prob(y = j \| x3, ϵ) dG(v, w, ϵ) (12) (12) where G(v, w, ϵ) is a discrete mixing distribution underlying unobserved heterogene- ity affecting age of onset of cannabis use, duration of use and opinions about cannabis policies. This approach is introduced by Heckman and Singer (1984) in order to con- trol for unobserved heterogeneity in hazard rates and used for example by Mroz (1999) to estimate the effects of dummy endogenous variables. The approach is equivalent to a correlated random effects model in which the main idea is that unobserved het- erogeneity affecting opinions about cannabis policies and unobserved heterogeneity affecting cannabis use dynamics can be correlated, i.e. they come from a joint mixing distribution. The assumption on support points of this joint distribution deﬁnes the 12 3 A. Palali, J. C. van Ours types of individuals regarding opinions and cannabis use. If, for example, conditional on observed characteristics there are two types of individuals in terms of uptake of cannabis and two types in terms of quitting cannabis, then there could be four types of individuals in terms of cannabis dynamics. If in addition to this conditional on unobserved characteristics there are two types of individuals in terms of opinions on cannabis policy, then there could be eight types of individuals in terms of cannabis dynamics and opinions on cannabis policy. However, it could also be that there are only two types of individuals in terms of cannabis dynamics: Individuals who are inclined to used cannabis and individuals who will never start using cannabis. If this is the case, then there could be four types of individuals: those who are more in favor of cannabis policies and more likely to use drugs, those who are less in favor of cannabis policies and more likely to use drugs, those who are more in favor of liberal drug policies and more likely to abstain from drugs, and those who are against liberal drug policies and more likely to abstain from drugs. 8 We assume that educational attainment represents ability since many individuals start using cannabis before ﬁnishing school. 4.2 Opinions on cannabis policy Finally, if there is a perfect correla- tion between unobserved heterogeneity behind cannabis use dynamics and opinions, only the ﬁrst and the last types are identiﬁed. An important advantage of using the functional form assumptions behind this approach is that identiﬁcation is achieved without relying on exclusion restrictions which can be very challenging to ﬁnd because cannabis use is likely to have the same determinants as opinions on cannabis policy. 5.1 Cannabis use dynamics The parameter estimates of the various models are obtained through the method of maximum likelihood. The parameter estimates of the mixed proportional hazard mod- els describing starting rates and quitting rates of cannabis use are presented in Table 3. The ﬁrst column presents the results for the starting rates. The parameter estimate for female is found to be negative and signiﬁcant. Thus, as in previous studies we ﬁnd that on average females start using cannabis at a later age. Religiosity of the parents during the childhood of respondents is found to be signiﬁcantly negative. So, as the parents are more religious, the age of initiation to cannabis use increases. Migrant status of the individual is found to be insigniﬁcant. Moreover, the degree of urbanization of the municipality has a signiﬁcant effect on the uptake of cannabis indicating that individuals residing in highly urban areas start using cannabis at ear- lier ages. The most likely reasons are that cannabis happens to be more available and living styles of individuals might make them more vulnerable to the risk of cannabis use in highly urban regions. Educational attainment does not seem to affect the uptake of cannabis.8 Finally, there is a clear cohort effect since age at the time of the survey is an important determinant of cannabis uptake. Older cohorts were less likely to start using cannabis. In line with Fig.1, age dependency parameters indicate 12 3 Cannabis use and support for cannabis legalization Cannabis use and support for cannabis legalization Table 3 Dynamics in cannabis use: parameter estimates of mixed proportional hazard models on starting age and quitting age of cannabis use Based on 2016 observations, likelihood ratio test 1 indicates that unobserved heterogeneity is not signiﬁcant in the quitting rates. 5.1 Cannabis use dynamics Note that d.o.f of the test is 2 since when ub = 0, α is not identiﬁed in the quitting rates estimation Likelihood ratio test 2 compares the independent models against the correlated model and shows that we cannot reject that the two processes are independent; absolute t-statistics in parentheses * and are for statistical signiﬁcance at 10 and 5%, respectively Bivariate Starting rates Quitting rates (1) (2) Female −0.68 (5.0) 0.33 (2.6) Religion −0.22 (5.2) 0.00 (0.1) Migrant −0.27 (1.3) 0.06 (0.3) Urban 1 0.92 (3.8) −0.76 (3.2) Urban 2 0.21 (1.0) −0.38 (1.8)* Urban 3 0.28 (1.2) −0.41 (1.8)* Secondary 1 −0.03 (0.1) 0.46 (1.7)* Secondary 2 0.08 (0.2) 1.14 (3.6) Vocational 1 −0.37 (1.1) 0.62 (2.4) Vocational 2 0.15 (0.5) 1.00 (3.6) University 0.31 (0.8) 1.06 (3.4) Age 25–34 −0.11 (0.6) −0.36 (1.7)* Age 35–44 −1.27 (6.1) −0.89 (4.5) Age 45+ −2.05 (7.3) −0.85 (2.4) Age of onset 0.76 (5.6) Age dependence Age dep. 21+ 1.39 (3.7) Age dep. 20 2.98 (8.2) Age dep. 19 2.50 (6.6) Age dep. 18 3.26 (9.7) Age dep. 17 2.72 (8.3) Age dep. 16 2.89 (9.9) Age dep. 15 1.86 (6.4) Age dep. 14 0.94 (3.0) Unobserved heterogeneity ua −2.49 (5.3) −3.11 (7.9) ub −5.95 (14.1) – α −1.25 (8.5)** – -Loglikelihood 2921.6 LR test 1 4.6 LR test 2 0.4 that there is a peak in the uptake of cannabis at age 17 and another peak at age 19. The mass point estimates of column 1 show that unobserved heterogeneity is indeed signiﬁcant in the data at hand. The estimate of −1.25 for α implies that 22% of the respondents have a high starting rate of cannabis use whereas 78% of them have a substantially lower starting rate. that there is a peak in the uptake of cannabis at age 17 and another peak at age 19. The mass point estimates of column 1 show that unobserved heterogeneity is indeed signiﬁcant in the data at hand. The estimate of −1.25 for α implies that 22% of the respondents have a high starting rate of cannabis use whereas 78% of them have a substantially lower starting rate. Column 2 of Table 3 presents the parameter estimates for quitting rates. We ﬁnd that females are not only more likely to start using cannabis at later ages but they are also more likely to quit early. 10 In countries where cannabis use is illegal, past users can still be affected by self-interest because the reason they do not use cannabis today can be illegality itself. In other words, they might have quit using cannabis because of the opportunity cost of being arrested. In such as case, they can still have a self- interest in supporting liberal policies, as they think liberal policies would remove the possible future costs 5.1 Cannabis use dynamics The parameter estimate for the ﬁrst urbanization 12 3 3 A. Palali, J. C. van Ours category is negative and signiﬁcant, indicating that those who live in highly urban areas quit using cannabis later. Lower educated individuals have a smaller quit rate and individuals from the older birth cohorts have a smaller quit rate as well. Finally, we ﬁnd that those who start using cannabis at a later age quit earlier, i.e. early starters quit late. Conditional on the observed characteristics we do not ﬁnd evidence of the presence of unobserved heterogeneity in the quit rate. If we estimate a joint model of cannabis starting and cannabis quitting, we ﬁnd that there is correlation between the two processes through unobserved heterogeneity. However, the second LR test reported at the bottom of the table compares the independent models of cannabis uptake and cannabis quits against the correlated model. As shown, we cannot reject the hypothesis that the two processes are independent. 9 The ﬁrst likelihood ratio test statistic has a value of 6.6 which is signiﬁcant with 1 degree of freedo (ϵb). 9 The ﬁrst likelihood ratio test statistic has a value of 6.6 which is signiﬁcant with 1 degree of freedom (ϵb). 10 In countries where cannabis use is illegal, past users can still be affected by self-interest because the 5.2 Opinions on cannabis use: baseline parameter estimates Table 4 presents our baseline parameter estimates for the ﬁrst policy statement that cannabis should be legalized. The results for the univariate ordered probit model are shown in the ﬁrst column. We ﬁnd no evidence of unobserved heterogeneity having a signiﬁcant effect. The parameter estimates of both past use and current use of cannabis are positive and signiﬁcant indicating that both self-interest and information may have an effect on opinions. However, the estimates in the ﬁrst column are based on the assumption that the decision to use cannabis is exogenous to the opinions regarding cannabis policies. The correlated models whose parameter estimates are presented in the second col- umn of Table 4 are obtained after controlling for possible endogeneity. Now, we are able to identify two mass-points in the ordered probit for opinions indicating that once we take correlation with cannabis use dynamics in account there is unobserved heterogeneity affecting opinions.9 Clearly, the parameter estimates of cannabis use status become smaller in absolute terms indicating that part of the effects found in the univariate model are due to the correlated unobserved heterogeneity. The remaining effect may be interpreted as causal. The second LR test shows that past and current cannabis use have signiﬁcant effects on opinion on cannabis legalization while the third LR test shows that the effect of past cannabis use is signiﬁcantly smaller than the effect of current cannabis use. The opinions of current cannabis users are expected to be affected by both self-interest and information. The opinions of past cannabis users are not affected by self-interest. Parameter estimates reﬂect only the effect of infor- mation about cannabis use. The results obtained for past users indicate that cannabis may not be as harmful as individuals originally thought or as non-users are inclined to think. This may be speciﬁc to countries like the Netherlands where cannabis use is legalized or quasi-legalized.10 12 3 Cannabis use and support for cannabis legalization pp g Table 4 Parameter estimates of the effect of past and current use of cannabis on the cannabis policy statement: Cannabis should be legalized Based on 2016 observations; LR test 1 tests the univariate model against the correlated model. Likelihood Ratio test 2 is for the joint signiﬁcance of cannabis use variables. LR test 3 is for equality of the parameters of past cannabis use and current cannabis use. 5.2 Opinions on cannabis use: baseline parameter estimates Absolute t-statistics in parentheses * and are for statistical signiﬁcance at 10 and 5%, respectively Univariate Correlated (1) (2) Past cannabis use 0.97 (16.0) 0.71 (5.7) Current cannabis use 1.60 (10.5) 1.35 (7.8) Female 0.01 (0.2) −0.03 (0.6) Religion −0.05 (3.1) −0.05 (3.6) Migrant −0.07 (1.0) −0.11 (1.6) Urban 1 −0.12 (1.2) −0.07 (0.7) Urban 2 −0.09 (1.2) −0.08 (1.0) Urban 3 −0.12 (1.4) −0.10 (1.1) Secondary 1 0.04 (0.1) −0.03 (0.2) Secondary 2 0.30 (2.2) 0.26 (2.1) Vocational 1 0.21 (1.6) 0.13 (1.2) Vocational 2 0.41 (3.1) 0.37 (3.2) University 0.56 (3.7) 0.53 (3.8) Couple without child −0.03 (0.5) −0.05 (0.5) Couple with child −0.27 (3.7) −0.28 (3.7) Single with child −0.06 (0.6) −0.09 (0.7) Other −0.33 (1.3) −0.39 (1.5) Age 25–34 −0.06 (0.7) −0.09 (1.4) Age 35–44 −0.24 (2.9) −0.29 (3.6) Age 45+ −0.20 (2.1) −0.25 (2.7) Left wing 0.28 (3.8) 0.26 (3.8) Center −0.31 (3.4) −0.29 (3.5) Others −0.20 (2.0) −0.18 (2.0) I don’t know 0.01 (0.2) 0.00 (0.0) ϵa 1.50 (7.3) 0.73 (3.0) ϵb 0.43 (2.3) α −1.26 (8.7) γ1 1.01 (52.7) 1.02 (50.8) μ2 [1.1] [1.0] γ2 0.82 (44.4) 0.81 (44.1) μ3 [1.8] [1.7] γ3 0.81 (41.2) 0.82 (41.6) μ4 [2.5] [2.4] -Loglikelihood 5783.2 5779.9 LR test 1 6.6 LR test 2 50.1 LR test 3 24.8 12 123 A. Palali, J. C. van Ours Since the parameter estimate for current users is larger in absolute terms than the parameter estimate for past users there is also evidence of self-interest inﬂuencing opinions. Indeed likelihood ratio test 3 in Table 4 shows that we strongly reject the hypothesis that current and past use of cannabis have the same effect on opinions. Current cannabis use has a substantial bigger effect on opinions than past cannabis use. In an unreported estimation (see for details Palali and van Ours 2014), we also ﬁnd that the frequency of current cannabis use matters. More frequent the cannabis use, i.e. more self-interest leads to a more favorable opinion about cannabis legalization. Casual users do not differ from past users. For individuals who rarely use cannabis, inside information is driving the results rather than self-interest, which is consistent with our interpretations of the results. This is particularly important because several studies, for example Caulkins and Pacula (2006) and Kilmer et al. Footnote 10 Continued of using cannabis. However, this is not the case for the population in the Netherlands where cannabis market is regulated for both supply and demand. Also note that society as a whole is constantly learning about negative effects or absence of negative effects of cannabis use through the accumulation of scientiﬁc evidence and its public presentation. All individuals have potential access to public and private signals about the self-harm effects of cannabis use. Own use (current or past) is not the only way to learn about this margin, but the most effective indeed since it is ﬁrst-hand information. Although all the other information is available to all individuals, ﬁrst-hand information is only available to cannabis users. 5.2 Opinions on cannabis use: baseline parameter estimates (2014), ﬁnd that even though they constitute a small group heavy users account for most of the cannabis transactions. 11 Note that for the second policy, we can identify a signiﬁcant unobserved heterogeneity in the univariate model although the estimated probability parameter is not well-identiﬁed. For this opinion, we can compare univariate models with unobserved heterogeneity and the correlated models through likelihood ratio tests. The results indicate that we fail to reject the correlated model against the univariate one. Therefore, there is further evidence supporting the ﬁndings of the previous LR tests that correlation between unobserved heterogeneity affecting opinions and cannabis use dynamics is signiﬁcant. 5.3.1 Sensitivity to policy statements Table 5 presents the results of a sensitivity analysis focusing on other statements on cannabis policy or drug policy in general. For reasons of comparison panel a replicates the main ﬁndings of Table 4. Panel b present the parameter estimates for the second policy statement on coffeeshops: It should be permitted to sell cannabis at coffeeshops. We obtain very similar results. Both current use of cannabis and past use of cannabis have positive and signiﬁcant effects on opinions. In panel b of column 2, similar to column 2 of Table 4, the parameter estimate of current use is found to be twice as large as the coefﬁcient estimate of past use.11 This suggests that the magnitude of the effect of self-interest is almost the same as that of inside information. For opinions on the ﬁrst and the second type of cannabis policy, there is a causal effect from past cannabis use to opinions. Individuals that used cannabis in the past changed their opinion after they had personal experience with cannabis toward a more liberal policy. They became 12 Footnote 10 Continued of using cannabis. However, this is not the case for the population in the Netherlands where cannabis market is regulated for both supply and demand. Also note that society as a whole is constantly learning about negative effects or absence of negative effects of cannabis use through the accumulation of scientiﬁc evidence and its public presentation. All individuals have potential access to public and private signals about the self-harm effects of cannabis use. Own use (current or past) is not the only way to learn about this margin, but the most effective indeed since it is ﬁrst-hand information. Although all the other information is available to all individuals, ﬁrst-hand information is only available to cannabis users. 11 Note that for the second policy, we can identify a signiﬁcant unobserved heterogeneity in the univariate model although the estimated probability parameter is not well-identiﬁed. For this opinion, we can compare univariate models with unobserved heterogeneity and the correlated models through likelihood ratio tests. The results indicate that we fail to reject the correlated model against the univariate one. Therefore, there is further evidence supporting the ﬁndings of the previous LR tests that correlation between unobserved heterogeneity affecting opinions and cannabis use dynamics is signiﬁcant. 123 123 Cannabis use and support for cannabis legalization pp g Table 5 Parameter estimates of the effect of past and current cannabis use on support for various types of cannabis policies The numbers of observations are 2016, 2015, 2015, 2014, 2012 and 2005, respectively The LR test 1 tests the univariate model against the correlated model. The likelihood ratio test 2 is for the joint signiﬁcance of cannabis use variables. The LR test 3 is for equality of the parameters of past cannabis use and current cannabis use. Absolute t-statistics in parentheses For opinions b & c, LR tests indicate that unobserved heterogeneity is signiﬁcant in opinions in the univariate models although the distribution is not well-identiﬁed. For these two opinions we also compared univariate models with unobserved heterogeneity and the correlated models through likelihood ratio tests. The results indicate that in both cases we fail to reject the correlated model against the univariate one, which supports the ﬁndings of the previous LR tests that correlation between unobserved heterogeneity affecting opinions and cannabis use dynamics is signiﬁcant * and are for statistical signiﬁcance at 10 and 5%, respectively Univariate (1) Correlated (2) a. Cannabis legalized Past cannabis use 0.97 (16.0) 0.71 (5.7) Current cannabis use 1.60 (10.5) 1.35 (7.8) LR Test 1 6.6 LR Test 2 50.1 LR Test 3 24.8 b. Coffeeshops permitted Past cannabis use 0.80 (13.1) 0.47 (4.0) Current cannabis use 1.33 (10.7) 1.05 (7.0) LR Test 1 11.8 LR Test 2 44.0 LR Test 3 21.1 c. No sale to youngsters Past cannabis use −0.54 (5.0) −0.04 (0.3) Current cannabis use −0.56 (2.3) −0.06 (0.3) LR Test 1 9.4 LR Test 2 0.6 LR Test 3 0.4 d. No coffeeshops near schools Past cannabis use −0.31 (4.9) 0.01 (0.3) Current cannabis use −0.67 (5.5) −0.41 (2.5) LR Test 1 6.8 LR Test 2 2.4 LR Test 3 12.0** e. Education campaigns Past cannabis use −0.13 (2.1)* −0.17 (1.6) Current cannabis use −0.32 (2.6) −0.35 (2.1) LR Test 1 0.2 LR Test 2 4.2 LR Test 3 2.1 f. Drugs education at schools Past cannabis use −0.06 (1.0) −0.05 (0.3) Current cannabis use −0.03 (0.2)** −0.01 (0.3) LR Test 1 0.2 LR Test 2 0.2 LR Test 3 0.6 more likely to support no-prohibition and more likely to support cannabis legalization. This suggests that cannabis use is not as harmful as they originally thought. 123 ore likely to support no-prohibition and more likely to support cannabis legalization. more likely to support no-prohibition and more likely to support cannabis legalization. This suggests that cannabis use is not as harmful as they originally thought. y pp p y pp g This suggests that cannabis use is not as harmful as they originally thought. Panels c–f of Table 1 give descriptive information about opinions on other drug policy statements which are not directly related to availability of cannabis. For all 12 3 A. Palali, J. C. van Ours these policies, the distribution of the responses is very skewed. Almost 90% of the individuals agree with the statements that coffeeshops should not sell cannabis to peo- ple below 18years old, the government should organize education campaigns against drugs, there should not be coffeeshops in the vicinity of schools and the government should ensure that schools organize education campaigns against drugs. The percent- ages of those who agree with a speciﬁc cannabis policy remain high irrespective of the user status. Nevertheless, to investigate whether there was a causal effect from cannabis use to opinions, we performed a similar type of analysis as for the two main policy statements that referred to cannabis prohibition and cannabis legalization. Since the following policy statements are not directly about availability of cannabis, they serve as falsiﬁcation analysis on the self-interest aspect of cannabis use. Panels c and d of Table5 present the parameter estimates of the effects of current and past cannabis use on restrictions on sale of cannabis. Concerning no sale of cannabis to youngsters, there is correlation with user status, but this disappears once correlated unobserved heterogeneity is taken into account. Apparently, individuals who started using cannabis did not change their opinion on no sale of cannabis to youngsters. They had a different opinion all along. They were less likely to support the idea of no sale of cannabis to youngsters anyway. The ﬁndings for the statement that there should be no coffeeshops near schools is somewhat different. 12 Making the same distinction in two main legalization policies whose results are given in Table4 did not make any changes. This distinction seems to make a difference only for this speciﬁc policy statement on the location of coffeeshops. 13 The exception to these ﬁndings is the negative of current cannabis use on support for the ban of cof- feeshops near schools. It is hard to imagine that this has a causal interpretation. 123 In order to explore the possible reason behind the signiﬁcant negative parameter estimate, we performed the same correlated analysis by dividing the current cannabis use variable into 3 parts: those who frequently (more than once) used cannabis in the last 30days, those who used cannabis only once in the last 30 days but started using cannabis at an earlier age and those who for the ﬁrst time used cannabis only once in the last 30days. Although not reported here, the results after this distinction show that the negative effect is located among those who used cannabis for the ﬁrst time in the last 30days, which has a very small number of observations. Once they are ignored, there is no effect. Moreover, since these people cannot accumulate inside information from one time use and they are mostly below 25years old, we argue that self-interest plays a small role.12 Panels e and f of Table 5 show the main parameter estimates for statements on education programs. For education campaigns, there is no big difference between the univariate model and the correlated model. This suggests that initially opinions were not different but once some individuals started using cannabis they changed their minds. Moreover, the fact that there is no difference between the effects of past use and current use suggests that it is inside information that is driving the results. Apparently, after personal experience with cannabis, some individuals were less inclined to support education campaigns because they thought there was less need for such campaigns. For drugs education at school, there is no difference in opinions according to user status. All in all, the ﬁndings for the speciﬁc types of drug policy show that cannabis use did not have a big effect on opinions and if it did such as is the case for education 12 3 Cannabis use and support for cannabis legalization campaigns, inside information is driving the results.13 As expected self-interest does not play a signiﬁcant role in shaping opinions about these policy statements. campaigns, inside information is driving the results.13 As expected self-interest does not play a signiﬁcant role in shaping opinions about these policy statements. 5.3.2 Placebo tests In order to investigate the robustness of our ﬁndings we performed a range of sensitivity checks of which we brieﬂy report the results (see for details Palali and van Ours 2014). First, by way of placebo analysis we investigate the effect of past and current cannabis use on opinions about policies unrelated to cannabis use. The ﬁrst placebo analysis is on the opinions about alcohol legislation. We use information about opinions on various types of alcohol policy all aiming at restricting access to alcohol. The proposed policies are: no sale of alcohol in supermarkets, banning alcohol advertisements, no sale of alcohol to youngsters under age 16, no happy hours in bars and discos, no sale of alcohol in places which are frequently visited by youngsters. Using the same correlated model approach, we ﬁnd that opinions about these alcohol policies are not different for cannabis users and non-cannabis users. The second placebo analysis is on the opinions about government policies which are completely unrelated to risky health behaviors. We use information about opinions on whether study grants should be replaced by study loans and opinions on citizens’ inﬂuence on government policies. Again there is no effect on these opinions of current or past cannabis use. 5.3.3 Sensitivity to model speciﬁcations 5.3.3 Sensitivity to model speciﬁcations In a further set of robustness checks, we investigate the sensitivity of our baseline results to various model speciﬁcations (see for details Palali and van Ours 2014). We remove education dummies from starting rate analysis cannabis use may affect educational attainment. We add political preference dummies in the starting rates. Again, our conclusions remain the same. One issue with retrospective responses about substance use is that there might be measurement errors due to recall bias. In order to investigate if our results are sensitive to such a recall bias, we restrict our sample to a much younger cohort as they are expected to recall more accurately. If we use information on individuals who were born after 1969 we lose almost half of our main sample but still obtain similar results. We also include several control variables regarding different aspects of cannabis use. Adding variables for opinions about the criminal aspect of cannabis use, peer use of cannabis and duration of cannabis use does not affect our main conclusions. Moreover, using ordered logit models instead of probit models yields the same results. As a ﬁnal robustness analysis we estimate the univariate and correlated models by reducing opinions variables to a three-point scale. The parameter estimates are very similar as before. This indicates that our ﬁndings are mainly driven by differences between individuals who agree or disagree to given policy statements and not by differences between those who agree (disagree) or deﬁnitely agree (deﬁnitely disagree). 12 3 3 A. Palali, J. C. van Ours Table 6 Simulations of the effects of past and current use of cannabis on opinions about cannabis legal- ization and policy of cannabis shops (percentages) Table 6 Simulations of the effects of past and current use of cannabis on opinions about cannabis legal- ization and policy of cannabis shops (percentages) Deﬁnitely disagree Disagree Indifferent Agree Deﬁnitely agree Total a. Cannabis should be legalized 1. Current use 1 5 13 37 44 100 2. Past use 6 12 22 38 22 100 3. Never use 20 23 25 26 6 100 b. It should be permitted to sell cannabis at coffeeshops 1. Current use 4 7 15 53 21 100 2. Past use 12 14 21 44 9 100 3. 5.3.3 Sensitivity to model speciﬁcations Never use 23 19 22 32 4 100 The simulations are based on the parameter estimates of the correlated models presented in Table 4; the reference person has sample mean values for all of the observable characteristics except for cannabis use The simulations are based on the parameter estimates of the correlated models presented in Table 4; the reference person has sample mean values for all of the observable characteristics except for cannabis use 5.4 Magnitude of the effects Toindicatethemagnitudeoftheeffectofpastandcurrentuseofcannabis,wesimulated the probabilities for each alternative in the ordered response variable. The simulation results are given in Table6. The ﬁrst panel presents the results for the ﬁrst policy statement (cannabis should be legalized). As shown there is a large effect of cannabis use. The estimated probability of agreeing with legalizing cannabis is 33% for our reference person who has never used cannabis (row (3)). If that reference person used cannabis in the past, support for no-prohibition increases to 60% and if this person is still using cannabis the support for no-prohibition jumps to 81%. Similarly, the second panel presents the simulation results for the second policy statement. The estimated probability of agreeing with the idea of selling cannabis at coffeeshops is 36% for a never user reference person. This probability increases to 53% if this reference person is a past-user and to 74% if the individual is a current user. Similarly the probability of disagreeing decreases from 43 to 26 and 11%, respectively. A comparison of these ﬁgures with the unconditional distribution given in Table 1 shows that even though differences between cannabis users and non-users decrease after controlling for observable and unobservable factors, they remain considerable. 6 Conclusions Previous studies show that cannabis users are more in favor of cannabis legalization than individuals who never used cannabis. Interesting as this may be in itself, it does not necessarily imply that opinions are inﬂuenced by cannabis use in a causal way. Individuals who are more likely to consume cannabis may also be more in favor of legalization without the personal experience affecting opinions. Knowing whether or not there is a causal effect from cannabis use is interesting because if so, it reveals how potential dangers of cannabis use are assessed. If cannabis use increases the support for legalizing cannabis, then this reveals that cannabis use may not be so harmful as Cannabis use and support for cannabis legalization individuals were inclined to think before they started using cannabis. However, such a causal effect may also have to do with self-interest, i.e. the expectation that cannabis legalization will induce easier access and perhaps lower prices. individuals were inclined to think before they started using cannabis. However, such a causal effect may also have to do with self-interest, i.e. the expectation that cannabis legalization will induce easier access and perhaps lower prices. We use data from a 2008 survey which includes detailed questions about cannabis use and opinions about cannabis policies in the Netherlands. From our analysis, we conclude that there is a causal effect of personal experience with cannabis use on the support given to more liberal cannabis policies. Those who currently use cannabis and those who used it in the past are more in favor of legalization. The opinion of current cannabis users may be driven by self-interest and inside information but the opinion of past users will be driven mainly by inside information about the dangers of cannabis use. From the signiﬁcance of the effect of past cannabis use, we conclude that cannabis use may not be as harmful as cannabis users originally thought it was and non-users are inclined to think. Our analysis suggests that as the share of cannabis users in the population goes up support for cannabis legalization will increase. 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https://openalex.org/W2122483069	https://europepmc.org/articles/pmc1717013?pdf=render	English	null	Microbial translocation is a cause of systemic immune activation in chronic HIV infection	Nature medicine	2,006	cc-by	275	BioMed Central BioMed Central Retrovirology Open Access Page 1 of 1 (page number not for citation purposes) Open Acc Oral presentation Microbial translocation is a cause of systemic immune activation in chronic HIV infection Jason M Brenchley* Address: Research Fellow, Vaccine Research Center, National Institutes of Health, Bethesda, Maryland, USA * Corresponding author from 2006 International Meeting of The Institute of Human Virology Baltimore, USA. 17–21 November, 2006 from 2006 International Meeting of The Institute of Human Virology Baltimore, USA. 17–21 November, 2006 Published: 21 December 2006 Retrovirology 2006, 3(Suppl 1):S98 doi:10.1186/1742-4690-3-S1-S98 Published: 21 December 2006 Retrovirology 2006, 3(Suppl 1):S98 doi:10.1186/1742-4690-3-S1-S98 Published: 21 December 2006 Retrovirology 2006, 3(Suppl 1):S98 doi:10.1186/1742-4690-3-S1-S98 © 2006 Brenchley; licensee BioMed Central Ltd. © 2006 Brenchley; licensee BioMed Central Ltd. Chronic activation of the immune system is a hallmark of progressive HIV infection and better predicts disease out- come than plasma viral load, yet its etiology remains obscure. Here, we show that circulating microbial prod- ucts, likely derived from the gastrointestinal tract, are a primary cause of HIV-related systemic immune activation. Circulating lipopolysaccharide, an indicator of microbial translocation, is significantly increased in chronically HIV-infected individuals and SIV-infected rhesus macaques. We show that monocytes are chronically stim- ulated in vivo by increased lipopolysaccharide, which also correlates with measures of innate and adaptive immune activation. Effective antiretroviral therapy appears to reduce microbial translocation. Furthermore, in non- pathogenic SIV infection of sooty mangabeys, microbial translocation does not seem to occur. These data establish a mechanism for chronic immune activation in the con- text of a compromised gastrointestinal mucosal surface and provide novel directions for therapeutic interventions that modify the consequences of acute HIV infection.
https://openalex.org/W3154950793	https://www.scielo.br/j/ibju/a/8WLxqk8kfnJ7LLYRfFwfPsG/?lang=en&format=pdf	English	null	Can extended upper pole ureterectomy prevent ureteral stump syndrome after proximal approach for duplex kidneys?	International Braz J Urol	2,021	cc-by	3,151	Vol. 47 (4): 821-826, July - August, 2021 doi: 10.1590/S1677-5538.IBJU.2020.0686 Vol. 47 (4): 821-826, July - August, 2021 doi: 10.1590/S1677-5538.IBJU.2020.0686 ORIGINAL ARTICLE Keywords: Materials and Methods: Forty-four consecutive patients with symptomatic duplex system were retrospectively classifi ed into two groups: those with limited ureteral excision after heminephrectomy (HN) (group-1) and those with extended ureterectomy after HN (group-2). Patients were followed-up for at least 36 months regarding outcomes of distal ureteral stump. _____________________ Submitted for publication: August 04, 2020 _____________________ Accepted after revision: January 17, 2021 _____________________ Published as Ahead of Print: February 28, 2021 Results: Overall complication was 20%. A total of 8 patients required unplanned further surgery in Group-1 (30%) whereas only 1 patient required unplanned surgery in group 2 (6%) (p=0.07). Subgroup analysis showed that Group-1 presented more DUSS requiring surgery during follow-up than group-2 (p=0.04). Factors possibly affecting complications incidence (such as ureterocele or ectopic ureter) did not differ between groups (p=0.72 and p=0.78). _____________________ Published as Ahead of Print: February 28, 2021 Conclusion: Upper pole nephrectomy should be performed with extended distal ureteral dissection to prevent ureteral stump complications. ARTICLE INFO ARTICLE INFO Bruno Nicolino Cezarino https://orcid.org/0000-0001-9161-7337 Keywords: Cakut [Supplementary Concept]; Ureter; Urinary Tract Infections Int Braz J Urol. 2021; 47: 821-6 _____________________ Submitted for publication: August 04, 2020 _____________________ Accepted after revision: January 17, 2021 _____________________ Published as Ahead of Print: February 28, 2021 ABSTRACT ARTICLE INFO Bruno Nicolino Cezarino https://orcid.org/0000-0001-9161-7337 Keywords: Cakut [Supplementary Concept]; Ureter; Urinary Tract Infections Int Braz J Urol. 2021; 47: 821-6 _____________________ Submitted for publication: August 04, 2020 _____________________ Accepted after revision: January 17, 2021 _____________________ Published as Ahead of Print: February 28, 2021 Introduction: Symptomatic duplex kidneys usually present with recurrent urinary tract infection due to ureteral obstruction (megaureter, ureterocele or ectopic ureter) and/or vesicoureteral refl ux. Upper-pole nephrectomy is a widely accepted procedure to correct symptomatic duplex systems with poor functioning moieties, also known as upper or proximal approach. The distal ureteral stump syndrome (DUSS) can be a late complication of this approach. There is no consensus upon the length of ureteral dissection and the better approach to symptomatic disease in duplex systems, so we aim to identify if extended ureteral dissection can prevent DUSS in top-down approach. Can extended upper pole ureterectomy prevent ureteral stump syndrome after proximal approach for duplex kidneys? Bruno Nicolino Cezarino 1, Roberto Iglesias Lopes 1, Ricardo Haidar Berjeaut 1, Francisco Tibor Dénes 1 Bruno Nicolino Cezarino 1, Roberto Iglesias Lopes 1, Ricardo Haidar Berjeaut 1, Francisco Tibor Dénes 1 1 Unidade de Urologia Pediátrica, Divisão de Urologia, Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo - FMUSP, SP, Brasil 1 Unidade de Urologia Pediátrica, Divisão de Urologia, Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo - FMUSP, SP, Brasil _____________________ Published as Ahead of Print: February 28, 2021 IBJU \| EXTENDED UPPER POLE URETERECTOMY cluded urinary tract infection associated to vesical ureteral junction obstruction, urethral obstruction due to ureterocele and/or urinary incontinence. En- doscopic incision was not performed in patients with asymptomatic ureteroceles and was only used to de- compress acutely infected ureteroceles. systems with poor functioning upper moieties. Al- ternatively, this condition can be managed with uretero-pyelic or uretero-ureteral anastomosis of the upper to the lower moiety, even in cases of low-function. Nowadays both procedures, also known as upper or proximal approaches, are usu- ally performed by laparoscopic technique, with sim- ple and straightforward techniques. Patients were classified into two groups: Group-1 underwent upper pole heminephrourete- rectomy (HN) with limited or no distal ureterec- tomy and Group-2 heminephroureterectomy (HN) with aggressive extended ureterectomy, well be- low iliac vessels. The ureteral stump was ligated or left open according to initial pathology of the affected moiety. The surgical access (laparoscopic vs. open) was chosen according to surgeon’s pre- ference and expertise, but favored extended ure- terectomy after 2008, after reviewing our initial results that showed a DUSS rate of 15.7% (9). Another option is the lower or distal ap- proach, which address directly the uretero-vesical junction (UVJ), whose main advantage is the more comprehensive correction of the anatomical abnor- malities of both ureters, albeit with a significantly more invasive reconstructive surgery, which is also prone to more complications (4). The distal ureteral stump syndrome (DUSS) can be a late manifestation of proximal approach and it is defined as any complication related to the upper pole ureteral stump: stump empyema or reflux, symptomatic febrile urinary tract infection (UTI), persistent bacteriuria or urethral discharge, hematuria or low quadrant pain, which frequently requires intervention (5, 6). Mean age at operation, baseline patho- logy, long-term complications related to ureteral stump and requirement for further surgical inter- vention were recorded for all cases. All patients were followed with US at 6 weeks and 6 months post-operatively, then on a yearly basis, with a minimum follow-up of 36 months. Only sympto- matic patients underwent a postoperative repeat VCUG. Statistical analysis consisted of chi-square test and Mann-Whitney, with p value <0.05 being statistically significant. There is no consensus regarding the best choice between the proximal and distal approaches in the treatment of symptomatic disease in duplex systems, as well as the adequate length of ureteral dissection in the later technique (7, 8). Operative technique detailsl Briefly, upper-pole nephrectomy involves proximal ureteral dissection of affected moiety, li- gation of polar vessels, division of two moieties and resection of the ischemic upper pole with electric or harmonic scalpel. Care is taken to remove all non- -viable renal and collector tissue to prevent post- -operative retention cysts. The extended distal urete- rectomy, performed in Group-2, can be challenging due to the common sheath of the duplicated ureters (10). Laparoscopic approach precludes the need of another open incision to perform distal dissection (Figure-1). The careful dissection is extended close to the UVJ, avoiding lesion to the functional lower pole ureter (Figure-1). As mentioned above, ligation of the distal stump in both groups is based on base- line pathology, being obligatory in cases of VUR and optional for obstructive cases (Figure-1). IBJU \| EXTENDED UPPER POLE URETERECTOMY This study aims to identify if extended ure- teral dissection can prevent DUSS in the upper or proximal approach. INTRODUCTION infection due to ureteral obstruction of the upper pole, megaureter, ureterocele or ectopic ureter, and/ or vesicoureteral refl ux to the lower pole, eventually compromising affected moiety function (2, 3). Duplex kidneys are the most common con- genital anomalies of the upper urinary tract, with an estimated incidence of 0.8% (1). Symptomatic duplex kidneys usually present with recurrent urinary tract Upper-pole nephrectomy is a widely per- formed procedure to correct symptomatic duplex 821 MATERIALS AND METHODS An ethics committee approved (research pro- tocol 1.895748) the study and a prospective database was maintained for consecutive patients undergoing surgery for duplex kidneys in our tertiary pediatric urology center between 2005 and 2017 and a re- trospective analysis was performed. Upper-pole ne- phrectomy has been the standard of care in our insti- tution for symptomatic non-functioning upper pole moieties in duplex kidneys. All patients underwent ultrasonography (US) and DMSA nuclear scan or computer tomography scan that confirmed the sig- nificantly impaired anatomy and function of the upper moiety, as well as a voiding urethrocystogram (VCUG) prior to surgery. Indications for surgery in- 822 IBJU \| EXTENDED UPPER POLE URETERECTOMY opic dissection of distal ureter in a 4 years-old girl with duplex kidney with non-functioning refl ux Figure 1 - Laparoscopic dissection of distal ureter in a 4 years-old girl with duplex kidney with non-functioning refl ux upper pole. A- Initial dissection of common sheath; B- Dissection of ureters below iliac vessels; C- Final aspect of the distal upper pole ureter dissection; D -Final dissection of Ureterovesical junction; E- Ligation of upper pole ureter; F- Final aspect of ureteral stump. A- Initial dissection of common sheath; B- Dissection of ureters below iliac vessels; C- Final aspect of the distal upper pole ureter dissection; D -Final dissection of Ureterovesical junction; E- Ligation of upper pole ureter; F- Final aspect of ureteral stump. A- Initial dissection of common sheath; B- Dissection of ureters below iliac vessels; C- Final aspect of the distal upper pole ureter dissection; D -Final dissection of Ureterovesical junction; E- Ligation of upper pole ureter; F- Final aspect of ureteral stump. RESULTS This symptomatic patient in group-2 was further investigated and a dysfunctional voiding pattern was diagnosed, being addressed with diet impro- vement and frequent voiding instructions after the surgical repair. No lesions of lower moiety ureter neither lower pole loss of function was identifi ed during or after surgery for both groups. A total of 44 upper pole nephrectomies were performed: 28 in Group-1 and 16 in group-2. Sex, diagnosis, indication for initial surgery and age at surgery were similar between groups. Two patients in group-1 and one patient in group-2 underwent ureterocele puncture before surgery and presented with secondary vesical ureteral re- fl ux to upper pole. Median follow-up was signifi - cantly shorter among patient submitted to exten- ded ureterectomy (42 months, with range of 36 - 108) compared to limited dissection (84 months, with a range of 72-184) (Table-1). Although the global rate of re-do surgeries did not reach a statistically signifi cant difference (p=0.07), subgroup analysis showed that Group-1 presented more DUSS (with at least one of pre- viously described symptoms) requiring surgery during follow-up than group-2 (p=0.04), requiring laparoscopic ureteral stump resection in 5 patients and open UVRS in one. Another patient from group-1 persisted with urethral obstruction (due to non-collapsed ureterocele) and was submitted to successfully UVRS. Detailed data and need for further surgery are presented in Table-1. Factors possibly affecting complications incidence, such as ureterocele or ectopic ureter, did not differ be- tween groups (p=0.72 and p=0.78). Overall complication was 20%. A total of 8 patients required unplanned further surgery in Group-1 (30%) whereas only 1 patient required unplanned surgery in group-2 (6%). One patient in each group presented with de novo refl ux to the lower remnant pole and were treated with endos- copic injection of bulking agent and uretero-ve- sical reconstructive surgery (UVRS), respectively. 823 IBJU \| EXTENDED UPPER POLE URETERECTOMY Table 1 - Demographics, follow-up, unplanned surgery diagnosis and need of further surgery. RESULTS Group 1- Limited ureteral extension 2- Extended Ureteral Dissection p value Sex F / M 26(92%) / 2(8%) 11(68%) / 4(32%) p=0.08 Diagnosis Upper pole ureterocele 14 (50%) 9 (60%) 0.72 Secondary vur after puncture 2(7%) 1(6%) 0.64 Primary UVJ obstruction 6(21%) 2(12%) 0.42 Ectopic ureter 6(21%) 4(24%) 0.78 Indication for initial surgery Urinary tract infection 17(60%) 9(60%) 0.63 Urinary incontinence 4 (14%) 1(6%) 0.45 Progressive hydronephrosis 6(21%) 4(24%) 0.69 Urethral obstruction 1(3,5%) 2(12%) 0.23 Mean age at surgery (months) / SD* 15.5 / 47.7 17/39 0.43 Median follow-up (months) 84 42.5 0.003 Unplanned surgery diagnosis 8 (30.7%) 1 (6%) 0.07 DE NOVO reflux to lower pole moiety 1 (12,5%) 1(100%) 0.64 Ureteral stump sindrome 6 (75%) 0 0.04 Persistent urethral obstruction (ureterocele) 1 (12.5%) 0 0.45 Need of further surgery Endoscopic injection to remnant moitey 1(12.5%) 0 Uretero-vesical reconstructive surgery 2(25%) 1(100%) Laparoscopic ureteral stump ressection 5(62.5%) 0 *Standard deviation. Table 1 - Demographics, follow-up, unplanned surgery diagnosis and need of further surgery. Classic upper pole nephrectomy is a widely accepted procedure to correct symptomatic duplex systems with poor functioning moieties, but seve- ral complications of this approach were described in literature: loss of function in the remaining lower pole as high as 8% (11), ureteral stump complications around 16% (9) and de novo reflux to lower pole in up to 25% (5, 12). Main advantages of this approa- ch when performed laparoscopically are those of the IBJU \| EXTENDED UPPER POLE URETERECTOMY minimally invasive approach, with reduced pain and faster recovery (10). complication rate after UVRS surgery. Their proximal data were published in a previous paper, however the length of ureteral dissection was not cited (5). The complication rate after UVRS was 5% whereas in the top-down approach was 21%. In our cohort, exten- ded distal ureteral dissection had a limited complica- tion rate of 6%, while the limited ureteral resection group reached 30%. These results are consonant to another paper that proposed bottom-up approach as well: Gran et al. showed 8% complication rate after UVRS (4). UVRS, on the other hand, may have a lower incidence of complication related to the ureteral ecto- pia, ureteral stump and ureterocele, which are known to be possible complications of top-down approach (5). This approach, however, is related to a longer urethral catheterization time, longer hospital stay, bladder spasms, hematuria and potentially increased risk of bladder dysfunction and ureteral obstruction (4, 13, 14), although recent papers reported lower in- cidence of these complication (15, 16). Leaving dys- plastic or hydronephrotic tissue with impaired func- tion in situ could be harmful as well. Husmann et al. (17) reported 5% incidence of persisting urinary tract infections despite de-obstruction, and anecdotal cases of hypertension, proteinuria and renal tumors were reported. Our study has several limitations. Although database was maintained prospectively, a retrospec- tive analysis was performed. The patient allocation to each group was not randomized and therefore the Group-2 had a shorter follow-up compared to Group-1. This shorter follow-up is based primarily on the surgeon’s observation that a longer ureteral dis- section could potentially prevent complications du- ring upper pole nephrectomy, so there was a surgical shift towards extending the distal ureteral dissection. Another limitation of this study is that Group-2 was operated later on, with an advanced knowledge and manage on bladder bowel dysfunctions, which could eventually lead to reduced chance of UTI and stum- pectomies. Further prospective randomized studies can corroborate our study hypothesis and maintain top-down approach as first-line option to treat du- plex system kidneys without increasing DUSS inci- dence, adding minimally invasive benefits to treat- ment. As both ureters in duplex system kidneys run inside the same sheath deep in the pelvis, especially below iliac vessels, classic open top-down approach may limit a successful dissection to the level above iliac vessels. IBJU \| EXTENDED UPPER POLE URETERECTOMY Development of laparoscopic knowled- ge and ability allows safe extended separation of the ureters, with low risk of lesion to the lower pole ure- ter, even when performed close to the UVJ. Numerous authors reported the need of com- plementary surgery in the proximal approach betwe- en 15 and 25%, either for DUSS or new onset reflux to lower pole, especially in patients with concurrent ureterocele (5, 17), but none of these authors perfor- med an extended distal ureteral dissection, nor ci- ted the length of ureteral dissection to prevent such complications. In fact, most of the papers cited only partial nephrectomy as the surgical technique, which probably resulted in a long ureteral stump left behind. CONCLUSIONS Upper pole nephrectomy performed with ex- tended distal ureteral dissection can prevent ureteral stump complications. Escolino et al. (10) studied the ureteral stump length after laparoscopic and retroperitoneosco- pic heminephroureterectomy for duplex kidneys in symptomatic patients. Laparoscopic ureteral stump measure varied between 3 to 7mm while retroperi- toneoscopic ureteral stump measured 20 to 50mm. Five years follow-up revealed no complications in the laparoscopic approach compared to two DUSS after retroperitoneoscopic surgery that required com- plementary surgery. DISCUSSION The management of symptomatic duplex kidney with severely impaired upper pole has been a recurrent issue in literature. Proximal approach with upper pole nephrectomy or distal (UVRS) approach, addressing the UVJ and preserving the upper moiety, are options to treat these patients (8). 824 IBJU \| EXTENDED UPPER POLE URETERECTOMY CONFLICT OF INTEREST None declared. None declared. IBJU \| EXTENDED UPPER POLE URETERECTOMY 2. Bastos JM Netto, Rondon AV, Machado MG, Zerati M Filho, Nascimento RLP, Lima SVC, et al. Brazilian consensus on vesicoureteral reflux-recommendations for clinical practice. Int Braz J Urol. 2020; 46:523-37. 10. Escolino M, Farina A, Turrà F, Cerulo M, Esposito R, Savanelli A, et al. Evaluation and outcome of the distal ureteral stump after nephro-ureterectomy in children. A comparison between laparoscopy and retroperitoneoscopy. J Pediatr Urol. 2016; 12:119.e1-8. 3. Andrioli V, Regacini R, Aguiar W. Primary Vesicoureteral reflux and chronic kidney disease in pediatric population. What we have learnt? Int Braz J Urol. 2020; 46:262-8. 11. Gundeti MS, Ransley PG, Duffy PG, Cuckow PM, Wilcox DT. Renal outcome following heminephrectomy for duplex kidney. J Urol. 2005; 173:1743-4. 4. Gran CD, Kropp BP, Cheng EY, Kropp KA. Primary lower urinary tract reconstruction for nonfunctioning renal moieties associated with obstructing ureteroceles. J Urol. 2005; 173:198-201. 12. De Caluwe D, Chertin B, Puri P. Fate of the retained ureteral stump after upper pole heminephrectomy in duplex kidneys. J Urol. 2002; 168:679-80. 5. Pearce R, Subramaniam R. Partial nephroureterectomy in a duplex system in children: the need for additional bladder procedures. Pediatr Surg Int. 2011; 27:1323-6. 13. Fung LC, McLorie GA, Jain U, Khoury AE, Churchill BM. Voiding efficiency after ureteral reimplantation: a comparison of extravesical and intravesical techniques. J Urol. 1995; 153:1972-5. 6. Malek RS, Moghaddam A, Furlow WL, Greene LF. Symptomatic ureteral stumps. J Urol. 1971; 106:521-8. 14. Sillén U. Bladder dysfunction in children with vesico-ureteric reflux. Acta Paediatr Suppl. 1999; 88:40-7. 15. de Kort LM, Klijn AJ, Uiterwaal CS, de Jong TP. Ureteral reimplantation in infants and children: effect on bladder function. J Urol. 2002; 167:285-7. 7. Hisamatsu E, Takagi S, Nakagawa Y, Sugita Y. Nephrectomy and upper pole heminephrectomy for poorly functioning kidney: Is total ureterectomy necessary? Indian J Urol. 2012; 28:271-4. 16. de Jong TP, Dik P, Klijn AJ, Uiterwaal CS, van Gool JD. Ectopic ureterocele: results of open surgical therapy in 40 patients. J Urol. 2000; 164:2040-3. 8. Keene DJB, Subramaniam R. Duplex systems: Top-down or bottom-up approach? J Pediatr Urol. 2020; 16:387. e1-387.e8. 17. Husmann D, Strand B, Ewalt D, Clement M, Kramer S, Allen T. Management of ectopic ureterocele associated with renal duplication: a comparison of partial nephrectomy and endoscopic decompression. J Urol. 1999; 162:1406-9. 9. Dénes FT, Danilovic A, Srougi M. Outcome of laparoscopic upper-pole nephrectomy in children with duplex systems. J Endourol. REFERENCES 1. Androulakakis PA, Stephanidis A, Antoniou A, Christophoridis C. Outcome of the distal ureteric stump after (hemi)nephrectomy and subtotal ureterectomy for reflux or obstruction. BJU Int. 2001; 88:586-9. Keene et al. (8) compared proximal to dis- tal approach and found statistically significant lower 825 825 IBJU \| EXTENDED UPPER POLE URETERECTOMY _______________________ Correspondence address: Bruno Nicolino Cezarino, MD Unidade de Urologia Pediátrica, Divisão de Urologia, Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo, SP, Brasil Av. Dr. Enéas Carvalho de Aguiar, 255, Cerqueira César, São Paulo, SP, 05403-000, Brasil E-mail: brunocezarino@yahoo.com.br Fax: + 55 11 3159-1921 IBJU \| EXTENDED UPPER POLE URETERECTOMY 2007; 21:162-8. _______________________ Correspondence address: Bruno Nicolino Cezarino, MD Unidade de Urologia Pediátrica, Divisão de Urologia, Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo, SP, Brasil Av. Dr. Enéas Carvalho de Aguiar, 255, Cerqueira César, São Paulo, SP, 05403-000, Brasil E-mail: brunocezarino@yahoo.com.br Fax: + 55 11 3159-1921 826
https://openalex.org/W2607789980	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0176167&type=printable	English	null	Correlation of histopathologic characteristics to protein expression and function in malignant melanoma	PloS one	2,017	cc-by	8,278	RESEARCH ARTICLE OPEN ACCESS Citation: Welinder C, Pawłowski K, Szasz AM, Yakovleva M, Sugihara Y, Malm J, et al. (2017) Correlation of histopathologic characteristics to protein expression and function in malignant melanoma. PLoS ONE 12(4): e0176167. https:// doi.org/10.1371/journal.pone.0176167 Editor: Michal Zmijewski, Medical University of Gdańsk, POLAND Metastatic melanoma is still one of the most prevalent skin cancers, which upon progression has neither a prognostic marker nor a specific and lasting treatment. Proteomic analysis is a versatile approach with high throughput data and results that can be used for characterizing tissue samples. However, such analysis is hampered by the complexity of the disease, het- erogeneity of patients, tumors, and samples themselves. With the long term aim of quest for better diagnostics biomarkers, as well as predictive and prognostic markers, we focused on relating high resolution proteomics data to careful histopathological evaluation of the tumor samples and patient survival information. Gdańsk, POLAND Received: October 31, 2016 Accepted: April 6, 2017 Published: April 26, 2017 Copyright: © 2017 Welinder 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. Copyright: © 2017 Welinder 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. * Gyorgy.Marko-Varga@bme.lth.se Background Editor: Michal Zmijewski, Medical University of Gdańsk, POLAND Correlation of histopathologic characteristics to protein expression and function in malignant melanoma Charlotte Welinder1,2, Krzysztof Pawłowski3,4, A. Marcell Szasz1,2,5, Maria Yakovleva2, Yutaka Sugihara1, Johan Malm2,4, Go¨ran Jo¨nsson1, Christian Ingvar6, Lotta Lundgren1,7, Bo Baldetorp1, Håkan Olsson1,7,8, Melinda Rezeli9, Thomas Laurell2,9, Elisabet Wieslander1, Gyo¨rgy Marko-Varga2,9,10* 1 Division of Oncology and Pathology, Dept. of Clinical Sciences Lund, Lund University, Lund, Sweden, 2 Centre of Excellence in Biological and Medical Mass Spectrometry “CEBMMS”, Biomedical Centre D13, Lund University, Lund, Sweden, 3 Faculty of Agriculture and Biology, Dept. of Experimental Design and Bioinformatics, Warsaw University of Life Sciences, Warszawa, Poland, 4 Dept. of Translational Medicine, Lund University, Malmo¨, Sweden, 5 2nd Dept. of Pathology, Semmelweis University, Budapest, Hungary, 6 Dept. of Surgery, Dept. of Clinical Sciences Lund, Lund University, Skåne University Hospital, Lund, Sweden, 7 Dept. of Oncology, Skåne University Hospital, Lund, Sweden, 8 Cancer Epidemiology, Dept. of Clinical Sciences, Lund University, Lund, Sweden, 9 Clinical Protein Science & Imaging, Biomedical Centre, Dept. of Biomedical Engineering, Lund University, BMC D13, Lund, Sweden, 10 First Dept. of Surgery, Tokyo Medical University, Tokyo, Japan a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Conclusion In this feasibility study, promising results show the feasibility of relating proteomics to histo- pathology and clinical outcome, and insight thus can be gained into the molecular processes driving the disease. The combined analysis of histological features including the sample cel- lular composition with protein expression of each metastasis enabled the identification of novel, differentially expressed proteins. Further studies are necessary to determine whether these putative biomarkers can be utilized in diagnostics and prognostic prediction of meta- static melanoma. Deep sequencing and clinicopathological analysis of lymph node metastatic melanoma Competing interests: The authors have declared that no competing interests exist. Results Competing interests: The authors have declared that no competing interests exist. In this feasibility study, several proteins were identified that positively correlated to tumor tissue content including IF6, ARF4, MUC18, UBC12, CSPG4, PCNA, PMEL and MAGD2. The study also identified MYC, HNF4A and TGFB1 as top upstream regulators correlating to tumor tissue content. Other proteins were inversely correlated to tumor tissue content, the most significant being; TENX, EHD2, ZA2G, AOC3, FETUA and THRB. A number of proteins were significantly related to clinical outcome, among these, HEXB, PKM and GPNMB stood out, as hallmarks of processes involved in progression from stage III to stage IV disease and poor survival. Patients and methods Data Availability Statement: Proteomics data were used, as deposited in the PRIDE database, under the dataset identifier PXD001725. Regional lymph node metastases obtained from ten patients with metastatic melanoma (stage III) were analyzed by histopathology and proteomics using mass spectrometry. Out of the ten patients, six had clinical follow-up data. The protein deep mining mass spectrome- try data was related to the histopathology tumor tissue sections adjacent to the area used for deep-mining. Clinical follow-up data provided information on disease progression which could be linked to protein expression aiming to identify tissue-based specific protein markers for metastatic melanoma and prognostic factors for prediction of progression of stage III disease. Funding: This study was supported by grants from Mrs. Berta Kamprad Foundation, Vinnova, Inga- Britt and Arne Lundbergs Foundation, The Crafoord Foundation, and European Research Council Advanced Grant. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 1 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0176167 April 26, 2017 PLOS ONE \| https://doi.org/10.1371/journal.pone.0176167 April 26, 2017 Deep sequencing and clinicopathological analysis of lymph node metastatic melanoma studies in the past years [14–20]. Most melanomas harbor alterations in the BRAF, NF1, RAS, MDM2 (KIT) genes, which result in activation of the MAPK and RAS pathways conferring survival advantage by reprogramming crucial cell cycle and apoptotic cellular functions [21]. Clinical and pathological properties partly reflect the identified (BRAF, RAS, NF1, triple wild type) genomic subtypes of melanoma, but from both clinical and genetic perspective the groups still are heterogeneous [21]. Newly developed drugs allowing targeted therapy such as kinase inhibitors or drugs modu- lating the immune response currently provide more promise to the patients [22–27]. However, some of these newer treatments have also been subjected to the development of resistance [28]. Recently, the Food and Drug Administration (FDA) approved nivolumab (Opdivo), a PD-1 immune checkpoint inhibitor, for the treatment of advanced (unresectable or metastatic) mel- anoma in adults, regardless of BRAF status. The drug was approved by FDA for the treatment of patients both with BRAF V600 wild-type unresectable and BRAF V600 mutation-positive metastatic melanoma in addition to combination treatment with ipilimumab (anti-CTLA-4 antibody). With several options requiring individualized treatment, there is a great demand for validated biomarkers that can support both the primary diagnosis and preferably predict the progression of disease and response to treatment of metastatic disease. Predictive biomark- ers of response to immune checkpoint inhibitors are currently under evaluation including but not limited to the lymphocytic infiltration of the tumor (= the immunoscore) [29], PD-L1 expression and or CTLA-4 expression [30]. Building upon our previous proteomics work [31] and related genomics studies [32–34], proteomics data for samples from 10 patients with lymph node metastatic melanoma (PRIDE dataset identifier: PXD001725) were referred to histopathological and clinical work-up and evaluation. Thus, in this feasibility study, in addition to the deep mining proteomic analysis utilizing high-resolution reversed phase nano-separation in combination with mass spectrometry, we also performed in depth pathological characterization (including tissue composition, tumor characteristics and lymphocytic infiltration). The pathological analyses were performed on tumor tissues sections adjacent to the areas used for deep mining. Clinical follow-up informa- tion of the patients was reviewed and the cases were grouped into prognostic sets. Hence, the protein expression could be related to the specific cellular composition of the sample. Based on identified protein signatures, molecular classification and clinicopathological characterization, emerging biological relevance could be assigned to several marker proteins. PLOS ONE \| https://doi.org/10.1371/journal.pone.0176167 April 26, 2017 Introduction Cutaneous metastatic melanoma (MM) is the sixth most common cancer worldwide with an increasing incidence especially in the Northern European countries and Australia [1, 2]. In Sweden it is the fifth most prevalent malignant neoplasm both in men (following prostate, colon, non-melanocytic skin and urinary tract tumors) and women (following breast, colon, non-melanocytic skin and uterine neoplasms) with an annual incidence of 5% [3, 4]. Malignant melanoma possessing a wide variety of cytomorphologic features can be consid- ered as a challenge from a diagnostic perspective, and is well-known for its metastatic spread, most common to the lungs, liver, brain, bone and skin but sometimes to any, and often unusual location in the body. Melanoma tumors commonly possess a broad toolkit to escape from the body surveillance system [5] thus facilitating further metastatic spread. Examples of such mechanisms are replicative immortality, genome instability and mutation, resistance to cell death, deregulated cellular energetics, sustaining proliferative signaling, evasion of growth suppressors, activation of invasion and metastasis, tumor promoting inflammation and means to avoid immune destruction [6]. Screening and early detection improve survival from MM and a better outcome can be expected once surgical removal of early primary lesion was performed, still about 15–25% of the sentinel lymph nodes contain metastatic melanoma cells [7]. Progression from the lymph nodes may occur with time, and metastatic melanoma has been inherently difficult to treat with a low survival rate (< 15% at 5 years) [8]. A phase 3 clinical trial (Multicenter Selective Lymphadenectomy Trial II, or MSLT-II) is designed and conducted to answer the question whether sentinel lymph node biopsy should routinely be followed by removal of the remaining regional lymph nodes (complete lymph node dissection) if the sentinel lymph node is positive for melanoma, or it should be followed-up by ultrasound [9] Factors determining prognosis routinely rely on clinicopathological characteristics of the patient and primary tumor [10–13], which have been supplemented with DNA sequencing PLOS ONE \| https://doi.org/10.1371/journal.pone.0176167 April 26, 2017 2 / 16 Materials and methods Clinical samples The location of primary tumors and progression of melanomas is displayed in Fig 1. This study was approved by the Regional Ethical Committee at Lund University, Southern Sweden, approval number: DNR 191/2007 and 101/2013. All patients within the study provided a written informed consent. The tumor tissues used were lymph node metastases from 10 mela- noma patients undergoing surgery at Lund University Hospital, Sweden. The clinical informa- tion on respective patients is summarized in Table 1. The fresh specimens were divided into two parts. One portion of the metastasis was fixed in formalin, embedded in paraffin (FFPE) and subsequently sectioned for histopathologic confirmation and the other part was snap fro- zen within minutes after removal and stored at -80˚C in the Biobank. All FFPE samples were routinely confirmed for diagnosis at the department of surgical pathology. The frozen speci- mens were used as described below for both protein expression analysis and further histologi- cal evaluations. 3 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0176167 April 26, 2017 Deep sequencing and clinicopathological analysis of lymph node metastatic melanoma Deep sequencing and clinicopathological analysis of lymph node metastatic melanoma Table 1. Clinical information of patient characteristics. Breslow thickness and Clarks refer to primary melanoma feature. Tumor Gender Age at met Age at primary Breslow class Clark Stage Status OS DSS MM35 Male 55 54 3 4 3 Alive 7960 - MM98 Male 75 73 4 4 3 Dead 519 519 MM504 Male 54 NA NA NA NA Dead 451 451 MM687 Male 74 72 1 2 3 Dead 591 591 MM787 Male 81 78 2 4 3 Dead 734 734 MM812 Male 51 NA NA NA NA Alive 5028 - MM813 Female 54 54 2 3 3 Alive 4913 - MM825 Female 66 64 2 4 3 Alive 4508 - MM829 Male 55 49 1 2 3 Alive 4431 - MM835 Female 36 32 3 3 3 Alive 4353 - NA–not available OS–Calculated from date of sample collection to 20160622. DSS–Calculated from date of sample collection to date of dead in melanoma disease. Table 1. Clinical information of patient characteristics. Breslow thickness and Clarks refer to primary melan Table 1. Clinical information of patient characteristics. Breslow thickne https://doi.org/10.1371/journal.pone.0176167.t001 power with dense melanin content); predominant cytoplasmic properties were assigned as unremarkable, eosinophilic or mixed. The lymphocytic infiltration in two dimensions (distri- bution: 0: no infiltration, 1: less than 25% of tumor infiltrated, 2: 25–50% of tumor infiltrated, 3: more than 50% of tumor infiltrated by lymphocytes; density was assessed on a 0–3 semi- quantitative scale) was assessed resulting in a combined (lymphocyte distribution + lympho- cyte density) = immunoscore (sum of components: 0, 2–6). Histological evaluation of tumors Frozen tissue samples were sectioned on a cryostat into 6 μm thick sections, placed upon glass slides, dried at 37˚C for 30 min and fixed with 100% methanol for 5 min. The sections were stained with hematoxylin and eosin (HE) [35]. Histological examination was performed on sections immediately neighboring on both sides those sections that were solubilized and sub- jected to LC-MS/MS investigation. The tumor samples were analyzed for their contents regarding ratio of neoplastic area, adjacent lymph node area, necrosis and connective tissue including fat, fibrous tissue or other material. The tumor parameters were grouped and scored as referenced [21]: tumor cell size was initially measured and assigned as < 20 um, 20–25 um, > 25 um; tumor cell shape was classified as epithelioid, spindle or mixed; pigmentation was scored as 0–3 (0 = no melanin pigment, 1 = slight melanin pigmentation visible at high power, 2 = moderate pigmentation visible at low power, 3 = high pigmentation readily visible at low Fig 1. Location of primary tumors on the body of melanoma patients and their route of progression. Red font represents poor prognosis (“non- survivors”), green font represents good prognosis (“survivors”). https://doi.org/10.1371/journal.pone.0176167.g001 Fig 1. Location of primary tumors on the body of melanoma patients and their route of progression. Red font represents poor prognosis (“non- survivors”), green font represents good prognosis (“survivors”). https://doi.org/10.1371/journal.pone.0176167.g001 Fig 1. Location of primary tumors on the body of melanoma patients and their route of progression. Red font represents poor prognosis (“non- survivors”), green font represents good prognosis (“survivors”). https://doi.org/10.1371/journal.pone.0176167.g001 https://doi.org/10.1371/journal.pone.0176167.g001 4 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0176167 April 26, 2017 Proteomics data Proteomics data were used, as deposited in the PRIDE database, under the dataset identifier PXD001725 [31]. At least two unique peptides were necessary for protein identification, and at the parameter Peptide Confidence in Proteome Discoverer was required to be “high”. Prognostic grouping of patients based on clinical follow-up data For prediction of occurrence of distant organ metastases and eventually, survival we have reviewed the clinical history and the follow-up information on all 10 patients. Overall survival (OS) and disease-specific survival (DSS) of the patients were available. Kaplan-Meier survival curve supported by log-rank test was constructed (Fig 2A). In order to relate protein expres- sion data to clinical outcome, we have identified a group of “survivors” (group 1 in Fig 2A, 2B and 2C), who did not develop any further recurrence of the neoplastic disease on a long-term follow-up period (range = 4353–7960 days) as compared to”non-survivors” (group 2 in Fig 2A, 2B and 2C) who have progressed to stage IV cancer and eventually succumbed to wide- spread malignant melanoma (range = 451–591 days). We excluded those cases, which had tumor tissue of less than 10% on the slides (Fig 2C), and one case which deceased without evi- dence of malignancy (Fig 2C). The characteristics of the tumors of the clinically and patholog- ically stratified patients are displayed in Table 2. Bioinformatics Initial functional analysis of protein lists used the DAVID and ConsensusPathDb tool sets [36, 37]. Enrichment of the lists in particular functional annotations was assessed by Fisher’s exact test with Benjamini correction for multiple tests. Gene Ontology annotations, SwissProt 5 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0176167 April 26, 2017 Deep sequencing and clinicopathological analysis of lymph node metastatic melanoma keywords, and KEGG pathways were used as annotation terms for the enrichment analysis. For lists of proteins with significantly changed expression, the background protein sets con- sisted of all proteins detected. Other visualizations, list manipulations and charts were con- ducted in Spotfire (Tibco Software, Inc., Palo Alto, CA, USA). Investigation of correlations between protein expression data and histopathology was per- formed for histological parameters: 1) tumor percentage, 2) connective tissue percentage, 3) adjacent lymph node area and 4) immunoscore. Herein, for protein expression, detection count was used as proxy for abundance. With triplicate proteomics analysis of every patient sample, detection count ranged from zero to three. Spearman rank correlation was used, in home-made scripts within the R environment. For statistical analysis of significant differences in protein occurrences between the clinical “survivors” and “non-survivors” patient sample sets, t-test was applied as implemented in the Perseus toolkit (http://www.coxdocs.org/doku.php?id=perseus:start). For the purpose of this test, for each protein total PSM count was used as proxy for protein abundance, thus the t-test compared 9 vs. 9 samples for every protein (3 patients per group times 3 replicates). For in-depth functional analysis of the set of differentially regulated proteins (“survivors” vs. “non-survivors”) and of the set of proteins correlated significantly with sample tumor con- tent, QIAGEN’s Ingenuity Pathway Analysis (Qiagen, Redwood City, CA, USA) was used to generate relationship networks, and perform additional functional analyses. Histological characterization The pathological evaluation is based on general directives of pathological assessment of tumor landmarks which is comparable to other studies previously performed [21], in addition to the Fig 2. (A) Overall survival of the prognostic groups, “survivors” (group 1) and “non-survivors” (group 2). (B) All ten cases were subjected to rigorous review both on histological and clinical grounds. (C) Those were omitted (lighter grey text), where the tumor content of the examined tissue was low (<10%) or the clinical follow-up data resulted in non-disease-specific outcome measures (one patient in group 2 died without evidence of malignancy). Abbreviations: l: left; r: right; LN met: lymph node metastasis; CNS: central nervous system; tx: therapy. Numbers in brackets mean lymph nodes containing metastatic melanomas / all lymph nodes dissected from either the groin or the axilla: nmetastatic/nall. Fig 2. (A) Overall survival of the prognostic groups, “survivors” (group 1) and “non-survivors” (group 2). (B) All ten cases were subjected to rigorous review both on histological and clinical grounds. (C) Those were omitted (lighter grey text), where the tumor content of the examined tissue was low (<10%) or the clinical follow-up data resulted in non-disease-specific outcome measures (one patient in group 2 died without evidence of malignancy). Abbreviations: l: left; r: right; LN met: lymph node metastasis; CNS: central nervous system; tx: therapy. Numbers in brackets mean lymph nodes containing metastatic melanomas / all lymph nodes dissected from either the groin or the axilla: nmetastatic/nall. 6 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0176167 April 26, 2017 Deep sequencing and clinicopathological analysis of lymph node metastatic melanoma Table 2. Histopathological properties of the melanoma metastases evaluated in this study. The ten tumors are grouped according to clinicopathologi- cal classification. GROUPING Clinicopathologic All Equivocal* "survivors" "non-survivors" Tumor cell size <20 microns 10 4 3 3 20–25 microns 0 0 0 0 >25 microns 0 0 0 0 Tumor cell shape epithelioid 7 2 2 2 Spindle 0 0 0 0 Mixed 3 1 1 1 Pigment score 0 2 1 1 0 1 2 0 0 1 2 2 1 1 0 3 4 1 1 2 Cytoplasm eosinophilic 10 4 3 3 Unremarkable 0 0 0 0 Mixed 0 0 0 0 Lymphocyte distribution 0 0 0 0 0 1 4 1 1 2 2 3 2 1 1 3 1 0 1 0 Lymphocyte density 0 0 0 0 0 1 6 2 1 2 2 2 1 1 1 3 0 0 0 0 Immunoscore (= lymphocyte density+ lymphocyte distribution) 0 0 0 0 0 2 3 1 1 1 3 4 0 1 1 4 0 1 0 0 5 1 0 1 0 6 0 0 0 0 Tumor (%) Avg 59.4 21.3 74.6 94.6 Adjacent lymph node area (%) Avg 27.0 50.5 21.8 0.5 Necrosis (%) Avg 0 0 0 0 Connective tissue content (%) avg 13.7 27.8 3.5 4.8 * equivocal cases represent those cases which had tumor tissue of less than 10% on the slides and one patient who deceased without evidence of ses, which had tumor tissue of less than 10% on the slides, and one patient who deceased without evidence of detailed pathophysiological disease status that each patient´s tissue represents. The first section of tumor tissue was stained with HE, the adjacent sections were subjected to proteomic analysis, and finally the last section was stained with HE and was also characterized by histology. The gen- eral tumor cell content of lymph nodes containing metastasis tissues was assessed, and percent- ages of identified areas were recorded. The melanoma metastases were also characterized for their cytological appearance: tumor cell size and shape, predominant cytoplasmic appearance in line with pigmentation. A pigment score and lymphocyte distribution and density were assessed resulting in a combined immunoscore as described in Materials and Methods (Table 2). PLOS ONE \| https://doi.org/10.1371/journal.pone.0176167 April 26, 2017 Correlation of protein expression to histological evaluation In this study, applying conservative criteria, more than 3000 proteins were detected in meta- static melanoma samples [31]. Investigation of correlations between protein expression data and histopathology was performed for histological parameters: 1) tumor percentage, 2) “back- ground” connective tissue percentage, 3) adjacent lymph node area and 4) tumor immuno- score. Herein, for protein expression, detection count was used as proxy for abundance. With triplicate proteomics analysis of every patient sample, the detection count ranged from zero to three. A downside to this approach was the fact that correlation could not be calculated for 159 proteins detected in each sample. The correlation of protein expression to tumor percentage and to immunoscore were them- selves significantly correlated (r = 0.57, p-value < 10−5). Among the 288 proteins significantly positively correlated to tumor percentage (shown in S1 Table), there were four proteins clearly labelled as melanoma markers: melanoma cell adhesion molecule (MUC18) [38], melanoma chondroitin sulfate proteoglycan (CSPG4) [39], melanocyte protein (PMEL, melan A), mela- noma-associated antigen D2 (MAGD2). Among melanoma markers and proteins linked to this disease, alpha-synuclein and PMEL were significantly correlated to tumor percentage while S100A1 had correlation close to significance at r = 0.61 (p = 0.06. However, S100B had a low correlation to tumor percentage at r = 0.32 (p = 0.37). As many as 71 proteins were nega- tively correlated to tumor content, however, these may be specific to non-tumor cell types (connective tissues and/or adjacent lymph node) rather than being specific to lack of tumor shown in S2 Table. An analysis of functional annotations over-represented among proteins correlated to tumor percentage was performed using the ConsensusPathDB and DAVID systems. Both analyses brought similar conclusions. Aminoacyl-tRNA- biosynthesis tRNA-charging was one of top pathways and biological processes significantly related to the protein set. This outcome was due to the fact that nine aminoacyl-tRNA-synthetases were significantly correlated to tumor percentage. These were those for Gln, Thr, Val, Cys, Asp, Ala, His, Arg and Ile, with the r correlation coefficient ranging from 0.67 to 0.81. Aminoacyl tRNA synthetases are believed to be involved in tumorigenesis presumably involving their additional domains which are not directly responsible to their biosynthetic activities [40]. Recently, one aminoacyl tRNA synthe- tase was used as a cancer drug target [41]. Deep sequencing and clinicopathological analysis of lymph node metastatic melanoma composed of generally small sized tumor cells (< 20 microns) with a predominantly eosino- philic cytoplasm (100%, for both properties). The shape of tumor cells was mostly epithelioid in 7 (70%) cases, while presence of admixed spindle morphology was noted in 3 (30%) cases. Melanin pigment was noted as follows; two score 0, two score 1, two score 2 and four score 3 cases were observed. Immunoscore could only be established in 8 cases, because the amount of tumor was too low (10%) in two cases for accurate assessment. The lymphocyte distribution was lower than 25% of the tumor in 4 cases, was more widespread (25–50%) in 3 cases and extensive (>50%) in 1 case. The density of lymphocytes was mostly low (score 1) in 6 cases and moderate (score 2) in 2 cases. The combined immunoscore was 2, 3 and 5 in three, four and one case, respectively (Table 2). Our previous genomics study using the same patient samples [32], identified “high immune” and the “pigmentation” subtypes of melanoma metastases. Here, we assessed the tumors independently of their intrinsic genomic subtype. All melanoma metastases were PLOS ONE \| https://doi.org/10.1371/journal.pone.0176167 April 26, 2017 7 / 16 Correlation of protein expression to histological evaluation Other pathways and processes manifested by the proteins correlated to tumor percentage, include mitochondrial proteins (more than 80 among significantly correlated) and N-cadherin signaling (including catenins alpha-1, beta-1 and delta-1). The set of proteins significantly correlated to tumor percentage was also subjected to Ingenuity Pathway Analysis (IPA) system. Among the “Canonical Pathways” significantly enriched in the protein set, tRNA-charging (p = 2,7E-08, 11 proteins out of 82) and Protein PLOS ONE \| https://doi.org/10.1371/journal.pone.0176167 April 26, 2017 8 / 16 Deep sequencing and clinicopathological analysis of lymph node metastatic melanoma Ubiquitination Pathway (p = 4,7E-08, the set included 18 proteins out of 259 pathway pro- teins) were most significantly related to the protein set. Other canonical pathways included Clathrin-mediated Endocytosis Signaling, Remodeling of Epithelial Adherens Junctions, Acute Phase Response Signaling, Fatty Acid β-oxidation I, EIF2 Signaling. Most of these path- ways were implicated in melanoma [42–44]. Ingenuity Pathway Analysis system also yields deduced upstream regulators of a gene set. In the case of proteins correlated to tumor content, the top upstream regulators were MYC, HNF4A and TGFB1, each regulating more than 50 proteins from the query set. For MYC and TGFB1, their roles in melanoma are well-estab- lished [45, 46]. Among the general functional annotations over-represented in the query pro- tein set, Cell Death and Survival, Cellular Growth and Proliferation, and Cellular Movement were most significant. Ubiquitination Pathway (p = 4,7E-08, the set included 18 proteins out of 259 pathway pro- teins) were most significantly related to the protein set. Other canonical pathways included Clathrin-mediated Endocytosis Signaling, Remodeling of Epithelial Adherens Junctions, Acute Phase Response Signaling, Fatty Acid β-oxidation I, EIF2 Signaling. Most of these path- ways were implicated in melanoma [42–44]. Ingenuity Pathway Analysis system also yields deduced upstream regulators of a gene set. In the case of proteins correlated to tumor content, the top upstream regulators were MYC, HNF4A and TGFB1, each regulating more than 50 proteins from the query set. For MYC and TGFB1, their roles in melanoma are well-estab- lished [45, 46]. Among the general functional annotations over-represented in the query pro- tein set, Cell Death and Survival, Cellular Growth and Proliferation, and Cellular Movement were most significant. Another application of IPA was “Core Analysis” that considers the overall network of rela- tionships between all human proteins and extracts sub-networks enriched in query proteins. Correlation of protein expression to histological evaluation Here, for the set of proteins correlated to tumor content, three top sub-networks, were enriched in molecules involved in Molecular Transport, Protein Trafficking, Cell Signaling (1st subnetwork, S1A Fig), DNA Replication, Recombination, and Repair, Cellular Assembly and Organization, Cell-To-Cell Signaling and Interaction (2nd subnetwork, S1B Fig) and Cancer, Organismal Injury and Abnormalities (3rd subnetwork, S1C Fig). PLOS ONE \| https://doi.org/10.1371/journal.pone.0176167 April 26, 2017 Correlation of protein expression to disease outcome In order to relate, in a currently performed pilot analysis, protein expression in metastatic lymph nodes to patient survival, a t-test was performed, by comparing for every protein, the sum of PSM counts (as proxy for protein abundance) between three survivors and three non- survivors. The results (see Fig 3A and S3 Table) showed a number of significant differences, suggesting that even for these very low patient numbers, some trends can be observed. As many as 36 proteins had the t-test p-value below 0.001, and 74 proteins had p-value below 0.01 (Fig 3B). Majority of significant proteins had higher expression in non-survivors. The only well-established melanoma marker in that group was PMEL (almost 8-fold more abundant in patients who progressed). However, many proteins from this list have previously been linked to melanoma in the literature (Fig 3C). Strikingly, most significant in relation to patient survival is HEXB, or Beta-hexosaminidase subunit beta. This lysosomal enzyme is involved in hydrolysis of gangliosides GM2 to GM3. A recent study by Tringali et al showed that poor survival in melanoma is significantly related to GM3 levels [47]. These data agree well with our preliminary finding of elevated HEXB levels in patients with poor survival. Another study reported that increased levels of a related ganglio- side GD3 increase malignant properties of melanoma cells [48], thus supporting the hypothesis that imbalance in ganglioside forms may contribute to melanoma severity and poor survival. The HEXB protein was also noted by Byrum et al as differing between primary melanoma, benign nevi and MM, but the direction of changes they reported was different [49]. Other proteins most significantly differing between survivors and non-survivors were GPNMB (glycoprotein non-metastatic melanoma protein B, a homologue of PMEL) and PKM (pyruvate kinase, muscle) hemopexin (HPX), complement factors (C3, C4B and CFH) and vinculin (VCL), Annexin A5 (ANXA5), V-type proton ATPase catalytic subunits (ATP6V1A, ATP6V1B2) and two aminoacyl tRNA ligases (VARS and IARS2). GPNMB was shown previ- ously to be linked to metastasis, and is in clinical trials for melanoma [50]. PKM controls gly- colysis, a process crucial for metabolism in malignant cells, and the balance of splicing-derived PKM isoforms is known to be related to cancer invasiveness [51, 52]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0176167 April 26, 2017 Correlation of protein expression to disease outcome The Complement C3a Receptor signaling has been very recently shown to contribute to melanoma tumorigenesis PLOS ONE \| https://doi.org/10.1371/journal.pone.0176167 April 26, 2017 9 / 16 Deep sequencing and clinicopathological analysis of lymph node metastatic melanoma Fig 3. (A) Volcano plot showing differences in approximate protein abundance between survivors a survivors. T-test p-value and mean fold difference shown (protein abundance evaluated as sum of P Fig 3. (A) Volcano plot showing differences in approximate protein abundance between survivors survivors. T-test p-value and mean fold difference shown (protein abundance evaluated as sum of Fig 3. (A) Volcano plot showing differences in approximate protein abundance between survivors and non- survivors. T-test p-value and mean fold difference shown (protein abundance evaluated as sum of PSMs). (B) Fig 3. (A) Volcano plot showing differences in approximate protein abundance between survivors and non- survivors. T-test p-value and mean fold difference shown (protein abundance evaluated as sum of PSMs). (B) 10 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0176167 April 26, 2017 Deep sequencing and clinicopathological analysis of lymph node metastatic melanoma As in (A), but only significant proteins shown (p<0.05). (C) Biological relationship network (IPA) for proteins differentiating between survivors and non-survivors (shown in B), and having literature links to melanoma or metastasis. Only significant proteins shown (T-test p-value below 0.01), excluding proteins having no IPA relationships within the presented set. https://doi.org/10.1371/journal.pone.0176167.g003 [53]. On the contrary, differential expression of vinculin between weakly and highly metastatic melanoma cell lines was noted more than 20 years ago [54]. The IPA analysis was performed for the sets of proteins that differed between survivors and non-survivors (S2 Fig and S3A–S3D Fig). Core IPA analysis revealed that most significant canonical pathway enriched in the set of proteins changed between survivors and non-survi- vors was glycolysis (p-value 9.8E-13) while cell death and survival, carbohydrate metabolism, cellular movement, and cellular growth and proliferation were the most significant biological functions overrepresented in this protein set. This is consistent with biological processes known to be related to metastatic properties of cancer. Despite a very small number of patients in this pilot evaluation, (three survivors and three non-survivors), a number of proteins were found to differentiate the two groups, and substan- tial number of these was previously linked to cancer in the literature (S2 Fig). Correlation of protein expression to disease outcome Among the pro- teins most significantly differentiating between the two patient groups, HEXB, PKM and GPNMB stood out, as hallmarks of processes involved in melanoma progression and responsi- ble for poor survival. PLOS ONE \| https://doi.org/10.1371/journal.pone.0176167 April 26, 2017 Deep sequencing and clinicopathological analysis of lymph node metastatic melanoma relatively limited number of patient samples. The mass spectrometry data represent the initial stage of a protein sequence database for metastatic melanoma. The combined knowledge of the tumor cellular composition with protein expression of each metastasis enables identifica- tion of novel, significantly regulated proteins, in melanoma tumor tissue sections. Upon fur- ther validation and development these proteins might serve as future diagnostic markers for this cancer. In Fig 4, the key upstream regulators, the major regulated canonical pathways and subnetworks according to analysis based on DAVID and IPA evaluation are summarized. Cellular functions and molecular fingerprints that provide survival advantage for malignant cells with promotion to further disease progression were highly represented in our overall analysis. In addition, several markers previously related to melanoma were seen to correlate to the tissue tumor cell content. Although the translational impact of this feasibility study is not immediate, future studies should confirm the relevance of such proteins as important mela- noma landmarks. Supporting information S1 Fig. (A) First, (B) second and (C) third most significant biological relationship subnet- works resulting from Ingenuity Pathways Analysis (IPA) for the proteins correlated to tumor content. Members of the original list of 359 proteins marked in grey. Magenta outline high- lights proteins implicated in cancer according to IPA database. (TIF) S2 Fig. Subcellular locations and biological relationship network for proteins differentiat- ing between survivors and non-survivors. Only significant proteins were shown (t-test, p-value below 0.01). Known cancer biomarkers marked by magenta outline (IPA). Proteins having no IPA relationships within the presented set are excluded. Red filling: proteins with higher expression in non-survivors. (TIF) S3 Fig. (A) First, (B) second, (C) third and (D) fourth, respectively, most significant biological relationship subnetworks resulting from Ingenuity Pathways Analysis for the proteins differ- entiating between survivors and non-survivors. Only significant proteins used in the IPA anal- ysis (T-test p-value below 0.01). Known cancer biomarkers marked by magenta outline. Red filling: proteins with higher expression in non-survivors. (TIF) S3 Fig. (A) First, (B) second, (C) third and (D) fourth, respectively, most significant biological relationship subnetworks resulting from Ingenuity Pathways Analysis for the proteins differ- entiating between survivors and non-survivors. Only significant proteins used in the IPA anal- ysis (T-test p-value below 0.01). Known cancer biomarkers marked by magenta outline. Red filling: proteins with higher expression in non-survivors. (TIF) S3 Fig. (A) First, (B) second, (C) third and (D) fourth, respectively, most significant biological relationship subnetworks resulting from Ingenuity Pathways Analysis for the proteins differ- entiating between survivors and non-survivors. Only significant proteins used in the IPA anal- ysis (T-test p-value below 0.01). Known cancer biomarkers marked by magenta outline. Red filling: proteins with higher expression in non-survivors. (TIF) S2 Table. Proteins significantly negatively correlated to tumor content. (XLSX) S3 Table. Correlation of protein expression to disease outcome. Student’s t-test p-value pro- vided for comparison of survivors vs non-survivors. PSM counts compared, three patients in a group, each in triplicate, hence 9 samples per group. (XLSX) S1 Table. Proteins significantly positively correlated to tumor content. (XLSX) S2 Table. Proteins significantly negatively correlated to tumor content. (XLSX) Conclusion An in depth pathological analysis of metastatic melanoma samples used for generating deep mining high-quality mass spectrometry data of protein expression is presented. This feasibility study provides an example how clinical outcome and detailed histopathology analysis related to proteomics discovery data can provide novel insights into the molecular processes driving the disease. The main implication of this study is that melanoma proteomics should be insepa- rable from histological evaluation performed in the most detailed manner. This unique approach is the main strength of this study while it has obvious limitations arising from Fig 4. Identified melanoma markers and pathobiological processes in tissue samples of melanoma lymph node metastases. The graphics displays key upstream regulators found according to analysis based on DAVID and IPA evaluation. The graph displays major regulated canonical pathways and subnetworks resulting in cellular functions and molecular fingerprints providing survival advantage for malignant cells with promotion to further progression and metastasis. https://doi.org/10.1371/journal.pone.0176167.g004 Fig 4. Identified melanoma markers and pathobiological processes in tissue samples of melanoma lymph node metastases. The graphics displays key upstream regulators found according to analysis based on DAVID and IPA evaluation. The graph displays major regulated canonical pathways and subnetworks resulting in cellular functions and molecular fingerprints providing survival advantage for malignant cells with promotion to further progression and metastasis. https://doi.org/10.1371/journal.pone.0176167.g004 11 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0176167 April 26, 2017 Writing – original draft: CW KP AMS EW GMV. Writing – review & editing: CW KP AMS EW GMV MR MY YS JM GJ CI LL BB HO TL. Writing – review & editing: CW KP AMS EW GMV MR MY YS JM GJ CI LL BB HO TL. Writing – review & editing: CW KP AMS EW GMV MR MY YS JM GJ CI LL BB HO T Acknowledgments We thank Dr. Dariusz Gozdowski for helpful discussions and advice in statistical issues. This study is dedicated to the memory of Dr. Thomas Edward Fehniger, our colleague and dear friend who left us far too early. 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https://openalex.org/W1427181711	https://hal.inria.fr/hal-01472229/document	English	null	Requirements Analysis and Definition for Eco-factories: The Case of EMC2	IFIP advances in information and communication technology	2,013	cc-by	3,579	To cite this version: Marco Taisch, Bojan Stahl. Requirements Analysis and Definition for Eco-factories: The Case of EMC2. 19th Advances in Production Management Systems (APMS), Sep 2012, Rhodes, Greece. pp.111-118, 10.1007/978-3-642-40352-1_15. hal-01472229 Marco Taisch, Bojan Stahl. Requirements Analysis and Definition for Eco-factories: The Case of EMC2. 19th Advances in Production Management Systems (APMS), Sep 2012, Rhodes, Greece. pp.111-118, 10.1007/978-3-642-40352-1_15. hal-01472229 Distributed under a Creative Commons Attribution 4.0 International License Requirements analysis and definition for eco- factories: the case of EMC2 Marco Taisch1, Bojan Stahl1 1Politecnico di Milano, Department of Management, Economics and Industrial En- gineering, Milan, Italy {marco.taisch, bojan.stahl}@polimi.it 1Politecnico di Milano, Department of Management, Economics and Industrial En- gineering, Milan, Italy {marco.taisch, bojan.stahl}@polimi.it Abstract. Climate change mitigation and the EU2020 strategy foster energy ef- ficiency in Europe’s future manufacturing landscape. These challenges make high demands to SMEs as well to MNCs. The paper gives insight to an ap- proach on Eco-factories based on the EU-funded FP7 project EMC2. Eco- factories will enable the quantum leap in integrating environmental issues in brownfield and greenfield factory planning and factory operation. The paper fo- cuses on the identification, structuring and definition of requirements for Eco- factory simulation approaches. Requirements for developing a simulation envi- ronment for integrating energy and material flows for detailed analysis but also wide user spectrum is presented. The paper shows that demands are twofold re- questing integrated, modular and detailed simulation solutions as well as em- phasize on user-friendliness and low complexity. Keywords: eco-factory, energy efficiency, sustainable manufacturing, simula- tion requirements, energy flow. HAL Id: hal-01472229 https://inria.hal.science/hal-01472229v1 Submitted on 20 Feb 2017 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License 1 Introduction Climate change mitigation and security of supply are the major drivers for rais- ing, keeping and enhancing the attention towards energy consumption on European level, addressing policy-makers, industry and society in the same way [1]. Energy consumption implies severe ecological consequences such as global warming and resource depletion on the one hand, and being dependent of supply from non- European countries may lead to potential economic and social risks. Industry is facing a strong economic pressure which might endanger the global competiveness. Energy and raw material prices are steadily increasing in the last years, and the public awareness for green products is steadily rising with more en- lightened customers. Consequently, awareness towards energy efficiency is strongly addressed by European policy-makers [2]. The European 2020 strategy defined sever- al eco-targets to be reached by 2020, i.e. the reduction of emissions by 20%, and the reduction in primary energy of 20% to be achieved by improving energy efficiency, while securing the competiveness of European companies at the same time. Production processes are the backbone of the industry in Europe regarding economic success but also regarding environmental and social impact. Resources and energy are the major inputs for the transformation processes to create value. However, creating value by the input of resources and energy leads also wastes in terms of losses, heat, and emissions. Together with households, commercial and transportation in the domain of energy consumption, manufacturing is considered as a main contributor, with being responsible for approximately 37% of primary energy consumption on global scale. At European level, industry is responsible for about 40% of the electricity consump- tion [3]. Future manufacturing paradigm should therefore be built on two pillars: avoiding material and energy waste through increased efficient production and avoid- ing energy and material consumption with harmful impact on the environment. Diverse studies have been carried out to highlight the improvement potential within industry. The international Energy Agency has stated in their Energy Efficiency Poli- cy Recommendation that potential savings sum up to 18.9 EJ/year and 1.6Gt CO2/year by 2030 [4]. A German-lead consortium of the Fraunhofer Gesellschaft highlights the major potential of increased production process efficiency to optimize the environmental as well as economic performance of companies [5]. A consultation group operating on European level has identified saving potentials of 10-40% in man- ufacturing in their “Smart Manufacturing” report [6]. 1 Introduction A similar study carried out on country level in Germany has likewise identified a saving potential of 10-30% on energy consumption [7]. Production system simulation incorporating environmental perspectives is seen as one appropriate tool in the design and optimization of manufacturing systems to- wards energy efficiency. The paper presents an approach for defining requirements on such simulation approaches by applying a requirements engineering approach. The aim is to gain a detailed insight into what developers, stakeholders and users see as main features to be implemented in order to support the successful application in in- dustry and research. 2 Methodology A requirement can be defined as a need that a specific product shows by at- tributes, characteristics or specific qualities that the end product should possess [8]. According to [9], requirements engineering regards establishing and documenting software requirements. Through this process it is possible defining what the system should be able to do and the constraints in which it needs to operate. This process consists of four major steps: elicitation, analysis, specification, verification and man- agement. The requirements management is represented by the planning and control- ling of the other steps of the requirements engineering phase. g p q g g p In the elicitation phase the problem to be solved is defined through the identi- fication of the boundaries and the stakeholders’ and goals’ definition. It is the first step of the requirements engineering. The information gathered in this phase need to be interpreted, analysed and validated [10]. Different techniques exist to perform the elicitation phase, e.g. interviews, scenarios creation, prototypes, meetings and obser- vation. The second phase, analysis, potential conflict by different requirements sources could imply need to be solved. Consequently this step deals with the require- ments’ conflicts detection and resolution. The conflict resolution is lead by negotia- tion. Furthermore, this is the phase in which the system bounds are discovered to- gether with the interactions with the environment [8]. In the documentation phase the requirements identified are specified and for- malized. It is important that the requirements can be traced in order to be easy to read, understood and navigated [10]. The output of this process is a requirements docu- ment. Finally, in the validation the requirements are checked in order to find out fur- ther omissions and conflicts. In this phase it is checked if the requirements document created is consistent and complete and requirements priorities are attributed. This is the step into which the quality of the model developed is validated [8]. In order to be complete, the process of requirements engineering should con- sider all the important sources of requirements from which the requirements are gained. These sources can be in conflict among them and part of the requirements engineering is, for this reason, represented by negotiation and prioritization activities. Each step is described in detail in the subsequent sections. 3 Elicitation In this step of the simulation requirements engineering, the requirements are collected from different sources and different stakeholders. It is the first phase of the requirements engineering and it helps the developers of the product in understanding the problem. The stakeholders from which the requirements are developed may be- long to different categories. They can be end-users, customers, regulators, developers, neighbouring systems and domain experts. In this case the requirements have been collected from three sources. The first one is represented by a classical literature re- view of papers dealing with simulation of energy in manufacturing, in order to take into account from them the general simulation requirements to be used in the model development. The second type of source is represented by the opinions of experts, stakeholders and users, represented by the partners and consortium of the EMC2- Factory project. The last source is represented by the developers themselves by hav- ing a visionary idea of building a holistic simulation model for combining energy and material flow simulation. Different works available in the literature have been reviewed according to their postulations of requirements for “integrating energy, material and building simu- lation which resulted in total in 52 requirements [12]-[31]. 42 requirements have been identified by the partners and consortium members. For this reason questionnaires were provided to the stakeholders in order to capture their opinion. The developers themselves provided 17 more requirements. Summarized, 111 requirements have been identified in total from the literature review, the stakeholder consultation and the internal development team. The require- ments present overlaps and conflicts which need to be managed in order to avoid repetitions and contradictions for the final requirements which is done in the next step. 3 4 Analysis In the second step of the requirements methodology applied, two main activi- ties are performed: the requirements classification and the requirements negotiation. In the classification of the requirements, they are divided into distinguishable catego- ries while in the negotiation the resolution of the conflicts has been conducted. The following paragraph identifies the categories in which the requirements are classified and divided. The simulation requirements were divided in four catego- ries: general, functional, non-functional and implementation requirements. General requirements are high level conceptual requirements. Implementation requirements focus on the system-side conceptualization of the simulator. Functional requirements define what the simulator should be able to perform. Non-functional requirements describe characteristics linked to the way in which the simulator performs its func- tions. According to the mentioned categories, the requirements listed in the require- ments elicitation step have been divided and categorised, each one in one of the above mentioned categories. Hence, the requirements with the same or similar meaning and scope have been joined together and the ones in conflicts have been resolved. Starting from the 111 requirements identified in the elicitation phase it has been possible to group and merge them according to the scope and the meaning they presented and to arrive until 33 final requirements. The meaning and the scope of each requirement has been identified by checking how each requirement fit with the simulation purpose. The set of requirements obtained at the end of the analysis process are listed and ex- plained in the documentation phase in which all the requirements identified are ex- plained and classified. 5 Documentation and Validation Documenting the requirements is the fundamental condition to the require- ments handling. In this phase the structure of the requirements document is devel- oped. The two tables below provide a short excerpt of the documentation of the re- quirements defined in the analysis step. The last phase of the requirements engineering is represented by the validation. This steps deals with the analysis of the document produce in the documentation phase in order to be sure that it represents the right requirements in the most complete way. In this phase the focus is on looking for mistakes, errors or lack of clarity. Ac- cording to [11], the requirements validation needs to be performed in order to be sure that the requirements are: unambiguous, allowing only a single interpretation; con- cise; finite; measurable; feasible to be implemented with the available technologies; traceable. This phase has been performed, however results are not explicitly men- tioned here. Table 1: Functional Requirements Requirements Description Assessing dif- The KPIs’ framework should include the evaluation of pro- Table 1: Functional Requirements Requirements Description Assessing dif- The KPIs’ framework should include the evaluation of pro- 5 ferent kind of KPIs duction, energy and environmental KPIs both real-time and post-processing in order to allow timely decisions oriented to the reduction of the energy costs and to the decrease of the environmental impact. Considering production assets and technical building services It means modeling and simulating not only the energy con- sumption of the production-related assets but also of the non- productive equipments, like the central TBS and the periphery systems, supporting their dimensioning and optimization. Supporting the dynamic connec- tions of elements and interrelations Representing in the simulation the dynamic connections ex- isting among production assets, periphery systems and central technical building services. Bringing inside the states based energy consump- tion calculations It underlines the importance of implementing energy calcu- lations during the simulation running basing them on the ener- gy consumption associated to entities’ states; representing the basis for non-productive states energy consumption reduction. Interfacing with production control policies It means supporting the connection of the simulation with production control policies at machines’ coordination level, oriented toward the energy consumption reduction and able to influence the equipments’ states. Preventing en- ergy peaks It underlines the important of intervening real time on the manufacturing through control policies when the energy con- sumption shows rapidly increases. 5 Documentation and Validation Including mul- ti-product per- spective Including product type control policies and supporting the different product type’s identification during the simulation. Supporting green oriented perspectives Enabling green oriented decisions in terms of scheduling, production configurations. Allowing dif- ferent levels of energy consump- tion calculations It underlines the importance for the simulation model to cal- culate energy consumptions starting from the machine level and arriving till the factory one. Considering different energy carriers and sup- ply systems It stresses the high contribution to the holistic perspective given by the consideration of different energy carriers and dif- ferent periphery systems. Table 2: Non-functional requirements Requirements Description Creating a sim- ple and controlla- ble solution It underlines the need of a manageable solution, easy to be controlled and customized. Integrated solu- tions It means integrating different tools together in order to de- velop an interoperable simulation approach. 5 Activating Plug-ins functions It sets the need to have functions which can be activated through plug-ins. Using graphical interface The functionalities of the simulation model are shown in a graphical interface. Optimizing ease of use The requirement stresses the relevance of having a tool easy to be used. Increasing speed It refers to the required increase of speed in the simulation running. Increasing the user-friendliness High user friendliness makes the model easier to be used and more attractive. Supporting step-by-step guid- ing procedures It underlines the importance for the model to be equipped with exhaustive descriptions. Including sto- chastic inputs and unpredictable events The model should allow the possibility to insert input data which are not deterministic. Increasing the level of data detail The model should support the highest possible level of data detail. 6 Summary and Outlook The definition of functional requirements for simulation of eco-factories is a first step to guide the way from traditional factory design and operations to eco- enabled competitive factories. The functional requirements build the backbone of the definition of a holistic reference model for eco-factories which serves on the one hand as a meta-model for future activities in the project itself and on the other hand as an enabler for a comprehensive guide or toolkit to support industrial implementation by hands on suggestions. The functional requirements need to be implemented into a discrete event simulator. This paper presented the requirement engineering process as an initial step be- fore the development of a discrete event simulation environment which combines energy and material flow simulation. Analyzing the requirements leads to the follow- ing conclusions. There is an increasing demand in adapting simulation applications to a larger user group, hence giving support by pre-defined objects and decreasing the modelling complexity. It is demanded to increase user-friendliness and keep solutions easy and simple. On the other side the integration of different tools and increase of level of detail of data are requirements which may result in a stress-field in accom- plishment. The functional requirements are widely focused on the increase of detail concerning energy considerations and widening the scope of traditional production asset simulations taking into account periphery systems and central technical building services. Finally it can be summarized that literature and stakeholders see an increasing interest in making energy simulations in manufacturing more comprehensive, de- tailed, but also simple. The requirement engineering process served as an initial step of a simulation environment development approach. The next major phase includes the conceptual design of a simulation environment which combines material flow and energy flow in production systems taking into account production assets as well as technical building service and being applicable on different scales in the factory. 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https://openalex.org/W4386134780	https://link.springer.com/content/pdf/10.1007/JHEP08(2023)166.pdf	English	null	Tree-level UV completions for NRSMEFT d = 6 and d = 7 operators	The Journal of high energy physics/The journal of high energy physics	2,023	cc-by	15,911	Received: July 3, 2023 Accepted: August 4, 2023 Published: August 24, 2023 Received: July 3, 2023 Accepted: August 4, 2023 Published: August 24, 2023 Received: July 3, 2023 Accepted: August 4, 2023 Published: August 24, 2023 Open Access, c⃝The Authors. Article funded by SCOAP3. Keywords: Baryon/Lepton Number Violation, SMEFT, Sterile or Heavy Neutrinos 3 Lepton number violation (LNV) 3.1 The black box theorem 3.2 A black box for NRSMEFT and Majorana neutrino masses 3.3 Neutrino masses in models derived from LNV d = 7 operators 4 Conclusions A Lagrangian 24 A Lagrangian Tree-level UV completions for NRSMEFT d = 6 and d = 7 operators JHEP08(2023)166 Rebeca Beltrán,a Ricardo Cepedellob and Martin Hirscha aInstituto de Física Corpuscular, CSIC-Universitat de València, C/Catedrático José Beltrán 2, Paterna 46980, Spain bInstitut für Theoretische Physik und Astrophysik, Universität Würzburg, Emil-Hilb-Weg 22, D-97074 Würzburg, Germany E-mail: rebeca.beltran@ific.uv.es, ricardo.cepedello@physik.uni-wuerzburg.de, mahirsch@ific.uv.es Abstract: We study ultra-violet completions for operators in standard model effective field theory extended with right-handed neutrinos (NRSMEFT). Using a diagrammatic method, we generate systematically lists of possible tree-level completions involving scalars, fermions or vectors for all operators at d = 6 and d = 7, which contain at least one right- handed neutrino. We compare our lists of possible UV models to the ones found for pure SMEFT. We also discuss how the observation of LNV processes via NRSMEFT operators at the LHC can be related to Majorana neutrino masses of the standard model neutrinos. Keywords: Baryon/Lepton Number Violation, SMEFT, Sterile or Heavy Neutrinos Keywords: Baryon/Lepton Number Violation, SMEFT, Sterile or Heavy Neutrinos ArXiv ePrint: 2306.12578 Open Access, c⃝The Authors. Article funded by SCOAP3. Open Access, c⃝The Authors. Article funded by SCOAP3. https://doi.org/10.1007/JHEP08(2023)166 Contents Contents 1 Introduction 1 2 Decompositions 3 2.1 Diagrammatic method basics 4 2.2 Dictionary for d = 6 7 2.3 Dictionary for d = 7 8 2.4 Baryon number violating operators 12 3 Lepton number violation (LNV) 13 3.1 The black box theorem 14 3.2 A black box for NRSMEFT and Majorana neutrino masses 15 3.3 Neutrino masses in models derived from LNV d = 7 operators 19 4 Conclusions 23 A Lagrangian 24 1 Introduction 2 Decompositions 2.1 Diagrammatic method basics 2.2 Dictionary for d = 6 2.3 Dictionary for d = 7 JHEP08(2023)166 2.4 Baryon number violating operators 3 Lepton number violation (LNV) 1 Introduction Experimental searches for heavy neutral leptons (HNLs) have gained a lot of momentum in the past few years, for recent reviews see for example [1, 2]. Minimal models of HNLs assume only that some nearly singlet fermion exists with a small coupling to gauge bosons. This setup is motivated experimentally as it gives a simple two parameter extension (one mixing angle and one mass) of the standard model (SM), describing the experimental sensitivity, which can then be easily compared among different searches. At the same time, there has also been a revival of interest in HNLs from theory. Here, the main motivation is usually the connection HNLs might have with the non-zero neutrino masses observed in oscillation experiments. In the minimal type-I seesaw [3– 7], Majorana right-handed neutrinos (NR’s) have only one coupling to SM particles: the Yukawa coupling to leptons and the Higgs field. After electro-weak symmetry breaking, this setup leads to Majorana neutrino masses for the active neutrinos and HNL states with a coupling suppressed by a small mixing angle, typically \|V \|2 ∝mν/MM, where MM is the mass of the right-handed neutrino. Compared to the expected experimental sensitivities this mixing is quite small, but prospects are much better in many non-minimal models: inverse [8] and linear seesaw [9, 10], also models with a new Z′ [11, 12] or leptoquarks [13], to mention a few examples. However, given the large number of possible BSM extensions involving NR’s and the absence (so far) of any BSM physics at the LHC, a more practical ansatz to parametrise the – 1 – phenomenolgy of NR’s is to use effective field theory, in particular NRSMEFT, i.e. standard model effective theory extended with right-handed neutrinos. The operator basis for NRSMEFT is known now up to d = 9 [14]. Lower dimensional operators have been studied in a number of papers earlier, for example d = 5 [15–17], d = 6 [15, 18, 19] and d = 7 [20, 21]. The phenomenology for NRSMEFT is also a very active area of research. Ref. [22] studied constraints on the four-fermion operators involving NR assuming a stable NR. For promptly decaying NR’s see [23], other decay modes have been studied in [24, 25]. NR’s as long-lived particles at the LHC in NRSMEFT have been discussed in [26–30]. The very recent paper [31] gives a reinterpretation of many previous HNL searches in terms of NRSMEFT operators. 1 Introduction Further collider studies of NRSMEFT include refs. [32–37]. JHEP08(2023)166 While different UV models for NRSMEFT operators at d = 5 and d = 6 have been mentioned in the literature, what is still lacking is a systematic decomposition of all d = 6 and d = 7 operators, i.e. an attempt to give the complete list of one and two particle extensions of the SM, that can generate the complete set of operators in the ultra-violet. To provide such a systematic particle “dictionary” constitutes the basic motivation for our current work. In this context we need to mention [38], which has some overlap with our paper. In [38] a list of leptoquark states for NRSMEFT operators at d = 6 has been derived and we agree with these results. Also important for us is [39]. Here, the authors have presented the complete dictionary of one field extensions of the SM particle content for pure SMEFT at d = 6. Recently, in [40] they also provide partial results of the one- loop dictionary for SMEFT at d = 6. Nevertheless, we will compare our results to [39] and comment on the differences between NRSMEFT and SMEFT dictionaries in section 2.2. The list of d = 7 operators and their tree-level completions is presented in section 2.3. At d = 7 level, all operators with NR’s violate lepton number by two units.1 Lepton number violation (LNV) in two units should always generate Majorana neutrino masses for the ac- tive neutrinos of the SM. The most famous example of this connection is the so-called “black box theorem” [41], where it was shown that the observation of neutrinoless double beta de- cay (0νββ decay) guarantees that Majorana neutrino masses are generated at some level in perturbation theory. Similarly, if LNV is observed in a process involving NR’s at the LHC, the active neutrinos must have Majorana masses. We will discuss this in detail in section 3. The rest of this paper is organised as follows. In the next section, we first discuss the basics of the diagrammatic method. We then give the list of d = 6 operators and their decomposition in section 2.2 and for d = 7 operators in 2.3. Baryon number violating operators are discussed separately in 2.4. Section 3 is then devoted to a discussion of LNV in NRSMEFT, before we close with a short summary. 1Assuming that the lepton number of NR is L(NR) = +1. There is a subtlety involved in this assumption, which is discussed in section 3. 1 Introduction JHEP08(2023)166 Name N E ∆1 ∆3 Σ Σ1 Irrep (1, 1, 0) (1, 1, −1) 1, 2, −1 2 1, 2, −3 2 (1, 3, 0) (1, 3, −1) d = 6 ◦ ◦ ◦ ◦ Name U D Q1 Q5 Q7 T1 T2 Irrep 3, 1, 2 3 3, 1, −1 3 3, 2, 1 6 3, 2, −5 6 3, 2, 7 6 3, 3, −1 3 3, 3, 2 3 d = 6 Table 2 New vector-like fermions contributing to d = 6 d = 7 NRSMEFT at tree-level Name N E ∆1 ∆3 Σ Σ1 Irrep (1, 1, 0) (1, 1, −1) 1, 2, −1 2 1, 2, −3 2 (1, 3, 0) (1, 3, −1) d = 6 ◦ ◦ ◦ ◦ Name U D Q1 Q5 Q7 T1 T2 Irrep 3, 1, 2 3 3, 1, −1 3 3, 2, 1 6 3, 2, −5 6 3, 2, 7 6 3, 3, −1 3 3, 3, 2 3 d = 6 Table 2 New vector-like fermions contributing to d = 6 d = 7 NRSMEFT at tree-level Table 2. New vector-like fermions contributing to d = 6, d = 7 NRSMEFT at tree-level. Name B B1 W W1 L1 L3 Irrep (1, 1, 0) (1, 1, 1) (1, 3, 0) (1, 3, 1) 1, 2, 1 2 1, 2, −3 2 d = 6 ◦ ◦ ◦ Name U1 U2 Q1 Q5 X Irrep 3, 1, −1 3 3, 1, 2 3 3, 2, 1 6 3, 2, −5 6 3, 3, 2 3 d = 6 ◦ ◦ ◦ Table 3. New vector bosons contributing to d = 6, d = 7 NRSMEFT at tree-level. Two fields, L1 and U1, are special cases, see text. Table 3. New vector bosons contributing to d = 6, d = 7 NRSMEFT at tree-level. Two fields, L1 and U1, are special cases, see text. 1 Introduction In the appendix we provide Lagrangians for all the UV models we discussed in the main text. – 2 – Name S S1 φ Ξ Ξ1 Irrep (1, 1, 0) (1, 1, 1) 1, 2, 1 2 (1, 3, 0) (1, 3, 1) d = 6 ◦ ◦ ◦ ◦ ◦ Name ω1 ω2 Π1 Π7 ζ Irrep 3, 1, −1 3 3, 1, 2 3 3, 2, 1 6 3, 2, 7 6 3, 3, −1 3 d = 6 ◦ ◦ ◦ Table 1. New scalars contributing to d = 6 and d = 7 NRSMEFT operators at tree-level. Only fields marked with a circle contribute to d = 6, the remaining ones appear in models for d = 7 operators. Field names follow the conventions of [39]. Table 1. New scalars contributing to d = 6 and d = 7 NRSMEFT operators at tree-level. Only fields marked with a circle contribute to d = 6, the remaining ones appear in models for d = 7 operators. Field names follow the conventions of [39]. Table 1. New scalars contributing to d = 6 and d = 7 NRSMEFT operators at tree-level. On fields marked with a circle contribute to d = 6, the remaining ones appear in models for d = operators. Field names follow the conventions of [39]. 2.1 Diagrammatic method basics The procedure of opening up or exploding2 EFT operators can be summarised in three main steps. First, given an effective operator with n light fields, one constructs all possible topologies that can generate the operator at some loop order. A topology is made up of n external legs connected through internal lines using only renormalisable interactions, i.e. 3- and 4-point interaction vertices. The number of internal lines depends on the topology and on loop order. In our case, we consider only tree-level openings and all internal lines are identified with BSM heavy fields. In the second step, one assigns each light field in the operator to an external leg of the topology, and Lorentz invariance fixes the Lorentz nature of the internal lines to either scalar (S), fermion (F) or vector (V ). All possible permutations of the external light fields give rise to different combinations for the internal fields. At this stage, the output consists of a set of diagrams where every line has definite Lorentz nature. JHEP08(2023)166 Finally, by imposing gauge invariance, in our case the SM symmetry SU(3)C×SU(2)L× U(1)Y , in all the interaction vertices, one determines the heavy fields’ quantum numbers, which are uniquely fixed for tree-level openings. The diagrams are then promoted to model diagrams and the lists of particles that can be accommodated as internal lines constitute our models. In summary, for each effective operator of dimension d, the output of the diagrammatic process is a set of models consisting of heavy BSM particles, each of which gives a tree-level opening of the operator. We collect in tables 1–3 all the BSM fields that appear in the model diagrams found for NRSMEFT d = 6 and d = 7 operators, classified according to their Lorentz nature. Fields contributing to d = 6 operators are marked, the remaining fields appear only in models for d = 7 operators. Some additional comments are in order. We have implemented the described method in a Mathematica code. The input for this code is the list of fields and the number of derivatives contained in the considered operator. Neither the Lorentz structure nor the field contractions have to be specified. 2This term was introduced in [48] and refers to the process of expanding an operator into a series of UV renormalisable models generating the initial operator at tree-level. 2 Decompositions In this section, we introduce the diagrammatic method used to systematically decompose NRSMEFT operators at tree-level. The same method has been used for studying the Weinberg operator at different loop orders in [42–45] and for 1-loop openings of SMEFT four-fermion operators in [46, 47]. We will therefore be brief in this description. Subse- quently, we apply the diagrammatic method to NRSMEFT operators which include at least one NR at both d = 6 (section 2.2) and d = 7 (section 2.3). Operators at d = 6 and d = 7 that violate baryon number are discussed separately in section 2.4. – 3 – 2.1 Diagrammatic method basics As a consequence, in cases where the basis of operators allow more than one operator with the same field content (and number of derivatives) the method yields a list of model diagrams, each of which will contribute to at least one operator in this set, but does not provide the information to which specific operator (or operators) the model will be matched. We choose to do so, because the model lists found in this way are complete, once one scans over all possible operators at a given level of d. After this has been done, many models are found more than once, reflecting the fact that in most cases BSM extensions will generate not only one specific operator, but contribute to several operators, when the correct matching for the model is calculated. Matching of tree-level models (as we consider here) could of course easily be calculated “by hand”, but recently the code Matchete [49] has been published, with which the matching can be done also automatically. – 4 – ψ2H3 (+h.c.) (RR)(RR) (LL)(RR) OLNH3 (LNR) ˜H(H†H) ONN (NRγµNR)(NRγµNR) OLN (LγµL)(NRγµNR) ψ2H2D (+h.c.) OeN (eRγµeR)(NRγµNR) OQN (QγµQ)(NRγµNR) ONH2D (NRγµNR)(H†i ←→ DµH) OuN (uRγµuR)(NRγµNR) (LR)(LR) (+h.c.) ONeH2D (NRγµeR)( ˜H†iDµH) OdN (dRγµdR)(NRγµNR) OLNLe (LNR)ϵ(LeR) (LR)(RL) (+h.c.) OduNe (dRγµuR)(NRγµeR) OLNQd (LNR)ϵ(QdR) OQuNL (QuR)(NRL) ONNNN (Nc RNR)(Nc RNR) OLdQN (LdR)ϵ(QNR) Table 4. NRSMEFT operators at d = 6 which can be generated at tree-level. Baryon number violating operators are discussed separately in section 2.4. JHEP08(2023)166 Table 4. NRSMEFT operators at d = 6 which can be generated at tree-level. Baryon number violating operators are discussed separately in section 2.4. Our code does not fix the operator list to any particular on-shell basis. In consequence, we do not consider diagrams with light bridges. Once the operator list, to which any given model is matched, is reduced to the on-shell basis, operators containing diagrams with light bridges will be automatically included. It is important to note, however, that diagrams with light bridges do not present new models, i.e. it is guaranteed that with the diagrammatic method, as discussed here, no models are lost. For tables 1–3, we follow the naming conventions of [39]. Most fields in our list of possible BSM particles have already appeared in that reference in a different context. 2.1 Diagrammatic method basics Note that (i) Ξ1 in neutrino physics is usually denoted with the symbol ∆and (ii) we use N for the heavy fermion F(1, 1, 0) to distinguish it from the light NR. However, there are two special cases, the vectors L1 and U1. The latter does not appear in pure SMEFT at d = 6 [39], but this field is listed in [38]. On the other hand, L1 is more subtle. One can choose to treat vector fields as either gauge vectors or not. For general vector fields, one can write down a general list of inter- actions obeying simply Lorentz and gauge symmetries of the model under consideration. However, for heavy gauge vectors a certain set of these interactions is not allowed, see the discussion in [50]. In pure SMEFT at d = 6, L1 can contribute to the matching only if the interaction term L† 1,µDµH is included [39]. Such a term is not allowed for a gauge vector [50]. However, in NRSMEFT a L1 will contribute to the matching, even if it is forced to be a gauge vector. We note that while we do not give the list of possible gauge groups for the vectors in table 3, L1 appears, for example, in 331 models [51–54]. Finally, all heavy fermions appearing in the models are considered to be vector-like under the SM gauge group, even though just one of the chiralities might be needed to open up an operator. This assumption is motivated by experimental data. The observation of a SM-like Higgs boson at ATLAS [55] and CMS [56] rules out the existence of a fourth chiral generation (in the minimal SM). – 5 – ψ2H3 : ψ4 : ψ2H2D : Figure 1. Operator classes at d = 6 and their respective tree openings at the diagram level. Solid lines correspond to fermions, dashed lines to scalars and wavy lines to vectors. Red lines are for heavy fields. JHEP08(2023)166 Figure 1. Operator classes at d = 6 and their respective tree openings at the diagram level. Solid lines correspond to fermions, dashed lines to scalars and wavy lines to vectors. Red lines are for heavy fields. 2.2 Dictionary for d = 6 2.2 In table 4 we show the list of operators at d = 6 that can be generated at tree-level. The operators are classified into three classes: ψ2H3, ψ2H2D, and ψ4.3 At d = 6 there is a fourth operator class, ψ2HX, whose operators can only be realised at loop level and hence are not listed here. For a complete basis of on-shell operators at d = 6, see [21]. In table 4 we show the list of operators at d = 6 that can be generated at tree-level. The operators are classified into three classes: ψ2H3, ψ2H2D, and ψ4.3 At d = 6 there is a fourth operator class, ψ2HX, whose operators can only be realised at loop level and hence are not listed here. For a complete basis of on-shell operators at d = 6, see [21]. Figure 1 shows all possible diagrams that generate the three operator classes mentioned above. The first class, ψ2H3, can be obtained through two different topologies, one of which contains two heavy fields (depending on their Lorentz nature we find three diagrams corresponding to the possibilities: SS, FF, SF), leading in most cases to two-particle extensions of the SM. The other topology includes a 4-point interaction vertex and it contains just one heavy scalar field. The four-fermion class, ψ4, can only be generated at tree-level with a scalar or a vector heavy propagator, depending on the chirality of the external fields. Finally, there are two openings for the operator class ψ2H2D with just one internal field, one of which contains the derivative in the V SS interaction vertex, and in the other, the derivative comes from the fermion propagator. Thus, all operators in table 4 can be generated at tree-level with one-particle SM extensions, except for the operator OLNH3 belonging to ψ2H3, for which some openings require two-particle models.f JHEP08(2023)166 Here, our approach differs from the philosophy followed in [39], since we insist on using only renormalisable vertices. The authors of [39], on the other hand, consider only one particle extensions of the SM. However, already at d = 6 in pure SMEFT there are some operators which require two BSM fields. 2.1 Diagrammatic method basics Models Operators S ONN, ONNNN S1 OLNLe, OeN φ OQuNL, OLNLe, OLNQd, OLN, OLNH3 ω1 OLNQd, OdN, OduNe ω2 OuN Π1 OLNQd, OQN ∆1 ONH2D, ONeH2D B ONH2D, ONN, OeN, OuN, OdN, OLN, OQN B1 ONeH2D, OeN, OduNe L1 OLN U1 OdN U2 OQuNL, OuN, OduNe Q1 OQuNL, OQN Table 5. One-particle decompositions for d = 6 NRSMEFT operators. We differentiate between scalar, fermion and vector models. The first column gives the particle, the second the operators that are generated at tree-level. ψ2H3 Two-particle models OLNH3 SS: (S, φ), (Ξ1, φ), (Ξ, φ) FF: (∆1, N), (∆1, Σ1), (∆1, Σ) FS: (N, S), (∆1, S), (∆1, Ξ1), (Σ1, Ξ1), (∆1, Ξ), (Σ, Ξ) Table 6. Two-particle decompositions for the d = 6 operator OLNH3. There are three types of models according to the Lorentz nature of the heavy fields. Table 6. Two-particle decompositions for the d = 6 operator OLNH3. There are three types of models according to the Lorentz nature of the heavy fields. – 6 – 4Our Mathematica code can handle also non-renormalisable interactions. We have checked for some concrete cases that we can reproduce the one-heavy-particle at a time results of [39], once we include d = 5 terms. 3The full list of four-fermion operators includes two additional operators that violate baryon number (B), these are discussed separately in section 2.4. 4 3The full list of four-fermion operators includes two additional operators that violate baryon numb (B) these are discussed separately in section 2 4 3The full list of four-fermion operators in 3The full list of four-fermion operators include (B), these are discussed separately in section 2.4. 4 2.2 Dictionary for d = 6 All operators a non-hermitian, (+h.c.) is implicitly assumed. can be automated with computer tools, such as Matchete [49]. We added a notebook with some example models as supplementary material to this paper. can be automated with computer tools, such as Matchete [49]. We added a notebook with some example models as supplementary material to this paper. 5Again, baryon number violating operators, in class ψ4H, are treated separately in section 2.4. 2.2 Dictionary for d = 6 The list of decompositions given in [39] for pure SMEFT is nevertheless complete, since [39] add also non-renormalisable operator (NRO) interactions at d = 5 to their Lagrangian.4 We present in table 5 the list of one-particle models found at d = 6 and the corre- sponding operators they open at tree-level, while the two-particle models for OLNH3 are shown separately in table 6, where we categorise the models based on the Lorentz nature of the involved fields. We note that the new field U1 contributes to only one d = 6 operator in NRSMEFT, OdN. This is due to the existence of a single interaction vertex involving light fields and the heavy vector, given by L ∝ NRγµdR Uµ† 1 .i The Lagrangian terms of heavy fields including a NR are given in appendix A. Further- more, we also collect there all gauge-invariant renormalisable terms that can be written down for U1 and the BSM fields appearing in tables 1–3, as well as for the vector L1. Recall, that the field L1 was also included in [39], but as a non-gauge vector. For NRSMEFT, we find additional renormalisable interactions of this vector. We also present them in ap- pendix A. The term containing solely light fields and L1, which contributes to the matching of the operator OLN, is given by L ∝ Nc RγµL Lµ 1. Given the complete UV Lagrangian, one could perform the tree-level matching onto the set of NRSMEFT operators. While we do not perform explicitly this matching, the process – 7 – ψ2H3D ψ4H ψ4H ONLH3D ϵij(Nc RγµLi)(iDµHj)(H†H) OLNLH ϵij(LγµL)(Nc RγµLi)Hj OLNeH (LNR)(Nc ReR)H ϵij(Nc RγµLi)Hj(H†i ←→ DµH) OQNLH ϵij(QγµQ)(Nc RγµLi)Hj OeLNH H†(eRL)(Nc RNR) ψ2H2D2 ϵij(QγµQi)(Nc RγµLj)H OQNdH (QNR)(Nc RdR)H ONeH2D2 ϵij(Nc R ←→ DµeR)(HiDµHj) OeNLH ϵij(eRγµeR)(Nc RγµLi)Hj OdQNH H†(dRQ)(Nc RNR) ONH2D2 (Nc R ←→ ∂µ NR)(H†←→ DµH) OdNLH ϵij(dRγµdR)(Nc RγµLi)Hj OQNuH (QNR)(Nc RuR) ˜H (Nc RNR)(DµH)†DµH OuNLH ϵij(uRγµuR)(Nc RγµLi)Hj OuQNH ˜H†(uRQ)(Nc RNR) ψ2H2X OduNLH ϵij(dRγµuR)(Nc RγµLi) ˜Hj OLNNH (LNR)(Nc RNR) ˜H ONeH2W (ϵτ I)ij(Nc RσµνeR)(HiHj)W I µν OdQNeH ϵij(dRQi)(Nc ReR)Hj ONLNH ˜H†(NRL)(Nc RNR) ONH2B (Nc RσµνNR)(H†H)Bµν OQuNeH (QuR)(Nc ReR)H ψ2H4 ONH2W (Nc RσµνNR)(H†τ IH)W I µν (QσµνuR)(Nc RσµνeR)H ONH4 (Nc RNR)(H†H)2 Table 7. NRSMEFT operators at d = 7 that admit tree-level decompositions. All operators are non-hermitian, (+h.c.) is implicitly assumed. JHEP08(2023)166 Table 7. NRSMEFT operators at d = 7 that admit tree-level decompositions. 2.3 Dictionary for d = 7 We next discuss the results for d = 7 NRSMEFT operators. There are eight different operator classes, but only five of them can be generated at tree-level. Operators with two fermions containing derivatives or strength field tensors can only come at tree-level if they have at least two scalars [50]. Operators with four fermions and a derivative cannot be generated either at tree-level, since the potential diagrams with four external legs (see diagrams for class ψ4 in figure 1) do not contain any derivative in the interaction vertices. Hence the classes ψ2H3D, ψ2H2D2 and ψ2H2X are tree-level generated, whereas ψ2HDX, ψ4D and ψ2X2 can only be opened via loops. The remaining classes generated at tree- level contain only fermions and scalars: ψ4H and ψ2H4. All the operators which can be decomposed at tree-level are shown in table 7.5 The complete list of on-shell operators at d = 7 can be found, for example, in [21].i The diagrams generating the listed operators are represented in figure 2, covering all possible configurations for the considered operators in NRSMEFT. As before, we adopt the convention of using solid, dashed, and wavy lines to denote fermion, scalar, and vector fields, respectively, and black for light fields, while red lines denote heavy fields. In most diagrams, there are now two or even three heavy propagators. However, the operator class ψ2H2D2 can be generated with just one heavy propagator. Consequently, at d = 7, we encounter models with up to three new particles. Regarding the Lorentz nature of the heavy fields appearing in the different models, we find that for the operators belonging – 8 – ψ2H3D : ψ2H2D2 : ψ2H2X : ψ4H : ψ2H4 : Figure 2. Diagrams generating different d = 7 operator classes at tree-level. External light fields appear as black lines and heavy propagators are marked in red. JHEP08(2023)166 Figure 2. Diagrams generating different d = 7 operator classes at tree-level. External light fields appear as black lines and heavy propagators are marked in red. to the ψ2H2X class, only heavy fermions are allowed and for the ones in ψ2H2D2 and ψ2H4, only scalars and fermions can be drawn as internal lines. In the remaining classes, ψ2H3D and ψ4H, the models consist of a pair of fields that are different combinations of scalars, fermions and vectors. Table 8. NRSMEFT d = 7 operators in classes ψ2H2D2, ψ2H2X and ψ4H and the corresponding models generating them at tree-level. 2.3 Dictionary for d = 7 As the number of internal lines increases in the opening of diagrams of higher di- mensional operators, so does the possible number of models. In total we count 224 UV decompositions for the d = 7 operators in table 7, but the same particle content can give rise to more than one d = 7 operator in many cases. The total number of different particle combinations for the listed operators is then found to be 112. We present the results for each operator individually in tables 8–9, classifying the models by the number of BSM particles they contain (one, two or three). Additionally, we group the models based on the Lorentz nature of the heavy propagators depicted in the diagrams. Among these models, there are only two one-particle models: the scalar S and the fermion ∆1. The number of two-particle models increases to 102, consisting of 42 FS models and 45 FV models. The remaining models include three FF, nine SS, and three SV configurations. As we indicated in figure 2, we did not find any V V model. Finally, there are eight three-particle models, which generate the operator ONH4. It is worth noting that subsets of particles within the three-particle models can also generate other operators. 2.3 Dictionary for d = 7 – 9 – ψ2H2D2 Models ψ2H2X Models ONeH2D2 F : ∆1 ONeH2W F : ∆1 ONH2D2 S : S ONH2B F : ∆1 F : ∆1 ONH2W F : ∆1 ψ4H Models OLNLH SS : (S1, φ) (φ, Ξ1) FS : (E, S1) (Σ1, Ξ1) (∆1, S1) (∆1, Ξ1) (N, φ) (Σ, φ) FV : (N, B) (Σ, W) (N, L1) (Σ, L1) (∆1, B) (∆1, W) (E, L1) (Σ1, L1) OQNLH SS : (ω1, Π1) (Π1, ζ) FS : (D, ω1) (T1, ζ) (∆1, ω1) (∆1, ζ) (N, Π1) (Σ, Π1) (U, Π1) (T2, Π1) FV : (U, U2) (T2, χ) (U, L1) (T2, L1) (N, B) (Σ, W) (N, Q1) (Σ, Q1) (∆1, U2) (∆1, χ) (D, L1) (T1, L1) (∆1, B) (∆1, W) (D, Q1) (T1, Q1) OeNLH SS : (S1, φ) FS : (N, S1) (∆1, φ) (∆3, S1) FV : (N, B) (N, B1) (∆3, L3) (∆3, L1) (∆1, B) (∆1, B1) (∆1, L3) (∆1, L1) OdNLH SS : (ω1, Π1) FS : (Q1, Π1) (∆1, Π1) (Q5, ω1) (N, ω1) FV : (N, B) (N, U1) (Q5, Q5) (Q5, L1) (∆1, B) (Q1, U1) (∆1, Q5) (Q1, L1) OuNLH SS : (ω2, Π7) FS : (Q7, Π7) (∆1, Π7) (N, ω2) (Q1, ω2) FV : (Q1, Q1) (Q1, L1) (N, B) (N, U2) (∆1, Q1) (Q7, L1) (∆1, B) (Q7, U2) OduNLH SS : (ω2, Π1) FS : (Q1, Π1) (∆1, Π1) (Q1, ω2) (E, ω2) FV : (E, B1) (E, U1) (Q1, Q1) (Q1, L1) (∆1, B1) (Q1, U1) (∆1, Q1) OdQNeH SS : (S1, φ) FS : (∆1, φ) (Q5, S1) (U, S1) FV : (U, U2) (U, U1) (Q5, Q5) (Q5, Q1) (∆1, U2) (∆1, U1) (∆1, Q5) (∆1, Q1) OQuNeH SS : (S1, φ) (ω1, Π1) (ω2, Π7) FS : (Q7, Π7) (∆1, Π7) (D, ω1) (∆1, ω1) (D, S1) (Q7, S1) (∆1, φ) (∆1, Π1) (Q7, Π1) (∆1, ω2) (D, ω2) JHEP08(2023)166 FV : (N, B) (N, U1) (Q5, Q5) (Q5, L1) (∆1, B) (Q1, U1) (∆1, Q5) (Q1, L1) SS : (ω2, Π7) Table 8. NRSMEFT d = 7 operators in classes ψ2H2D2, ψ2H2X and ψ4H and the corresponding models generating them at tree-level. Table 9. Continuation of table 8. NRSMEFT d = 7 operators in ψ2H3D, ψ2H4, ψ4H and their tree-level decompositions. 2.3 Dictionary for d = 7 – 10 – JHEP08(2023)166 ψ2H3D Models ONLH3D SV : (S, L1) (Ξ, L1) (Ξ1, L1) FS : (N, S) (Σ, Ξ) (Σ1, Ξ1) (∆1, S) (∆1, Ξ) (∆1, Ξ1) FV : (N, B) (Σ, W) (Σ1, W1) (∆1, B) (∆1, W) (∆1, B1) (∆1, W1) FF : (N, ∆1) (∆1, Σ) (∆1, Σ1) ψ2H4 Models ONH4 S : S SS : (S, φ) (S, Ξ1) (S, Ξ) FS : (N, S) (Σ, Ξ) (Σ1, Ξ1) (∆1, S) (∆1, Ξ) (∆1, Ξ1) (∆1, φ) FF : (N, ∆1) (∆1, Σ) (∆1, Σ1) SSS : (S, φ, Ξ1) (S, φ, Ξ) FFS : (∆1, Σ1, Ξ1) (N, ∆1, S) (∆1, Σ, Ξ) FSS : (∆1, S, φ) (∆1, φ, Ξ) (∆1, φ, Ξ1) ψ4H Models OLNeH SS : (S, φ) (S1, φ) FS : (E, S) (E, S1) (∆1, φ) (∆1, S) (∆1, S1) OeLNH SS : (S, φ) FS : (∆1, S) (∆1, φ) (E, S1) FV : (E, B1) (∆1, L1) (∆1, B1) OQNdH SS : (S, φ) (ω1, Π1) FS : (∆1, φ) (D, ω1)(∆1, ω1) (Q1, Π1) (∆1, Π1) (Q1, S) (D, S) OdQNH SS : (S, φ) FS : (∆1, φ) (Q1, S) (D, S) FV : (∆1, U1) (∆1, Q1) (D, U1) (Q1, Q1) OQNuH SS : (S, φ) (ω2, Π1) FS : (Q1, Π1), (∆1, Π1), (U, ω2), (∆1, ω2), (U, S), (Q1, S) OuQNH SS : (S, φ) FS : (∆1, φ) (U, S) (Q1, S) FV : (Q1, Q1) (U, U2) (∆1, Q1) (∆1, U2) OLNNH SS : (S, φ) FS : (N, S) (∆1, S) (∆1, φ) ONLNH SS : (S, φ) FS : (N, S) (∆1, S) (∆1, φ) FV : (N, B) (∆1, B) (∆1, L1) 2 3 2 4 4 JHEP08(2023)166 – 11 – ψ4 (d = 6) Models OQQdN εij Qc iQj dc RNR S : ω1 V : Q1 OuddN uc RdR dc RNR S : ω1, ω2 ψ4H (d = 7) Models OQNddH εij QiNR dRdc R ˜Hj SS : (ω2, Π1) FS : (U, ω2) (∆1, ω2) (Q1, Π1) FV : (Q1, Q1) (Q1, U1) (∆1, Q1) (U, U1) OQNQH εij QiNR QjQc H SS : (ω1, Π1) (Π1, ζ) FS : (D, ω1) (∆1, ω1) (T1, ζ) (∆1, ζ) (D, Π1) (T1, Π1) OQNudH QNR (uRdc R) H SS : (ω1, Π1) FS : (D, ω1) (∆1, ω1) (Q5, Π1) (Q1, Π1) FV : (Q1, Q1) (Q1, U1) (Q5, Q5) (Q5, U2) (∆1, Q1) (D, U1) (∆1, Q5) (D, U2) Table 10. 2.3 Dictionary for d = 7 Baryon number violating operators of d = 6 and d = 7 in NRSMEFT and their tree-level decompositions. Models are classified in terms of the Lorentz nature of the fields. JHEP08(2023)166 Table 10. Baryon number violating operators of d = 6 and d = 7 in NRSMEFT and their tree-level decompositions. Models are classified in terms of the Lorentz nature of the fields. Table 10. Baryon number violating operators of d = 6 and d = 7 in NRSMEFT and their tree-level decompositions. Models are classified in terms of the Lorentz nature of the fields. 6When defining the operators, SU(3) contractions have been omitted. In all cases there should be the total antisymmetric tensor in colour indices, εαβσ, contracted with the three quark fields’ colour indices. 2.4 Baryon number violating operators However, if we impose B conservation, either yω1 Nd or yω1 ud could be non-zero, depending on which B(ω1) is assigned. In this case, ω1 can appear in the decomposition of other NRSMEFT operators, relevant for collider experiments. The previous discussion can also be extended to decompositions for d = 7 operators, although additional subtleties arise as a larger number of couplings enter in the matching relations. In summary, in two particle models for d = 7 operators there are three couplings entering the matching conditions and usually it is sufficient to forbid one of the three in order to ensure baryon number conservation.l Finally, we briefly comment on lepton number violation in BNV operators. The above discussion has not assumed any value for the lepton number of NR, see also the discussion in the next section. If we use the standard assignment of L(NR) = 1, one can easily see that d = 6 BNV operators violate B + L, while conserving B −L. Operators at d = 7, on the other hand, violate B −L. This follows exactly the same pattern as found in SMEFT. 2.4 Baryon number violating operators Note that again we have suppressed flavour indices; in full generality the Wilson coefficients are matrices in flavour space. This operator triggers the decay process p →π+N.7 Experimental limits on the proton lifetime impose stringent constraints on the combination yω1 Nd yω1 ud/Λ2. For the current experimental bound on this proton decay mode [57] the upper limit is roughly where we assumed mω1 = Λ. Note that again we have suppressed flavour indices; in full generality the Wilson coefficients are matrices in flavour space. This operator triggers the decay process p →π+N.7 Experimental limits on the proton lifetime impose stringent constraints on the combination yω1 Nd yω1 ud/Λ2. For the current experimental bound on this proton decay mode [57] the upper limit is roughly yω1 Nd yω1 ud ≲10−26 Λ TeV 2 . (2.3) (2.3) Clearly, this constraint renders OuddN unobservable for any foreseeable collider experiment. This does not, however, imply that the particle ω1 can not appear in other operators. Consider the following. In this simple model, BNV arises from the simultaneous presence of the two couplings, yω1 Nd and yω1 ud, since there is no baryon number (B) that can be assigned to ω1 in a way that preserves B in both interaction terms. For example, if we assign B(ω1) = 1/3, so that B is respected in the terms of the first row of eq. (2.1), the yukawa interactions of the second row violate B. However, if we impose B conservation, either yω1 Nd or yω1 ud could be non-zero, depending on which B(ω1) is assigned. In this case, ω1 can appear in the decomposition of other NRSMEFT operators, relevant for collider experiments. Clearly, this constraint renders OuddN unobservable for any foreseeable collider experiment. This does not, however, imply that the particle ω1 can not appear in other operators. Consider the following. In this simple model, BNV arises from the simultaneous presence of the two couplings, yω1 Nd and yω1 ud, since there is no baryon number (B) that can be assigned to ω1 in a way that preserves B in both interaction terms. For example, if we assign B(ω1) = 1/3, so that B is respected in the terms of the first row of eq. (2.1), the yukawa interactions of the second row violate B. 7While ω1 can trigger decays such as p →π+N and p →K+N, ω2 can only cause the decay p →K+N because the coupling yω2 dd is anti-symmetric in flavour space. 2.4 Baryon number violating operators In this subsection we comment on baryon number violation (BNV) in NRSMEFT. We give the decompositions for the BNV operators here only for completeness, since proton decay constraints (see below) usually render these operators uninteresting for collider phe- nomenology. The BNV operators at d = 6 and d = 7 that can be opened at tree-level are presented in table 10.6 Next to each operator, we provide the list of decompositions, classified according to the Lorentz nature of the fields. At d = 6, there are two four-fermion operators that can be opened at tree-level. At d = 7 there are five BNV operators, two of them belong to the ψ4D class and can only be generated at loop level, hence we don’t list them. The three remaining operators are in the ψ4H category and can be opened through three different diagrams, as was shown in figure 2.i For the d = 6 operators we find three one-particle decompositions: the two scalars ω1, ω2, and the vector Q1. Regarding the models for d = 7 operators, we found 22 two-particle models (3 SS, 10 FS and 9 FV models). Out of these 22, 17 models appear also in the opening of other d = 7 NRSMEFT operators, while 3 FS and 2 FV models are new. These are (D, Π1), (T1, Π1), (Q5, Π1) and (Q5, U2), (D, U2), respectively. Note that the non-observation of proton decay sets stringent limits on parameters of the models that allow for baryon number violating processes. Let us discuss this first for – 12 – a specific example. For simplicity, we consider the scalar decomposition ω1 of the d = 6 operator OuddN. The relevant part of the UV Lagrangian for our discussion reads a specific example. For simplicity, we consider the scalar decomposition ω1 of the d = 6 operator OuddN. The relevant part of the UV Lagrangian for our discussion reads L ∝yω1 ue uc ReR ω† 1 + yω1 Nd dc RNR ω† 1 + yω1 QL QcL ω† 1 + yω1 ud uc RdR ω1 + yω1 Q QcQ ω1 + h.c. + . . . (2.1) (2.1) The contribution to the operator OuddN is given by the matching relation cuddN = yω1 Nd yω1 ud Λ2 , (2.2) (2.2) JHEP08(2023)166 where we assumed mω1 = Λ. 3 Lepton number violation (LNV) In this section we will discuss lepton number violation in NRSMEFT and how it is connected to observable LNV processes with charged leptons at the LHC and to the Majorana masses – 13 – ν ν e e 0νββ e e d ¯d W W ¯d ¯d u u u u L L eR eR 0νββ eR eR dR dR H H dR dR Q Q uR uR H H Figure 3. Black box theorem of 0νββ decay graphically [41]: whatever is the underlying mechanism generating a non-zero neutrinoless double beta decay amplitude, will also generate a Majorana mass term for at least one of the SM neutrinos. Cutting the diagrams at the thinner lines leaves an operator contributing to 0νββ decay. To the left, diagram in the mass eigenstate basis. To the right, example diagram for the operator O9 ude = u2 RdR 2e2 R in the gauge basis. L L eR eR 0νββ eR eR dR dR H H dR dR Q Q uR uR H H eR Figure 3. Black box theorem of 0νββ decay graphically [41]: whatever is the underlying mechanism generating a non-zero neutrinoless double beta decay amplitude, will also generate a Majorana mass term for at least one of the SM neutrinos. Cutting the diagrams at the thinner lines leaves an operator contributing to 0νββ decay. To the left, diagram in the mass eigenstate basis. To the right, example diagram for the operator O9 ude = u2 RdR 2e2 R in the gauge basis. JHEP08(2023)166 of the SM neutrinos. Before we turn to NRSMEFT operators, however, it is instructive to discuss the “black box theorem” of neutrinoless double beta decay (0νββ decay) [41]. We will recapitulate the basics in section 3.1. In subsection 3.2, using very similar arguments, we will discuss how the observation of LNV @ LHC in processes involving a NR and Majorana neutrino masses are related at the operator level. For NRSMEFT at d = 7 we have given the possible decompositions at tree-level in section 2.3. In subsection 3.3 we examine Majorana neutrino masses in the renormalisable UV models, found in section 2.3. 8The mass mechanism of 0νββ decay corresponds actually to a d = 11 operator: O11 ¯ Q2Q2L2H2. 3.1 The black box theorem It is known for a long time that the observation of 0νββ decay will guarantee that at least one neutrino has a Majorana mass term [41]. This is usually called the “black box theorem”, since the mechanism (or “model”) underlying the 0νββ decay amplitude needs not to be known for this conclusion to hold, see figure 3, left: at the quark level 0νββ decay is a d = 9 operator of the form ( ¯d ¯duuee). As the figure shows, this operator can always be “dressed” with W-bosons to draw a diagram that generates a radiative contribution to the Majorana neutrino mass of a SM neutrino. 0νββ decay is a low-energy process, thus the black box diagram is usually drawn in the mass eigenstate basis, as done in figure 3, left. It is instructive, however, to discuss the black box theorem in terms of SMEFT operators. The neutrino Majorana mass matrix is gener- ated from the famous Weinberg operator, OW = LLHH, while contributions to the 0νββ decay amplitude start at the d = 9 level. ∆L = 2 operators (disregarding operators with derivatives or field strength tensors) have been listed up to d = 11 in [58]. At d = 9 there are six operators relevant for 0νββ decay.8 The simplest six fermion SMEFT operator for 0νββ decay is O9 u ¯de ∝u2 RdR 2e2 R. The operator and its black box connection is shown in figure 3, right. The SM Yukawa couplings ¯LeRH, ¯QdRH and uRQH are used to close the loops. This example results in a 4-loop diagram, the same as the black box diagram in the mass basis. The other five d = 9 operators will result either in 2-loop or in 3-loop black box diagrams. – 14 – Note that black box diagrams are in general divergent. Thus, one expects that neutrino masses are generated at a lower loop level than indicated by the respective black box diagram. However, at the level of NROs it is not possible to decide at which level neutrino masses are indeed generated, for this one needs to know the underlying UV model. We will come back to this question in subsection 3.3. 3.1 The black box theorem A 4-loop diagram will give only a tiny contribution to the neutrino mass [59] and, thus, the guaranteed, but minimal contribution from the 0νββ decay black box diagram to the neutrino masses is numerically much smaller than what is required to explain neutrino oscillation data. However, given the finite number of operators at d = 9, one can open up the 0νββ decay operator(s) in all possible ways, say, at tree-level [60]. The list of possible UV “models” found in this exercise can then be examined one-by-one and neutrino mass models from the tree- to the 4-loop level emerge [61]. Whether any particular of these models can or can not be the main contribution to the neutrino masses — as required for an explanation of oscillation data — is then mainly a question of the loop level at which the neutrino masses are generated in the respective model, but in all cases a non-zero Majorana mass for the SM neutrinos is guaranteed. JHEP08(2023)166 10At the LHC l = e, µ, τ, in 0νββ decay only l = e, of course. We will not discuss flavour in detail. 9NRSMEFT operators put the chirality of NR as right-handed. In the mass eigenstate basis N has bot left- and right-handed components, so we do not add the subscript R for mass eigenstates. 10l 3.2 A black box for NRSMEFT and Majorana neutrino masses JHEP08(2023)166 di-lepton plus jets)/#events(opposite-sign di-lepton plus jets) is equal to one. Same-sign di-lepton events are obviously ∆(L) = 2 processes, formally the same as a Majorana neu- trino mass. The diagram for this LHC process is shown in figure 4 on the left. From this diagram, one can cut off the quarks and draw the 2-loop Majorana neutrino mass diagram shown on the right of figure 4. Clearly, if the diagram on the left is present in the model, the diagram on the right must also exist. Thus, the observation of this LNV process at the LHC guarantees that neutrinos are Majorana states for this particular model. Several comments are in order. First of all, the connection between the LHC process and Majorana neutrino masses in this particular example can be trivially understood. The model as discussed is, after all, a type-I seesaw and type-I seesaw of course generates Majorana neutrino masses. However, Majorana neutrino masses in the seesaw are generated at tree-level, thus the 2-loop diagram in figure 4 just represents a “minimal link” between the LHC process and the existence of a Majorana neutrino mass. Numerically it is only a very minor correction to the total neutrino mass in this model. This is very similar to the black box theorem for 0νββ decay discussed above. But, different from the black box theorem for 0νββ decay, the discussion as presented so far is model dependent, since we have assumed that the Lagrangian terms of eq. (3.1) exist. To make statements as model-independent as possible, we should not a priori assume that N has a coupling to SM W-bosons,11 nor that the mass of the N is of Majorana type, but instead consider EFT operators. In the mass eigenstate basis, after electro-weak symmetry breaking, the simplest purely fermionic operator one can write down for the production of a fermion singlet is either ¯duNe or ¯duNce. Assigning lepton number to N as L(N) = 1 (L(N) = −1), the former (latter) operator is lepton number conserving, while the latter (former) represents a LNV object. If both types of operators are present, LNV processes will be generated-independently of the nature of the mass term of N. However, N needs to be a massive particle, its mass term could be either Dirac, i.e. lepton number conserving (LNC), or Majorana (LNV), as discussed above. 3.2 A black box for NRSMEFT and Majorana neutrino masses Before turning to NRSMEFT operators, let us briefly discuss the renormalisable Lagrangian for a simple model that adds a NR to the SM. In the minimal type-I seesaw, there is a Majorana mass term and the Lagrangian involving NR contains also a Yukawa coupling: LType−I = −yνNRLH −1 2MMNc RNR + h.c. (3.1) (3.1) Note that the Majorana nature of the mass term is specific for the type-I seesaw. The mass term for NR could also be of Dirac type, the best-known example is the inverse seesaw [8]. Adding a second Weyl fermion to the model, call it NL, one can write a mass term MRNLNR instead of MM in eq. (3.1). It is obvious that, assigning lepton number L(NR) = 1 (and L(NL) = 1), the Yukawa term and MR conserve lepton number, while MM violates L by ∆(L) = 2. However, in the absence of yν one could assign L(NR) = 0 and this would eliminate LNV from MM. This may seem trivial, but we would like to stress that LNV is always related to a mismatch in the lepton number assignment of two or more terms in the Lagrangian in any model, NRSMEFT is no exception to this statement.i After electro-weak symmetry breaking, the simple model, defined by eq. (3.1), intro- duces a non-zero coupling of the (mostly) singlet state N9 to the gauge bosons, gVlN, where VlN is the mixing angle between active and sterile states. At the LHC W-bosons are produced, which can then decay to N + l via this non-zero coupling.10 N will decay itself via an off-shell W to another lepton plus jets. For a Majorana neutrino, the probability to decay to either lepton or anti-lepton is the same (at tree-level), thus the final state for the whole process will contain two leptons and two jets and the ratio R = #events(same-sign – 15 – d e e d u u W N W e N e W ν ν Figure 4. Lepton number violation at the LHC in a minimally extended variant of the SM (left) and a 2-loop neutrino mass diagram that will necessarily be generated at the same time. u Figure 4. Lepton number violation at the LHC in a minimally extended variant of the SM (left) and a 2-loop neutrino mass diagram that will necessarily be generated at the same time. 11We will come back to this point near the end of this subsection. 3.2 A black box for NRSMEFT and Majorana neutrino masses Thus, overall, one can find ∆(L) = 2 processes either with two LNC opera- tors and a Majorana propagator or with one LNV and one LNC operator, along with a LNC propagator (no mass flip). For the discussion to be complete, we need to cover both options. – 16 – ν ν e e d d u u u u d d e e N N W W M pp →jje+e+ Figure 5. Lepton number violation at the LHC and a 4-loop neutrino mass diagram. Different from figure 4, this diagram does not assume that N has couplings to the W-boson. Instead, only electric charge conservation and the existence of some operator ¯duNe is assumed. Again, the diagram is drawn in the mass eigenstate basis and the origin of the LNV is assigned to the Majorana mass M. JHEP08(2023)166 Figure 5. Lepton number violation at the LHC and a 4-loop neutrino mass diagram. Different from figure 4, this diagram does not assume that N has couplings to the W-boson. Instead, only electric charge conservation and the existence of some operator ¯duNe is assumed. Again, the diagram is drawn in the mass eigenstate basis and the origin of the LNV is assigned to the Majorana mass M. Figure 5 shows, as an example, a 4-loop Majorana neutrino mass diagram, combining two LNC four-fermion operators with a LNV Majorana mass insertion in the N propagator. The LHC LNV process pp →l+l+jj is contained in the inside of the diagram, just cutting the diagram at the thin lines. Thus, the two observables are always either both present in the theory or none of them is, exactly as in the originally black box theorem for 0νββ decay. Note that, simple power counting shows that this 4-loop diagram is divergent. Clearly, a lower order diagram contributing to the neutrino mass should exist, in order to provide a counter term for this infinity, and — apart from some very fine-tuned special cases — that lower order diagram will give a larger contribution to the neutrino mass than the 4-loop diagram. However, given only the effective operator and not the full UV complete model, it is not possible to decide at which loop level Majorana masses will appear. We will come back to discuss this point in section 3.3.f Figure 5 shows the connection between different LNV observables in the mass eigen- state basis. 3.2 A black box for NRSMEFT and Majorana neutrino masses The underlying physics, however, is again more clearly visible in the weak eigenstate basis. Figure 6 shows an example based on the simplest d = 6 NRSMEFT oper- ator, OduNe. Here, the diagram gives actually a 4-loop realisation of the Weinberg operator, OWbg ∝LLHH (which will generate the neutrino mass matrix after symmetry breaking). Similar diagrams can be drawn for all other single-NR four-fermion operators in NRSMEFT. In the above discussion it was assumed that the source of the LNV is the Majorana propagator of the NR. However, the same black box connection between LNV @ LHC and Majorana neutrino mass can be established also in the case that the necessary LNV is due to a LNV operator. Let’s discuss this in the weak eigenstate basis directly. We choose as the LNV d = 7 operator the example of OdLNH. (For all other LNV operators the discussion is very similar.) For the LNC d = 6 operator we choose OdQNL. Figure 7 shows the resulting 2-loop diagram for OWbg. As before, cutting open the loops defines a – 17 – L L eR eR dR Q uR uR uR uR Q dR eR eR NR NR H H M H H Figure 6. Lepton number violation at the LHC and a 4-loop realisation of the Weinberg operator. Different from figure 5, this diagram is drawn in the electro-weak basis, assuming the d = 6 operator OduNe is non-zero. L L eR eR dR Q uR uR uR uR Q dR eR eR NR NR H H M H H JHEP08(2023)166 H Figure 6. Lepton number violation at the LHC and a 4-loop realisation of the Weinberg operator. Different from figure 5, this diagram is drawn in the electro-weak basis, assuming the d = 6 operator OduNe is non-zero. Figure 6. Lepton number violation at the LHC and a 4-loop realisation of the Weinberg operator. Different from figure 5, this diagram is drawn in the electro-weak basis, assuming the d = 6 operator OduNe is non-zero. LNV process for the LHC: one could, for example, produce the NR via OdQNL, while the final state is produced from the decay of NR via OdLNH. 12Only final states without missing energy can be used to determine lepton number experimentally, of course. 3.2 A black box for NRSMEFT and Majorana neutrino masses This decay could either contain two jets plus a Higgs (or Z0) boson and missing momentum, or two jets plus a W and a charged lepton.12 The additional bosons (relative to the “standard” lljj signal) could actually be used to distinguish the two possibilities (Majorana propagator versus d = 7 operator) experimentally — at least in principle.i One can easily check that the chiralities in the diagram in figure 7 are such, that the momentum /q is picked from the neutrino propagator (no mass flip). Again, the resulting integral is divergent, indicating that a lower order contribution to the neutrino mass should exist in any UV completion generating this diagram at low energies. However, whether the neutrino mass is tree-level or 1-loop can not be decided at the level of effective operators only. While we have concentrated here on a specific combination of operators, the same conclusions can easily be reached for all other possible combinations: observation of LNV @ LHC guarantees the existence of Majorana neutrino masses for the SM neutrinos. Let us return to the comment, stated above, that for generality of our argument we should not assume that N has a non-zero coupling to gauge bosons. This statement is motivated by the fact that experimentally it might not be possible to show that the vertex e-N-W exists. However, consider the following: H and L can always be coupled to a SU(2) singlet, thus, if a NR is present in the theory one can always write down a yukawa term yνNRLH — which is equivalent to a non-zero VlN in the broken phase. One might attempt – 18 – L L dR dR NR q/ NR dR dR Q H H Figure 7. Lepton number violation at the LHC and a 2-loop realisation of the Weinberg operator. Different from figure 6, in this diagram the origin of LNV is the d = 7 operator OdLNH. L L dR dR NR q/ NR dR dR Q H H JHEP08(2023)166 Figure 7. Lepton number violation at the LHC and a 2-loop realisation of the Weinberg operator. Different from figure 6, in this diagram the origin of LNV is the d = 7 operator OdLNH. to forbid this coupling via some extra symmetry beyond those of the SM. An example could be a Z2 symmetry, under which the NR is odd, such as in the famous “scotogenic” neutrino mass model [62]. [ ] 14To simplify the discussion below, we assume the d = 7 operators violate L, see previous section. 3.2 A black box for NRSMEFT and Majorana neutrino masses However, such an extra symmetry is incompatible with the existence of any of the single-NR operators in tables 4 and 7. This is easy to see: consider, for example OQuNL. We can take this operator and replace QuR by H (up type yukawa coupling do exist after all), thus writing down a term proportional to NRLH. Since similar replacements can be done for any of the single-NR operators, the observation of any of these will guarantee that some (although maybe very small) coupling to gauge bosons should be present in the model as well. We stress, however, that as with the black box, this argument is purely qualitative. It does not allow to fix the numerical value of yν. In particular, both production and decay of NR at the LHC could easily be dominated by NRO operators.l We close this subsection with a brief comment about flavour. While in double beta decay the charged leptons are always electrons, the LHC can produce, in principle, any lepton flavour. Just as 0νββ decay guarantees that the (m)ee entry of the neutrino mass matrix is non-zero, the observation of different flavour combinations (α, β) in LNV processes at the LHC would then be related to the Majorana neutrino mass matrix entry (m)αβ in the gauge basis. 13Once a particular model is specified, neutrino fits can be easily done using, for example, the formul in [63]. 3.3 Neutrino masses in models derived from LNV d = 7 operators In this subsection, we will briefly discuss neutrino mass generation at the renormalisable level. The aim of this discussion is not to provide a detailed fit of neutrino masses and mixing angles to experimental data,13 but rather to demonstrate that all models generating LNV operators with NR also generate active neutrino masses.14 Given this connection, one might be tempted to think that d = 7 operators are not observable in accelerator experiments, due to the smallness of the observed neutrino masses. However, as we will – 19 – L L H NR L N ϕ† L L L NR H Ξ† 1 ϕ† L L L NR H S† 1 ϕ† L L H L NR E S1 Figure 8. Four example decompositions for OLNLH. The two examples shown in the top row will give tree-level contributions to the active neutrino masses via type-I (left) or type-II seesaw (right). The decomposition in the bottom generate radiative neutrino masses at 1-loop (left) and 2-loops (right). The quantum numbers for the new fields are given in tables 1 and 2. JHEP08(2023)166 Figure 8. Four example decompositions for OLNLH. The two examples shown in the top row will give tree-level contributions to the active neutrino masses via type-I (left) or type-II seesaw (right). The decomposition in the bottom generate radiative neutrino masses at 1-loop (left) and 2-loops (right). The quantum numbers for the new fields are given in tables 1 and 2. discuss now, such a conclusion holds only for a very specific subset of UV decompositions and not in general. First of all, with NR being a complete SM singlet, all models giving rise to single- NR d = 6 operators necessarily allow to write down also a neutrino yukawa coupling, as discussed above. If, in addition, lepton number is violated, also a Majorana mass for NR is allowed. In fact, the Majorana mass term is mandatory in this case: one can easily show that in all NR models with LNV, MM is generated radiatively via diagrams with divergent integrals. Thus, for consistency, all these models require also the presence of a tree-level MM as a counter term. A seesaw type-I contribution to the neutrino masses is therefore unavoidable in all models with a NR and LNV. 3.3 Neutrino masses in models derived from LNV d = 7 operators For the discussion in this subsection, however, this contribution to the neutrino masses is irrelevant, since it does not place any restrictions on the Wilson coefficients of the d = 7 operators.f As we pointed out in section 2.3, we found 112 different models for d = 7 operators. We will not discuss all these models in detail, but instead focus on just four decompositions for the example operator OLNLH, see figure 8. These four examples are sufficient to cover essentially all relevant aspects of neutrino mass generation in the 112 models: the remaining models could be discussed in much the same way, with adequate replacements for the corresponding model parameters.i In figure 8 we show in the top row two example decompositions for OLNLH containing N and Ξ1. These will give a tree-level seesaw contribution of type-I and type-II to the neutrino masses (note that one can replace N by Σ, and obtain a decomposition with a type-III seesaw too). We note in passing that out of the 112 models (14, 8, 8) contain (N, Σ, Ξ1), respectively. The decompositions in the bottom row, on the other hand, will generate neutrino masses radiatively. – 20 – Let us discuss the tree-level cases first. Consider the example decomposition shown in figure 8, top left. The Lagrangian contains the terms: L ∝yφ NL NRL φ + yφ NL NL φ + yNL NL H + 1 2MN N cN + h.c. (3.2) (3.2) where N and φ are “heavy” copies of NR and H.15 The Wilson coefficient generated from this diagram is matched via where N and φ are “heavy” copies of NR and H.15 The Wilson coefficient generated from this diagram is matched via cLNLH = −1 4 yφ NLyφ NLyNL MN m2φ . (3.3) (3.3) JHEP08(2023)166 On the other hand, N must be a Majorana field, otherwise the diagram can not be closed. Thus, N gives a contribution to the neutrino mass á la seesaw type-I: mν ∝y2 NL v2 MN . (3.4) (3.4) We can use this equation to put an upper limit on the coefficient cLNLH: cLNLH ≲10−6 yφ NLyφ NL Λ3 Λ v 1/2 mν 0.1 eV , (3.5) (3.5) where we have assumed MN ≃mφ ≃Λ. This estimate shows that production of a NR via this operator in a lepton collider is completely negligible for this model. 15Notice that we have omitted the explicit contraction of indices. To simplify the notation we omit them in the rest of the manuscript. 3.3 Neutrino masses in models derived from LNV d = 7 operators L H H L eR L ϕ S1 L H H L L NR L eR S1 S1 E Figure 9. One-loop (left) and two-loop (right) neutrino mass diagrams, based on the decomposition of OLNLH containing the BSM particles (S1, φ) or (E, S1). JHEP08(2023)166 where we have assumed mΞ1 = mφ = κΞ1φ = Λ. The SM ρ-parameter puts an upper limit on the induced vacuum expectation value of the triplet, vΞ1, of roughly vΞ1 ≲2 GeV [57], which motivates the stringent constraint in eq. (3.9). Neutrino oscillation data, however, allow vΞ1 as small as vΞ1 ∼0.1 eV. Obviously, no numerically relevant constraint on \|cLNLH\| can be derived in this case.i Let us turn now to decomposition #3 in figure 8, (S1, φ). The same particle content appears also in decompositions of the operators OeNLH, OLNeH, OdQNeH and OQuNeH. In neutrino physics this combination of BSM particles is known as the Zee model [64]. Neutrino masses are generated at 1-loop level, see figure 9 to the left. The Lagrangian of the Zee model contains the terms: L ∝yS1 L LcL S1 + yS1 Ne Nc ReR S1 + yφ eL (eRL) φ† + κS1φS† 1Hφ + h.c. + . . . (3.10)l L ∝yS1 L LcL S1 + yS1 Ne Nc ReR S1 + yφ eL (eRL) φ† + κS1φS† 1Hφ + h.c. + . . . (3.10) (3.10) Disregarding flavour indices for simplicity, the neutrino mass in the Zee model can be estimated as:  2  mZee ν ≃− 1 16π2 yS1 L mτyφ eL √ 2vκS1φ m2 h+ 2 −m2 h+ 1 log   m2 h+ 2 m2 h+ 1  . (3.11) (3.11) Here, hi are the two mass eigenstates formed by S1 and the charged component in φ. mτ is the mass of the τ lepton and we have neglected terms proportional to mµ,e, which are not relevant for this discussion. Thus, neutrino masses will put a stringent constraint on the product yS1 L yφ eL(κS1φ/Λ), where Λ ≃mS1 ≃mφ. Logically, this combination could be small because one, two or all three parameters are suppressed. The matching of the operators OLNLH, OeNLH, OLNeH, on the other hand, will be proportional to: cLNLH ∝yS1 L yφ NL κS1φ Λ ceNLH ∝yS1 Neyφ eL κS1φ Λ cLNeH ∝yS1 Neyφ NL κS1φ Λ . 3.3 Neutrino masses in models derived from LNV d = 7 operators We can also estimate the partial decay width of a NR via OLNLH given this constraint. We find the decay length is roughly (cτ) ∼ Λ TeV 5 mNR 10 GeV −5 0.1 eV mν 2 108 m, (3.6) (3.6) for yφ NL = yφ NL = 1. A decay length this large would render the NR essentially stable for collider experiments, unless it is much heavier than indicated in eq. (3.6). Note that similar arguments can be presented for all decompositions of d = 7 operators containing either N or Σ. for yφ NL = yφ NL = 1. A decay length this large would render the NR essentially stable for collider experiments, unless it is much heavier than indicated in eq. (3.6). Note that similar arguments can be presented for all decompositions of d = 7 operators containing either N or Σ. For decompositions containing Ξ1, the situation is slightly more complicated. If we allow for LNV, the Lagrangian for a model with a Ξ1 field contains the terms: L ∝yΞ1 L LcL Ξ1 + κΞ1HHΞ† 1 + κΞ1φΞ† 1Hφ + . . . (3.7) (3.7) The simultaneous presence of both terms will lead to a seesaw type-II contribution to the active neutrino mass matrix. We can use this to rewrite the Wilson coefficient for the decomposition shown in figure 8, top right as: The simultaneous presence of both terms will lead to a seesaw type-II contribution to the active neutrino mass matrix. We can use this to rewrite the Wilson coefficient for the decomposition shown in figure 8, top right as: \|cLNLH\| ≲yφ NL mν vΞ1 1 Λ3 (3.8) ≲10−10yφ NL mν 0.1 eV GeV vΞ1 1 Λ3 , (3.9) (3.8) (3.9) 15Notice that we have omitted the explicit contraction of indices. To simplify the notation we omit them in the rest of the manuscript. 15Notice that we have omitted the explicit contraction of indices. To simplify the notation we omit them in the rest of the manuscript. – 21 – – 21 – L H H L eR L ϕ S1 L H H L L NR L eR S1 S1 E Figure 9. One-loop (left) and two-loop (right) neutrino mass diagrams, based on the decomposition of OLNLH containing the BSM particles (S1, φ) or (E, S1). 3.3 Neutrino masses in models derived from LNV d = 7 operators (3.12) (3.12) In case all three parameters entering the neutrino mass are small, yS1 L ∼yφ eL ∼(κS1φ/Λ) ∼ ϵ, all of the coefficients in eq. (3.12) will be suppressed. However, in the more optimistic case, where either yS1 L or yφ eL are suppressed by ϵ3, while the other two parameters are order O(1), either cLNLH or ceNLH and cLNeH can be large. However, given the constraint from neutrino masses, it is impossible that all three operators are observable at the same time. – 22 – Consider now decomposition #4 in figure 8, (E, S1). This model allows to write the following terms in the Lagrangian: L ∝yS1 L LcL S1+yS1 Ne Nc ReR S1+yLE LE H +yS1 NEL NRE S1+mEEE+. . . (3.13) L ∝yS1 L LcL S1+yS1 Ne Nc ReR S1+yLE LE H +yS1 NEL NRE S1+mEEE+. . . (3.1 (3.13) Note that the vertex proportional to yS1 Ne does not appear in figure 8, but it is contained in a decomposition for OLNeH with the same particle content. Also, yS1 Ne is necessary for the 2-loop diagram in figure 9. In this diagram, from the NR propagator PR( /q + MNR)PL the momentum term survives. LNV is due to the simultaneous presence of yS1 Ne and yS1 NEL. Similar to the discussion for the Zee model, either yS1 Ne or yS1 NEL (or both) must be small, to fulfill the neutrino mass constraint. Thus, either OLNLH or OLNeH can have unsupressed Wilson coefficients, but not both operators at the same time. JHEP08(2023)166 We close this section summarising: (1) Models for LNV in d = 7 NRSMEFT operators will always also lead to active neutrino masses either at tree-, 1- or 2-loop level. (2) For decompositions leading to seesaw type-I or type-III contributions to the neutrino mass, the Wilson coefficients will be severely suppressed, rendering the corresponding operators phenomenologically irrelevant for accelerator experiments. (3) For decompositions leading to radiative neutrino masses, the Wilson coefficients for some of the corresponding oper- ators can be large, but the same UV decomposition contributes typically to more than one operator and neutrino mass constraints exclude the possibility that all corresponding operators are observable at the same time. 4 Conclusions Right-handed neutrinos with electro-weak scale masses have recently attracted a lot of attention in the literature. From the theoretical point of view, NR’s represent the simplest extension of the standard model that can explain the active neutrino masses as observed in oscillation experiments (via some variant of the seesaw mechanism). From the experimental side, in the past few years a number of new experiments have been proposed to search for long-lived particles with unprecedented sensitivities. NR’s with masses around (1-100) GeV are prime candidates for long-lived particles, due to the smallness of the active neutrino masses.f If new physics exists, but at a mass scale outside the reach of the LHC, effective field theory is the correct tool to study BSM. The relevant EFT involving right-handed neutrinos is NRSMEFT and a number of recent papers have studied the phenomenology of NRSMEFT. In this work we discussed a systematic tree-level decomposition of NRSMEFT opera- tors at d = 6 and d = 7, using a diagrammatic method. The resulting lists of BSM particles provide a complete dictionary of models, which can be used for studying NR phenomenol- ogy. We have also briefly compared our lists of particles to the Granada dictionary for tree-level UV completions for SMEFT at d = 6 [39]. Our lists of BSM particles are given in tables 1–3. In the appendix we give the Lagrangian terms for the resulting models for all terms involving NR. These Lagrangians were calculated with Sym2Int [65, 66]. In the – 23 – supplementary material files added to this paper, we give all remaining terms involving the BSM fields necessary for the calculation of the matching of the UV models onto NRSMEFT. The matching can be done automatically with the help of, for example, Matchete [49] and we added an example notebook for a number of models as supplementary material to this paper. We also discussed lepton number violation, that unavoidably appears if d = 6 and d = 7 operators are present in the theory at the same time. LNV is always linked to Majorana neutrino masses and LNV in NRSMEFT is no exception, as we discussed in detail. We also discussed possible constraints on d = 7 operators from the observed neutrino masses. 4 Conclusions While some of the possible UV models for d = 7 operators must have tiny Wilson coefficients, due the neutrino mass constraint, there exist many models for which d = 7 NRSMEFT operators could be observable in future LLP experiments. JHEP08(2023)166 p 17Explicit SU(2) and SU(3) index contractions have been omitted in the Lagrangians of this appendix. A Lagrangian , (A.4) LNR F F V,(A) = gB NNR NRγµNR Bµ + gB NNL Nc RγµNL Bµ + gB1 NER NRγµER Bµ 1 + gB1 NEL Nc RγµEL Bµ 1 + gL1 N∆1R NRγµ∆1R Lµ 1 + gL1 N∆1L Nc Rγµ∆1L Lµ 1 + gL3 N∆3R NRγµ∆3R Lµ† 3 + gL3 N∆3L Nc Rγµ∆3L Lµ† 3 + gW NΣR NRγµΣR Wµ + gW NΣL Nc RγµΣL Wµ + gW1 NΣ1R NRγµΣ1R Wµ 1 + gW1 NΣ1L Nc RγµΣ1L Wµ 1 + gU1 NDR NRγµDR Uµ† 1 + gU1 NDL Nc RγµDL Uµ† 1 + gU2 NUR NRγµUR Uµ† 2 + gU2 NUL Nc RγµUL Uµ† 2 + gQ1 NQ1R NRγµQ1R Qµ† 1 + gQ1 NQ1L Nc RγµQ1L Qµ† 1 + gQ5 NQ5R NRγµQ5R Qµ† 5 + gQ5 NQ5L Nc RγµQ5L Qµ† 5 + gχ NT2R NRγµT2R χµ† + gχ NT2L Nc RγµT2L χµ† + h.c. , (A.5) LNR F F V,(B) = gB NN NRγµNR Bµ + gB1 Ne NRγµeR Bµ 1 + gL1 NL Nc RγµL Lµ 1 + gU1 Nd NRγµdR Uµ† 1 + gU2 Nu NRγµuR Uµ† 2 + gQ1 QN Nc RγµQ Qµ† 1 + h.c. A Lagrangian The Lagrangian describing renormalisable interactions among the SM fields, the singlet NR, and the new BSM fields introduced in tables 1–3, can be expressed as LUV = Llight + Lmixed + Lheavy . The first term includes interactions involving only the SM fields and the NR. The second term describes the interactions between these light fields and the heavy BSM fields, while the last term contains the interactions of the heavy fields among themselves. The first term includes interactions involving only the SM fields and the NR. The second term describes the interactions between these light fields and the heavy BSM fields, while the last term contains the interactions of the heavy fields among themselves. In this appendix we write down all renormalisable terms in which the light singlet NR is involved. Additionally, we provide the Lagrangian terms that include the new vector field U1 and new interactions for the vector L1 not considered in ref. [39]. Finally, we present the interaction terms belonging to Lheavy that contribute to the model diagrams leading to NRSMEFT operators at d = 7. The Lagrangian Llight comprises the renormalisable SM Lagrangian, the mass term for NR,16 and the allowed Yukawa interaction for the singlet. It is expressed as17 Llight = LSM −1 2MMNc RNR −yν NRL H + h.c. (A.1) (A.1) The interactions involving NR in Lmixed can be classified into two categories: fermion- fermion-scalar (FFS) and fermion-fermion-vector (FFV ) interactions. Each category in- cludes two types of terms: A) those with one light field (NR) and two heavy fields, and B) those with two light fields (either one or two NR) and one heavy field. We gather all these interactions in LNR mixed, which is given by LNR mixed = LNR F F S,(A) + LNR F F S,(B) + LNR F F V,(A) + LNR F F V,(B) , (A.2) (A.2) 16Note that we have only written a Majorana mass term for NR. Further discussion regarding this aspect provided in section 3. 17Explicit SU(2) and SU(3) index contractions have been omitted in the Lagrangians of this appendix. A Lagrangian – 24 – – 24 – where JHEP08(2023)166 where LNR F F S,(A) = yS NNR Nc RNR S + yS NNL NRNL S + yS1 NER Nc RER S1 + yS1 NEL NREL S1 + yφ N∆1R Nc R∆1R φ + yφ N∆1L NR∆1L φ + yΞ NΣR Nc RΣR Ξ + yΞ NΣL NRΣL Ξ + yΞ1 NΣ1R Nc RΣ1R Ξ1 + yΞ1 NΣ1L NRΣ1L Ξ1 + yω1 NDR Nc RDR ω† 1 + yω1 NDL NRDL ω† 1 + yω2 NUR Nc RUR ω† 2 + yω2 NUL NRUL ω† 2 + yΠ1 NQ1R Nc RQ1R Π† 1 + yΠ1 NQ1L NRQ1L Π† 1 + yΠ7 NQ7R Nc RQ7R Π† 7 + yΠ7 NQ7L NRQ7L Π† 7 + yζ NT1R Nc RT1R ζ† + yζ NT1L NRT1L ζ† + h.c. , (A.3) LNR F F S,(B) = yS NN Nc RNR S + yS1 Ne Nc ReR S1 + yφ NL NRL φ + yω1 Nd Nc RdR ω† 1 + yω2 Nu Nc RuR ω† 2 + yΠ1 QN QNR Π1 + yN∆1R Nc R∆1R H + yN∆1L NR∆1L H+ h.c. A Lagrangian (A.6) LNR F F S,(A) = yS NNR Nc RNR S + yS NNL NRNL S + yS1 NER Nc RER S1 + yS1 NEL NREL S1 + yφ N∆1R Nc R∆1R φ + yφ N∆1L NR∆1L φ + yΞ NΣR Nc RΣR Ξ + yΞ NΣL NRΣL Ξ + yΞ1 NΣ1R Nc RΣ1R Ξ1 + yΞ1 NΣ1L NRΣ1L Ξ1 + yω1 NDR Nc RDR ω† 1 + yω1 NDL NRDL ω† 1 + yω2 NUR Nc RUR ω† 2 + yω2 NUL NRUL ω† 2 + yΠ1 NQ1R Nc RQ1R Π† 1 + yΠ1 NQ1L NRQ1L Π† 1 + yΠ7 NQ7R Nc RQ7R Π† 7 + yΠ7 NQ7L NRQ7L Π† 7 JHEP08(2023)166 (A.3) (A.4) (A.5) + gχ NT2R NRγµT2R χµ† + gχ NT2L Nc RγµT2L χµ† + h.c. , (A.5) LNR F F V,(B) = gB NN NRγµNR Bµ + gB1 Ne NRγµeR Bµ 1 + gL1 NL Nc RγµL Lµ 1 + gU1 Nd NRγµdR Uµ† 1 + gU2 Nu NRγµuR Uµ† 2 + gQ1 QN Nc RγµQ Qµ† 1 + h.c. (A.6) In the previous Lagrangian, we assumed all new heavy fermions are Dirac spinors, although there are two cases, N and Σ, which could be Majorana fermions. In the case of those fields being Majorana, one can replace one of the Weyl fermions by its charged conjugated In the previous Lagrangian, we assumed all new heavy fermions are Dirac spinors, although there are two cases, N and Σ, which could be Majorana fermions. In the case of those fields being Majorana, one can replace one of the Weyl fermions by its charged conjugated – 25 – counterpart, since ψL ≡ψc R (and ψR ≡ψc L). In fact, in one particular model discussed in section 3, the Majorana nature of N was required to draw one model diagram. Nevertheless, in this appendix we keep the notation of two distinct chiral components for each fermion. A Lagrangian These are Ldiag heavy = yS ∆1 ∆1L∆1R S + yΞ ∆1 ∆1L∆1R Ξ + yΞ1 ∆1L ∆c 1L∆1L Ξ1 + yΞ1 ∆1R ∆c 1R∆1R Ξ1 + h.c. + µSΞ1 SΞ† 1Ξ1 + µSΞ (SΞΞ) + µS (SSS) . (A.9) + yΞ1 ∆1L ∆c 1L∆1L Ξ1 + yΞ1 ∆1R ∆c 1R∆1R Ξ1 + h.c. (A.9) + µSΞ1 SΞ† 1Ξ1 + µSΞ (SΞΞ) + µS (SSS) . (A.9) The complete set of interactions in LUV can be found in the supplementary material added to this paper. A Lagrangian The Lagrangian terms that involve the new vector U1 and contain at least one light field are LU1 mixed = gU1 NDL Nc RγµDL Uµ† 1 + gU1 NDR NRγµDR Uµ† 1 + gU1 Nd NRγµdR Uµ† 1 + gU1 eU ec RγµUL Uµ† 1 + gU1 LQ1 LcγµQ1R Uµ† 1 + gU1 LQ5 LγµQ5L Uµ† 1 + gU1 uE uc RγµEL Uµ† 1 + gU1 Q∆1 Qcγµ∆1R Uµ† 1 + gU1 uD uc RγµDL Uµ 1 + gU1 dU dc RγµUL Uµ 1 + gU1 QQ1 QcγµQ1R Uµ 1 + gU1 dNL dc RγµNL Uµ† 1 + gU1 dNR dRγµNR Uµ 1 + h.c. (A. JHEP08(2023)166 (A.7) For completeness, we have written in the first line the interaction terms containing NR, which were already introduced in LNR F F V .i The renormalisable interactions of the vector L1 with the light fields are given by LL1 mixed = gL1 N∆1R NRγµ∆1R Lµ 1 + gL1 N∆1L Nc Rγµ∆1L Lµ 1 + gL1 NL Nc RγµL Lµ 1 + gL1 e∆1 ∆1RγµeR Lµ 1 + gL1 LNL NLγµL + gL1 LNR N c RγµL Lµ 1 + gL1 e∆3 (eRγµ∆3R) Lµ 1 + gL1 LΣL ΣLγµL Lµ 1 + gL1 LΣR Σc RγµL Lµ 1 + gL1 LE LγµEL Lµ 1 + gL1 LΣ1 LγµΣ1L Lµ 1 + gL1 QT2 T2LγµQ Lµ 1 + gL1 uQ7 Q7RγµuR Lµ 1 + gL1 dQ1 Q1RγµdR Lµ 1 + gL1 QU ULγµQ Lµ 1 + gL1 QD QγµDL Lµ 1 + gL1 QT1 QγµT1L Lµ 1 + gL1 dQ5 dRγµQ5R Lµ 1 + gL1 uQ1 (uRγµQ1R) Lµ 1 + h.c. (A.8 (A.8) Again, we have included in the first line the three interactions with NR, which we already presented in LNR F F V . The remaining terms involve at least one SM field.i Finally, we write down the 3-point interaction terms among heavy BSM fields that are needed in different opening diagrams of the operator ONH4. Acknowledgments We would like to thank José Santiago for help with the tool MatchMakerEFT [67] and Javier Fuentes-Martín for help with Matchete [49]. M.H. and R.B. acknowledge support – 26 – by grants PID2020-113775GB-I00 (AEI/10.13039/501100011033) and CIPROM/2021/054 (Generalitat Valenciana). R.B. also acknowledges financial support from the Generalitat Valenciana (grant ACIF/2021/052). R.C. is supported by the Alexander von Humboldt Foundation Fellowship. Open Access. This article is distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits any use, distribution and reproduction in any medium, provided the original author(s) and source are credited. References JHEP08(2023)166 JHEP08(2023)166 [1] D. 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https://openalex.org/W2086272531	https://research.vu.nl/files/2762586/10.1007%20s12110-010-9098-9.pdf	English	null	How Grandparents Matter	Human nature	2,010	cc-by	8,161	VU Research Portal How grandparents matter. Support for the cooperative breeding hypothesis in a contemporary Dutch population Kaptijn, R.; Thomese, G.C.F.; van Tilburg, T.G.; Liefbroer, A.C. Link to publication in VU Research Portal citation for published version (APA) Kaptijn, R., Thomese, G. C. F., van Tilburg, T. G., & Liefbroer, A. C. (2010). How grandparents matter. Support for the cooperative breeding hypothesis in a contemporary Dutch population. Human Nature : An Interdisciplinary Biosocial Perspective, 21(4), 393-405. https://doi.org/10.1007/s12110-010-9098-9 General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owner and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. 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Oct. 2024 Take down policy Take down policy If b li th t th Take down policy f 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. Take down policy f 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 y this document breaches copyright please contact us providing details, and we will remove access to the work immediate ur claim E-mail address: vuresearchportal.ub@vu.nl E-mail address: vuresearchportal.ub@vu.nl Download date: 24. Oct. 2024 Hum Nat (2010) 21:393–405 DOI 10.1007/s12110-010-9098-9 R. Kaptijn () Faculty of Social Sciences, VU University Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands e-mail: r.w.j.kaptijn@vu.nl F. Thomese: T. G. van Tilburg VU University Amsterdam, Amsterdam, The Netherlands A. C. Liefbroer Netherlands Interdisciplinary Demographic Institute, VU University Amsterdam, Amsterdam, The Netherlands R. Kaptijn () Faculty of Social Sciences, VU University Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands e-mail: r.w.j.kaptijn@vu.nl F. Thomese: T. G. van Tilburg How Grandparents Matter Support for the Cooperative Breeding Hypothesis in a Contemporary Dutch Population Although these policies seem to be moderately effective in raising fertility (Gauthier 2005; Hoem 2008; McDonald 2000), they are limited in their focus on the child’s parents. Evolutionary theory suggests that kin other than the child’s parents are also important in human fertility decisions (Hawkes 2004; Hawkes et al. 1998; Hrdy 2009; Turke 1989). Current low fertility in modern societies affects many social arrangements. In the long term, societies with low fertility face the risk of a shrinking working population. As a consequence, the sustainability of collective pension, social insurance, and care systems is under debate (Grant 2004; McDonald 2006). Low fertility rates reflect women’s difficulties in combining work and motherhood. Fertility tends to be higher in countries where work and motherhood are more compatible (Hoem 2008; McDonald 2000). Policies that aim to increase the compatibility of work and motherhood, such as maternity leave, parental leave, and formal childcare, have become increasingly popular (Gauthier 2005). Although these policies seem to be moderately effective in raising fertility (Gauthier 2005; Hoem 2008; McDonald 2000), they are limited in their focus on the child’s parents. Evolutionary theory suggests that kin other than the child’s parents are also important in human fertility decisions (Hawkes 2004; Hawkes et al. 1998; Hrdy 2009; Turke 1989). Because relatives share common genes by descent, natural selection can favor genes that enable individuals to help their relatives to reproduce successfully (e.g., Hamilton 1964). This type of selection, known as kin selection (Maynard Smith 1964), may have played a crucial role in the evolution of the life history characteristics of the human species. In contrast to those of the other great apes, the life history of contemporary hunter-gatherers is characterized by short birth intervals and a long postmenopausal life phase for women. Human infants are weaned before they can take care of their own feeding. The evolution of such a life history strategy could only be possible when human mothers had reliable sources of help to take care of their children (Robson et al. 2006). The child’s father is one of these sources of help (Gurven and Walker 2006; Marlowe 2001, 2003), but the wider kin group also appears to be an important resource for the mother. How Grandparents Matter Support for the Cooperative Breeding Hypothesis in a Contemporary Dutch Population Ralf Kaptijn & Fleur Thomese & Theo G. van Tilburg & Aart C. Liefbroer Published online: 11 November 2010 # h A h ( ) 2010 hi i l i Published online: 11 November 2010 # The Author(s) 2010. This article is published with open access at Springerlink.com. Published online: 11 November 2010 # The Author(s) 2010. This article is published with open access at Springerlink.com. Abstract Low birth rates in developed societies reflect women’s difficulties in combining work and motherhood. While demographic research has focused on the role of formal childcare in easing this dilemma, evolutionary theory points to the importance of kin. The cooperative breeding hypothesis states that the wider kin group has facilitated women’s reproduction during our evolutionary history. This mechanism has been demonstrated in pre-industrial societies, but there is no direct evidence of beneficial effects of kin’s support on parents’ reproduction in modern societies. Using three-generation longitudinal data anchored in a sample of grandparents aged 55 and over in 1992 in the Netherlands, we show that childcare support from grandparents increases the probability that parents have additional children in the next 8 to 10 years. Grandparental childcare provided to a nephew or niece of childless children did not significantly increase the probability that those children started a family. These results suggest that childcare support by grand- parents can enhance their children’s reproductive success in modern societies and is an important factor in people’s fertility decisions, along with the availability of formal childcare. Keywords Allomothers . Cooperative breeding . Grandparents . Kin selection . Life history theory. Reproductive success F. Thomese: T. G. van Tilburg VU University Amsterdam, Amsterdam, The Netherlands 394 Hum Nat (2010) 21:393–405 Current low fertility in modern societies affects many social arrangements. In the long term, societies with low fertility face the risk of a shrinking working population. As a consequence, the sustainability of collective pension, social insurance, and care systems is under debate (Grant 2004; McDonald 2006). Low fertility rates reflect women’s difficulties in combining work and motherhood. Fertility tends to be higher in countries where work and motherhood are more compatible (Hoem 2008; McDonald 2000). Policies that aim to increase the compatibility of work and motherhood, such as maternity leave, parental leave, and formal childcare, have become increasingly popular (Gauthier 2005). How Grandparents Matter Support for the Cooperative Breeding Hypothesis in a Contemporary Dutch Population Several studies have found that the presence of a grandmother has a positive effect on the survival chances of the child (Hrdy 2005; Sear and Mace 2008), and positive effects of grandfathers and older siblings on the child’s survival have also been reported (Sear and Mace 2008). Although who provides support differs from society to society, and relatives may compete with one another for scarce resources, making successful reproduction more difficult, the pattern of relatives providing care is consistent (Sear 2008; Sear and Mace 2008). The importance of these other caregivers led Hrdy (2005, 2009) to conclude that humans can be characterized as cooperative breeders—a species in which individuals help to care for young that are not their own. According to the cooperative breeding hypothesis, grandmothers are among the most important caregivers besides the parents. When older women in hunter-gatherer groups turn incapable of reproducing themselves, they frequently make valuable contributions to the survival of their grandchildren by providing care, food, or accumulated medical knowledge related to newborn children (Crittenden and Marlowe 2008; Hrdy 2005, 2009). This supportive role of grandmothers for their grandchildren during our evolutionary past may have contributed to the evolution of the long postmenopausal life phase (Hawkes 2003, 2004; Robson et al. 2006). The combination of the long postmenopausal life phase in hunter-gatherers (Blurton Jones et al. 2002; Hawkes 2004; Robson et al. 2006) with the reported positive effects of grandmothers on their grandchildren’s survival (reviewed in Sear and Mace 2008) supports the idea that this long postmenopausal life phase has evolved Hum Nat (2010) 21:393–405 395 395 because long-lived grandmothers could enhance the successful reproduction of their children. Moreover, a simulation model shows that a positive effect of grandmothers on their adult children’s fertility is necessary for menopause to evolve as an adaptive strategy (Shanley and Kirkwood 2001). because long-lived grandmothers could enhance the successful reproduction of their children. Moreover, a simulation model shows that a positive effect of grandmothers on their adult children’s fertility is necessary for menopause to evolve as an adaptive strategy (Shanley and Kirkwood 2001). In this study, we focus not on the evolutionary process that shaped human life history characteristics, but on a possible outcome of this process: the importance of the wider kin group for human fertility decisions under modern conditions. In modern populations, supportive grandparents may make a difference in their children’s reproductive success. How Grandparents Matter Support for the Cooperative Breeding Hypothesis in a Contemporary Dutch Population Grandparents frequently assist their children by taking care of the grandchildren. In the United States, 23% of children under 5 years of age are cared for by their grandparents weekly; in contrast, only 3% of children are cared for by a sibling (Johnson 2005). In Europe, 58% of grandmothers and 49% of grandfathers took care of at least one of their grandchildren in the preceding year (Hank and Buber 2009). By taking care of their grandchildren, grandparents could ease the women’s dilemma of combining paid employment and motherhood. Mothers who work get more childcare support from their parents (Gray 2005; Vandell et al. 2003). Furthermore, a simulation model suggests that assistance from grandparents allows the mother to increase her labor force participation (Cardia and Ng 2003). Childcare support from grandparents may also allow women to have more children. Grandparental childcare support could decrease the burden for women to combine paid employment and motherhood and thus have a positive effect on their fertility. In western Germany, a woman’s chance of experiencing a first birth is higher when her parents live in the same town (Hank and Kreyenfeld 2003). Furthermore, women whose parents are still alive have higher fertility in Italy (Del Boca 2002). Although these results are in accordance with the hypothesis that supportive grandparents can enhance the reproductive success of their children, these results could also be explained by different mechanisms and only form indirect support for this hypothesis. To our knowledge, no studies have focused on the effects of grandparental support on children’s fertility in a modern society. The aim of this study is to explore the effect of childcare support from grandparents on their children’s reproductive success. In line with the cooperative breeding hypothesis, we expect that childcare by the grandparents positively affects their children’s reproductive success. Because previous studies have emphasized the differential impact of different types of kin—grandmothers and maternal kin more frequently make a positive contribution to the survival of the grandchildren than grandfathers and paternal kin (Sear and Mace 2008)—we also explore whether grandparental childcare from these different types of kin has a different effect on the fertility of the children. In addition, we explore whether the effect of grandparental childcare differs with women’s paid employment status. Data The data for this study come from the survey on Living Arrangements and Social Networks of Older Adults (LSN; Knipscheer et al. 1995) and the Longitudinal Aging Study Amsterdam (LASA; Deeg et al. 2002). The LSN survey is a 396 Hum Nat (2010) 21:393–405 representative sample of the Dutch older population. Face-to-face interviews took place in 1992 with respondents aged 54 to 84. LASA is a followup study of the LSN survey. In this paper we use data from the fourth LASA wave, which was collected between 2000 and 2002. representative sample of the Dutch older population. Face-to-face interviews took place in 1992 with respondents aged 54 to 84. LASA is a followup study of the LSN survey. In this paper we use data from the fourth LASA wave, which was collected between 2000 and 2002. The data consist of information on the respondents, their children, and their grandchildren. Information on the children and grandchildren was collected during the interviews with the grandparent. The units of analysis are the children of the respondents. In 1992, a random sample of the grandparents in the LSN survey were asked about the childcare they gave to each individual grandchild. y g g We analyzed two groups of children separately. The first group consists of the children of the respondents who already had children themselves in 1992. For this group we know whether the child received support from the grandparent in the form of childcare for the grandchildren. We examined whether children of this group who received childcare support in 1992 were more likely to have had another child during the next 8 to 10 years than children who did not receive childcare support in 1992. The second group consists of the children of the respondents who did not have children themselves in 1992, but were aunts or uncles at that time. Because the children in this group are childless, this group could not receive support from the grandparent in the form of childcare. However, for this group we do know whether a grandparent took care of the child’s nephew or niece. So for this group we examined whether children whose niece or nephew was looked after by a grandparent (i.e., a grandparent who gave childcare support to the siblings of these children) were more likely to start a family than children whose siblings did not receive childcare support from a grandparent. Data For both groups we expect that grandparental childcare support has a positive effect on the fertility of the children. Of the 826 grandparents who were interviewed about their grandchildren in 1992, 474 could not be reinterviewed in the LASA followup. The main reason was the death of the respondent (73%). Other reasons were refusal of the respondent (15%) and frailty of the respondent (10%). Finally, some of the respondents could not be contacted (2%). The 352 grandparents who were interviewed on both occasions had 1,242 children. Because kin selection theory refers to biological kin, we excluded adoptive children and stepchildren from the analysis. Furthermore we only included children who were younger than 40 in 1992 because after this age the likelihood of starting or adding to a family becomes very small. For the children who had children themselves, an additional inclusion criterion was that the youngest grandchild was younger than 5 in 1992. We only included families with young grandchildren because those with older grandchildren are much more likely to be completed. These inclusion criteria left 616 children eligible for our analysis. Owing to missing values our final sample consists of 572 children; 265 of them already had children themselves in 1992, and 307 were childless at that time. Measures Measures Grandparental Childcare Grandparental Childcare For each individual grandchild under the age of 17 the grandparent was asked: “How often did you take care of . . . in the past 12 months?” Possible answer categories Hum Nat (2010) 21:393–405 397 397 were: “never,” “seldom,” “sometimes,” and “often.” Although this question was answered for each individual grandchild, there was no variance at the grandchild level in our data. Each grandparent took care of all grandchildren from the same child equally often. For the children who already had children themselves, we aggregated this variable to the child level. We analyzed grandparental childcare as a categorical variable with “never” as the reference category. The number of cases in the category “seldom” was quite small. Because this would produce a very wide confidence interval for the effect of this category, we decided to combine the categories of “seldom” and “sometimes” into the category “occasionally.” For the children who were childless in 1992 we aggregated information on caring for grandchildren to the grandparent level. There was not much variance in grandparental childcare on the child level. Most children who were childless either had only one sibling with children (49%) or had a grandparent who divided his or her childcare equally over his or her children (22%). For these cases the aggregate score is the same as the siblings’ scores. For the remaining cases (29%) we aggregated grandparental childcare to the grandparent level by taking the maximum frequency of grandparental childcare. Fertility We do not have data on the exact number of grandchildren born after 1992. In the LASA wave that was collected between 2000 and 2002 only the age of the youngest grandchild for each specific child was asked. Because the age of each individual grandchild in 1992 was known we could, by comparing the ages at both waves, construct a dummy variable which indicates for each specific child if one or more grandchildren were born. For the children who already had children themselves in 1992, this variable indicates a family addition. For the children who were childless in 1992, this variable indicates the start of a family. Control Variables A number of possible confounding factors are taken into account: at the grandparent level, the grandparent’s sex, the number of children that the grandparent ever had, educational attainment of the grandparent in number of years, and the time span between the two interviews in years. At the child level: the child’s sex, paid employment by the child, the travel time for the grandparent to the child (in minutes, log transformed), the age of the child, the age of the youngest grandchild, and the child’s family size. Because almost all men are in paid employment, we combined child’s sex and paid employment status into one categorical variable which contrasts employed women and unemployed women with men. The age of the youngest grandchild and the child’s family size are only applicable to the children who already had children themselves in 1992. Because the age of the child and the age of the youngest grandchild are strongly correlated, we used the standardized residual of the age of the youngest grandchild (after regression on the age of the child) for the children who already had children themselves. Family size was transformed in a dummy variable indicating whether the child already had two or more children in 1992. Descriptive statistics of the final sample are shown in Table 1. On the whole Hum Nat (2010) 21:393–405 398 Table 1 Descriptive statistics there are no major differences between the two groups, except that the childless group also includes younger children and the childless women are more often employed than the women with children. Control Variables Table 1 Descriptive statistics Children who have children themselves Childless children Meana SDb Rangeb Meana SDb Rangeb Grandparent level Grandmother 58% 60% Number of children 3.95 1.97 1–15 4.06 1.63 2–9 Education (years) 9.40 3.34 5–18 9.20 3.11 5–18 Time span T0−T1 (years) 9.61 .56 8.05–10.35 9.63 .56 8.05–10.34 Grandparental care for nephew or niece Never – 26% Occasionally – 43% Often – 31% n grandparents 182 171 Child level Child’s sex and work status Man 52% 55% Unemployed woman 28% 8% Employed woman 20% 37% Travel time (minutes, log) 3.14 1.22 0–7.27 2.59 1.75 0–7.27 Age (years) 32.83 3.35 23–39 29.50 5.10 9–39 Age youngest grandchild (years) 1.95 1.23 0–4 – Residual age youngest grandchild 0 1 −1.82–1.96 – Two or more grandchildren 68% – Grandparental care for grandchildren Never 31% – Occasionally 45% – Often 24% – Family addition 48% – Started family – 50% n children 265 307 a Percentage shown if variable is dichotomous b Not shown if variable is dichotomous there are no major differences between the two groups, except that the childless group also includes younger children and the childless women are more often employed than the women with children. Statistical Analysis Statistical Analysis Our data have a hierarchical structure. The children are nested within the grandparents. The dependent variable, fertility, is dichotomous. We conducted a multilevel logistic regression analysis to test our hypotheses using the Markov Chain 399 399 Hum Nat (2010) 21:393–405 Monte Carlo (MCMC) method to estimate the parameters of the model. MCMC is the preferred estimation method for multilevel logistic models since it generates reliable interval estimates of the parameters of nonlinear multilevel models (Draper 2008; Rasbash et al. 2004). The models have two levels, with children at level one and grandparents at level two. The models have a random intercept, and all dependent variables were entered to the model as fixed effects. The independent variables were centered at their means. To ease the interpretation of our models we calculate the predicted probability to experience a family addition or to start a family using the formula P ¼ 1= 1 þ eZ ð Þ, where Z is the predicted log-odds from the regression equation (Liao 1994). We added interaction terms to the models to determine whether the effect of grandparental childcare differs by type of kin and women’s employment status. In some cases the models with an interaction term did not converge. In those cases we ran a stratified analysis and tested whether the regression coefficients of the independent variable significantly differed using the z-test for the equality of the regression coefficients proposed by Brame et al. (1998). Results Most of the children who had children themselves received childcare support from the grandparent. Also, for a majority of the childless children a grandparent provided care to the child’s nephew or niece. Tables 2 and 3 show the frequency of grandparental childcare by type of kin and daughter’s employment status for the children who already had children themselves and for the childless children, respectively. In both groups of children, there is a tendency for grandmothers to provide more care than grandfathers. Maternal grandparents also tend to provide more care than paternal grandparents, and daughters with paid employment tend to receive more care than daughters without paid employment. The results of the multilevel logistic regression are shown in Table 4. In these models, we test the effect of grandparental childcare on children’s fertility controlling for the variables described in the previous section. For the children who have children themselves, receiving frequent childcare support from the grandparent has a positive effect on the likelihood of a family addition compared with receiving no grandparental childcare support. The predicted probability for adding to the family for a child who never receives grandparental childcare support and with average scores on all control variables is 35%. For a child who often receives grandparental childcare support and has average scores on all control variables, the predicted probability is 66%. The effect of occasional grandparental childcare support compared with never receiving grandparental childcare support is not significant. None of the control variables on the grandparent level has a significant effect on the likelihood of having another child. On the child level, the chance of a family addition decreases with the child’s age. Children who already have two or more children themselves also have a smaller chance of experiencing a family addition. For the childless children, grandparental care for a nephew or niece does not increase their chances to start a family. Results On the grandparent level, none of the control 400 Hum Nat (2010) 21:393–405 Table 2 Cross-tabulation of the frequency of grandparental childcare by the grandparent’s sex, lineage, and the daughter’s paid employment status for children who have children themselves Table 2 Cross-tabulation of the frequency of grandparental childcare by the grandparent’s sex, lineage, and the daughter’s paid employment status for children who have children themselves Grandparental childcare Grandfather Grandmother Paternal grandparent Maternal grandparent Unemployed daughter Employed daughter Never 32 49 51 30 20 10 28% 33% 37% 23% 27% 19% Occasionally 63 57 62 58 37 21 55% 38% 45% 45% 49% 40% Often 20 44 24 40 18 22 17% 29% 18% 31% 24% 42% Total 115 150 137 128 75 53 100% 100% 100% 100% 100% 100% variables has a significant effect on the likelihood of starting a family. On the child level, the likelihood decreases with the child’s age. The variance of the random intercept of the model for the childless children is somewhat smaller than the variance of the random intercept of the model for the children who already have children themselves. This difference might be explained by the larger number of explanatory variables on the grandparent level for the childless children. In the model for the children who already have children themselves, grandparental childcare is an explanatory factor on the child level. In the model for the childless children, grandparental childcare is an explanatory factor on the grandparental level. For both groups of children, we tested whether the effects of grandparental childcare on the children’s fertility differ for grandfathers and grandmothers, paternal grandparents and maternal grandparents, and for daughters with paid employment compared with daughters without paid employment. We found no significant differences in the effect of grandparental childcare by type of kin or by the daughters’ employment status (results not shown). variables has a significant effect on the likelihood of starting a family. On the child level, the likelihood decreases with the child’s age. The variance of the random intercept of the model for the childless children is somewhat smaller than the variance of the random intercept of the model for the children who already have children themselves. This difference might be explained by the larger number of explanatory variables on the grandparent level for the childless children. Results able 3 Cross-tabulation of the frequency of grandparental childcare by the grandparent’s sex, lineage, d the daughter’s paid employment status for childless children Table 3 Cross-tabulation of the frequency of grandparental childcare by the grandparent’s sex, lineage, and the daughter’s paid employment status for childless children Grandparental childcare Grandfather Grandmother Paternal grandparent Maternal grandparent Unemployed daughter Employed daughter Never 33 47 45 35 9 26 29% 24% 26% 26% 38% 23% Occasionally 57 74 78 53 11 42 50% 38% 46% 39% 46% 37% Often 24 72 47 49 4 45 21% 37% 28% 36% 17% 40% Total 114 193 170 137 24 113 100% 100% 100% 100% 100% 100% 401 401 Hum Nat (2010) 21:393–405 Table 4 Logistic regression estimates of a multilevel model explaining the likelihood of adding to a fami for children who have children themselves and the likelihood of starting a family for the childless children Children who have children themselves Childless children Children who have children themselves Children who have children themselves Childless children Grandparent level Grandmother −.131 .241 Number of children .015 .065 Education (years) −.018 −.007 Time span T0−T1 (years) .237 .185 Grandparental care for nephew or niece Occasionally – .591 Often – −.174 Child level Child’s sex and work status Unemployed woman −.264 −.461 Employed woman −.549 .014 Travel time (minutes, log) .048 .140 Age (years) −.272*** −.073* Residual age youngest grandchild −.372 – Two or more grandchildren −2.635*** – Grandparental care for grandchildren Occasionally .434 – Often 1.286* – Intercept −.118 −.002 Variance of intercepta 2.208 .091 n grandparents 182 171 n children 265 307 a Asterisks are not shown because MCMC confidence intervals of variance parameters always exclude 0 (Hox 2002) p<0.05, p<0.01, *p<0.001 a Asterisks are not shown because MCMC confidence intervals of variance parameters always exclude (Hox 2002) p<0.05, p<0.01, *p<0.001 Results In the model for the children who already have children themselves, grandparental childcare is an explanatory factor on the child level. In the model for the childless children, grandparental childcare is an explanatory factor on the grandparental level. For both groups of children, we tested whether the effects of grandparental childcare on the children’s fertility differ for grandfathers and grandmothers, paternal grandparents and maternal grandparents, and for daughters with paid employment compared with daughters without paid employment. We found no significant differences in the effect of grandparental childcare by type of kin or by the daughters’ employment status (results not shown). variables has a significant effect on the likelihood of starting a family. On the child level, the likelihood decreases with the child’s age. The variance of the random intercept of the model for the childless children is somewhat smaller than the variance of the random intercept of the model for the children who already have children themselves. This difference might be explained by the larger number of explanatory variables on the grandparent level for the childless children. In the model for the children who already have children themselves, grandparental childcare is an explanatory factor on the child level. In the model for the childless children, grandparental childcare is an explanatory factor on the grandparental level. For both groups of children, we tested whether the effects of grandparental childcare on the children’s fertility differ for grandfathers and grandmothers, paternal grandparents and maternal grandparents, and for daughters with paid employment compared with daughters without paid employment. We found no significant differences in the effect of grandparental childcare by type of kin or by the daughters’ employment status (results not shown). Discussion Although most demographic research has focused on the role of formal childcare in easing the dilemma between labor force participation and motherhood (Hoem 2008; McDonald 2000), evolutionary theory points to the importance of the wider kin group for human fertility decisions (Hawkes 2004; Hrdy 2005). In this study we sought to explore the effect of grandparental childcare support on the children’s reproductive success. Children who already had children themselves more frequently experienced a family addition in the next 8 to 10 years if they had a helping grandparent who often looked after their grandchildren compared with those who received no grandparental childcare support. Childless children were not more likely 402 Hum Nat (2010) 21:393–405 to start a family if a grandparent looked after their nephew(s) or niece(s). These results are in line with the cooperative breeding hypothesis. Grandparents can enhance the reproductive success of their children by looking after the grand- children, though only the children who actually receive childcare support, and not their siblings, have a higher fertility. For the children who already had children themselves, only children who received frequent grandparental childcare support were more likely to experience a family addition. Occasional grandparental childcare did not have a significant effect, though children who received occasional grandparental childcare support also tended to have a larger chance of experiencing a family addition compared with children who did not receive any grandparental childcare support. Our measure of grandparental childcare was based on reports from the grandparent on a subjective scale. The answers may therefore also reflect the effort grandparents themselves think they put in their (grand)children, rather than the actual support children receive. Further research could focus on the threshold indicating when grandparental childcare is frequent enough to make a difference for the children’s fertility. Grandparental support for a child’s siblings did not have a positive effect on the child’s fertility. Grandparental support provided to a child’s siblings may be uninformative for assessing the assistance a child can expect when he or she starts a family. However, it may also be that opposing mechanisms are operative. On the one hand, grandparental support to a child’s siblings may indicate that the grandparent is willing to look after future grandchildren. Discussion On the other hand, grandparental support to a child’s siblings may also indicate that the grandparent is too occupied with supporting the child’s siblings and will not be available to assist the child by looking after his or her future children. Further research on the topic of how grandparental support is influenced by the family constellation is needed. g p pp y y Research on grandchildren’s survival has shown that the beneficial effect of having a grandparent on the grandchild’s survival is not unconditional. Having a maternal grandparent matters more than having a paternal grandparent, and grandfathers are much less beneficial than grandmothers (Sear and Mace 2008). In our study, descriptive statistics suggested that grandmothers tend to give more childcare than grandfathers, and maternal grandparents tend to give more care than paternal grandparents. These results are in line with several studies which have shown that maternal grandparents invest more than paternal grandparents and grandmothers invest more than grand- fathers (e.g., Crittenden and Marlowe 2008; Euler and Weitzel 1996; Pollet et al. 2006, 2009; Scelza 2009). We also tested whether the childcare of different types of grandparents had differential effects on children’s fertility. We did not find any significant differences, though our sample was quite small. Taken together, these results suggest that the beneficial effect of having a grandparent is mediated by the amount of care that a specific type of grandparent gives. Maternal grandmothers might invest more than paternal grandmothers and grandfathers, but the effect of each unit of investment seems to be equal for all types of grandparents. The distinction between grandfathers and grandmothers may be less relevant in modern societies, however, where men and women have come to share more of care and work activities, in comparison to early modern societies in which gender roles were more clearly separate (Bianchi et al. 2006; Settersten 2007). This could make it more difficult to distinguish each grandparent’s investments when considered as Hum Nat (2010) 21:393–405 403 403 couples, as both grandparents may act together when taking care of their grandchildren. To our knowledge, there have been no such studies on the caregiving activities of grandfathers and grandmothers. Further research could test hypotheses on differences between maternal and paternal grandparents and between grand- mothers and grandfathers in various living arrangements. In this study, we focused on the effect of practical support from the grandparent on children’s fertility. Discussion But in addition to providing practical support, such as childcare, grandparents can also encourage their children to reproduce. Grandparents can communicate that they would welcome grandchildren, and that they would like their children to become parents. Such normative social influence may also have a positive effect on the fertility of the children (Newson et al. 2005, 2007; Newson and Richerson 2009). Although in many cases practical support and normative social influence may come together, more research with proper measures of both types of support is needed to disentangle the effects of practical support and normative social influence. A limitation of our study that needs to be addressed is the attrition in our longitudinal data. We found a strong positive effect of frequent grandparental childcare on children’s fertility for the children who already have children themselves. Although the direction of this effect is not likely to be influenced by attrition, the strength of this effect might be. In our longitudinal dataset, attrition was in large part due to the death or frailty of the grandparent. If the children’s fertility decisions are influenced by the anticipated childcare for the future rather than the existing childcare at the time of the first interview, support from more vigorous grandparents at the time of the first interview probably has a stronger effect than support from less vigorous grandparents. Therefore, if children’s fertility decisions are primarily influenced by anticipated support, the attrition in our sample may have enlarged the effect of grandparental childcare that we found because vigorous grandparents are overrepresented in our sample of survivors. The effect of grandparental childcare support may be affected by the national welfare regime. We found a strong effect of grandparental childcare support on children’s fertility in the Netherlands, and mothers who were in paid employment tended to receive more grandparental childcare than mothers who were not. The Dutch care regime is characterized by a predominantly private responsibility in the case of childcare in combination with large public investments in the care for older adults (Bettio and Plantenga 2004). This combination is likely to make the role of older adults as informal caregivers especially important (Kohli 1999). This suggests the interesting hypothesis that the effect of parental support on children’s fertility behavior will be stronger in countries with low levels of public childcare than in countries with high levels of public childcare. Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. 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Wheelock, J., & Jones, K. (2002). “Grandparents are the next best thing parents in urban Britain. Journal of Social Policy, 31, 441–463. References Ralf Kaptijn is a PhD student of sociology at the VU University Amsterdam. His research is focused on the evolutionary causes and consequences of aging societies. Fleur Thomese is an assistant professor of sociology at the VU University Amsterdam. Her research interests include life course, older adults and life history theory. Theo van Tilburg is a professor in social gerontology at the VU University Amsterdam. His research focuses on social aspects of aging in particular on social integration of older adults in relation to their emotional, physical and cognitive functioning. Aart Liefbroer is head of the Department of Social Demography at the Netherlands Interdisciplinary Demographic Institute and professor of demography of young adults and intergenerational transmission at the Department of Social Research Methodology of the VU University Amsterdam. His research interests include fertility and family formation.
https://openalex.org/W4396593150	https://www.researchsquare.com/article/rs-3216664/latest.pdf	English	null	Antiplatelet Therapy to Prevent Ischemic Events in Giant Cell Arteritis: protocol for a systematic review and meta-analysis	Research Square (Research Square)	2,024	cc-by	4,863	Antiplatelet Therapy to Prevent Ischemic Events in Giant Cell Arteritis: protocol for a systematic review and meta-analysis Jean-Paul Makhzoum Research Article Posted Date: May 2nd, 2024 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/14 Abstract Background: Giant cell arteritis (GCA) is the most common systemic vasculitis in adults. Presenting features include new-onset headaches, constitutional symptoms, jaw claudication, polymyalgia rheumatica, and visual symptoms. Arterial inflammation with subsequent stenosis and occlusion may cause tissue ischemia, leading to blindness, strokes, and myocardial infarction. Oral antiplatelet therapy has been hypothesized to reduce GCA-related ischemic events. However, previous studies have demonstrated conflicting results regarding the efficacy of antiplatelet agents in GCA. The objective of this systematic review is to assess the safety and efficacy of antiplatelet therapy for the prevention of these events in adults with giant cell arteritis. Background: Giant cell arteritis (GCA) is the most common systemic vas features include new-onset headaches, constitutional symptoms, jaw cla rheumatica, and visual symptoms. Arterial inflammation with subsequen cause tissue ischemia, leading to blindness, strokes, and myocardial infa has been hypothesized to reduce GCA-related ischemic events. However demonstrated conflicting results regarding the efficacy of antiplatelet ag this systematic review is to assess the safety and efficacy of antiplatelet these events in adults with giant cell arteritis. Methods: In this systematic review, we will include randomized controlled trials (RTCs), quasi- randomized trials, non-randomized intervention studies, cohort studies, and case-control studies on patients with new-onset or relapsing GCA. The intervention of interest will be pre-existing use or initiation of an oral antiplatelet medication (aspirin, clopidogrel, prasugrel or ticagrelor) at GCA onset or relapse. The comparator of interest will be the absence of antiplatelet therapy. Endpoints will be evaluated after 1 year of follow-up. The primary outcome will be GCA-related ischemic events, including permanent blindness, stroke, myocardial infarction, and ischemic event-related deaths. Adverse events such as major bleeding and death caused by a bleeding event will be assessed. Discussion: GCA-related ischemic events are catastrophic, sudden, often irreversible, and lead to significant morbidity. Antiplatelet agents are affordable, accessible, and could be effective for prevention of these events. Nevertheless, potential benefits of platelet aggregation inhibition must be weighed against their associated risk of bleeding. Assessing the efficacy and safety of antiplatelet therapy in GCA is therefore clinically important. Systematic review registration: Our systematic review protocol was registered with the International Prospective Register of Systematic Reviews (PROSPERO, registration number CRD42023441574 Background Moreover, these agents are affordable and accessible. Nevertheless, potential benefits of platelet aggregation inhibition must be weighed against their associated risk of bleeding.(20) Commercially available and commonly prescribed antiplatelet medications include aspirin, clopidogrel, ticagrelor and prasugrel. Assessing the efficacy of antiplatelet therapy to prevent these events is critically important. Moreover, these agents are affordable and accessible. Nevertheless, potential benefits of platelet aggregation inhibition must be weighed against their associated risk of bleeding.(20) Commercially available and commonly prescribed antiplatelet medications include aspirin, clopidogrel, ticagrelor and prasugrel. Previous small and mostly retrospective studies have demonstrated conflicting results regarding the efficacy of antiplatelet agents to reduce GCA-related permanent vision loss.(1, 11, 17, 18) Previous small and mostly retrospective studies have demonstrated conflicting results regarding the efficacy of antiplatelet agents to reduce GCA-related permanent vision loss.(1, 11, 17, 18) A Cochrane systematic review attempted to assess the safety and effectiveness of low-dose aspirin, as an adjunctive therapy, in the treatment of GCA.(21). However, observational studies were excluded, and only randomized controlled trials (RCTs) directly comparing outcomes of GCA with and without concurrent adjunctive use of low‐dose aspirin were eligible for inclusion, and therefore no studies met the inclusion criteria. Furthermore, the review was conducted before results of major therapeutic clinical trials in GCA were available. The objective of this systematic review and meta-analysis is to assess the safety and efficacy of antiplatelet therapy in the treatment of adults with GCA. For efficacy assessment, we will focus on the incidence of specific GCA-related ischemic events including permanent blindness, stroke, myocardial infarction, and ischemic event related deaths. The safety outcomes will be clinically significant adverse events such as major bleeding events and death caused by a bleeding event.. Background Giant cell arteritis (GCA) is the most common systemic vasculitis in adults.(1) GCA causes inflammation of large arteries and has an incidence of 15–20 cases per 100,000 people in those over 50 years of age. (2) Common features include new-onset headaches, constitutional symptoms, jaw claudication, visual symptoms, and polymyalgia rheumatica.(3, 4, 5, 6) GCA is treated with oral glucocorticoids which are usually tapered over a period of 12 to 18 months.(1) Depending on the clinical phenotype, additional immunosuppressive therapy is sometimes required.(7, 8) Symptomatic relapses are common and occur in 40–80% of patients during glucocorticoid tapering or discontinuation.(9, 10) Page 2/14 Page 2/14 Page 2/14 Complications of GCA can be severe. Arterial inflammation with subsequent stenosis and occlusion may cause GCA-related ischemic events. These include permanent blindness in 15–20% of patients, strokes in 3–8% of patients and myocardial infarction in 2–4% of patients.(11, 12, 13) Moreover, GCA often affects patients over 70 years of age, and older age is recognized as a non-modifiable risk factor for ischemic events.(14) GCA-related ischemic events, which are often irreversible, typically occur within one year following disease onset or relapse,(10) and may happen despite appropriate glucocorticoid or immunosuppressive therapy.(15) Complications of GCA can be severe. Arterial inflammation with subsequent stenosis and occlusion may cause GCA-related ischemic events. These include permanent blindness in 15–20% of patients, strokes in 3–8% of patients and myocardial infarction in 2–4% of patients.(11, 12, 13) Moreover, GCA often affects patients over 70 years of age, and older age is recognized as a non-modifiable risk factor for ischemic events.(14) GCA-related ischemic events, which are often irreversible, typically occur within one year following disease onset or relapse,(10) and may happen despite appropriate glucocorticoid or immunosuppressive therapy.(15) Pathophysiology of ischemic events in GCA is complex and incompletely understood. Arterial inflammation, myointimal thickening and endothelial dysfunction can trigger platelet activation and aggregation.(16) This potentially leads to the narrowing and/or thrombotic occlusion of the inflamed arteries, leading to organ ischemia. Potential benefits of oral adjunctive antiplatelet therapy have been hypothesized to reduce ischemic events since they inhibit platelet aggregation and thrombus formation. (17) Furthermore, antiplatelet agents may have immune-mediated effects by supressing interferon- gamma transcription in arterial tissue resident cells.(18) The addition of antiplatelet therapy to oral glucocorticoids may therefore provide a synergistic effect in the treatment of GCA.(19) Assessing the efficacy of antiplatelet therapy to prevent these events is critically important. Research questions Page 3/14 Page 3/14 The aim of this systematic review is to evaluate the safety and efficacy of antiplatelet therapy, in addition to standard of care, compared to no antiplatelet therapy, in adult patients with new-onset or relapsing GCA. The proposed systematic review will aim at answering the following questions: 1. In adult patients with new-onset or relapsing GCA, does antiplatelet therapy reduce GCA-related ischemic complications (permanent blindness, stroke, myocardial infarction, and ischemic event related deaths)? 1. In adult patients with new-onset or relapsing GCA, does antiplatelet ther 1. In adult patients with new-onset or relapsing GCA, does antiplatelet th 1. In adult patients with new-onset or relapsing GCA, does antiplatelet therapy reduce GCA-related ischemic complications (permanent blindness, stroke, myocardial infarction, and ischemic event related deaths)? 2. In adult patients with new-onset or relapsing GCA, does antiplatelet therapy increase the risk of major bleeding events? 2. In adult patients with new-onset or relapsing GCA, does antiplatelet therapy increase the risk of major bleeding events? Eligibility criteria major bleeding events? Eligibility criteria Eligibility criteria for studies based on patient population, intervention or exposure, comparator and methods are presented in Table 1. We will include studies irrespective of the reported outcomes. We will include randomized controlled trials (RTCs), quasi-randomized trials and non-randomized intervention studies. For observational studies, we will include cohort studies and case-control studies. We will exclude cross-sectional, case series and case reports. Page 4/14 Table 1 – Eligibility Criteria for Studies Population Inclusion criteria • Adult patients (≥ 18 years), with new-onset or relapsing GCA based on one of the official ACR classification criteria (version 1990 or version 2022). * Exclusion criteria • Patients with systemic vasculitides other than GCA. Intervention/ Exposure Inclusion criteria • Administration of an oral antiplatelet medication in addition to GCA standard of care. • Accepted antiplatelet medications: aspirin (≥ 80mg daily), clopidogrel (75mg daily), ticagrelor (90mg twice a day) or prasugrel (10mg daily). • Accepted timing of antiplatelet initiation: o Within 8 weeks of GCA onset or relapse. o Already administered at the time of GCA onset or relapse. Exclusion criteria • Antiplatelet medication initiated in a patient with inactive GCA. • Use of oral, intravenous, or subcutaneous anticoagulants. Comparator Inclusion criteria • Absence of adjunctive antiplatelet therapy. Exclusion criteria • Use of oral, intravenous, or subcutaneous anticoagulants. Method/ Design Inclusion criteria • RTCs, quasi-randomized trials, non-randomized intervention studies, cohort studies and case-control studies. • Study duration of at least 6 months. Exclusion criteria • Cross-sectional, case series and case reports. GCA: giant cell arteritis; ACR: American College of Rheumatology; RCT: randomised controlled trial. * New-onset GCA is defined as a diagnosis of active GCA in a participant with no previous history of GCA. A relapsing GCA is defined as a diagnosis of active GCA in a patient with a history of GCA in remission (asymptomatic). Table 1 – Eligibility Criteria for Studies Table 1 – Eligibility Criteria for Studies – Eligibility Criteria for Studies Population Inclusion criteria • Adult patients (≥ 18 years), with new-onset or relapsing GCA based on one of the official ACR classification criteria (version 1990 or version 2022). * Exclusion criteria • Patients with systemic vasculitides other than GCA. Intervention/ Exposure Inclusion criteria • Administration of an oral antiplatelet medication in addition to GCA standard of care. • Accepted antiplatelet medications: aspirin (≥ 80mg daily), clopidogrel (75mg daily), ticagrelor (90mg twice a day) or prasugrel (10mg daily). Outcomes The primary composite efficacy outcome is the incidence (proportion) of GCA-related ischemic events occurring during the follow-up period, which include ischemic strokes, permanent blindness, myocardial infarction, or ischemic event-related deaths. The definition of ischemic events is based on the official American Heart Association (AHA) and American Stroke association (ASA) consensus. Ischemic stroke is defined as an episode of neurological dysfunction caused by focal cerebral, spinal, or retinal infarction. Permanent blindness is a permanent loss of sight, whether it be a full or partial loss. Myocardial infarction is defined as an episode of myocardial injury (elevated cardiac troponin values at least above the 99th percentile upper reference limit) with clinical evidence of at least one acute myocardial ischemia manifestation (symptoms of cardiac ischemia, new ischemic ECG changes, new pathological Q waves, compatible cardiac imaging, or coronary thrombus on angiography). The main secondary efficacy outcome is the incidence (proportion) of ischemic strokes, permanent blindness, myocardial infarction, and death, measured separately and occurring during the follow-up period. The main safety outcome is the incidence (proportion) of major bleeding events. A major bleeding event is defined based on the classification of the International Society of Thrombosis and Hemostasis as either: fatal bleeding, and/or symptomatic bleeding in a critical area or organ, such as intracranial, intraspinal, intraocular, retroperitoneal, intra-articular or pericardial, or intramuscular with compartment syndrome, and/or bleeding causing a fall in hemoglobin levels of 20 g/L or more, and/or leading to a transfusion. The main secondary safety outcome is the incidence (proportion) of death due to a bleeding event. Information sources Eligibility criteria • Accepted timing of antiplatelet initiation: o Within 8 weeks of GCA onset or relapse. o Already administered at the time of GCA onset or relapse. Exclusion criteria • Antiplatelet medication initiated in a patient with inactive GCA. • Use of oral, intravenous, or subcutaneous anticoagulants. Comparator Inclusion criteria • Absence of adjunctive antiplatelet therapy. Exclusion criteria • Use of oral, intravenous, or subcutaneous anticoagulants. Method/ Design Inclusion criteria • RTCs, quasi-randomized trials, non-randomized intervention studies, cohort studies and case-control studies. • Study duration of at least 6 months. Exclusion criteria • Cross-sectional, case series and case reports. GCA: giant cell arteritis; ACR: American College of Rheumatology; RCT: randomised controlled trial. * New-onset GCA is defined as a diagnosis of active GCA in a participant with no previous history of GCA. A relapsing GCA is defined as a diagnosis of active GCA in a patient with a history of GCA in remission (asymptomatic). Page 5/14 Study duration of at least 6 months will be required for study inclusion. End points will be evaluated at 6 and 12 months. Study duration of at least 6 months will be required for study inclusion. End points will be evaluated at 6 and 12 months. We will include trials irrespective of the language of publication or format in which they were reported. If we identify studies with unpublished data, they will be considered for inclusion. There will be no restriction by type of setting (academic hospital, community hospital, inpatient or outpatient). Information sources Published studies and unpublished gray literature will be searched. We will search the following electronic bibliographic databases: MEDLINE (Ovid interface, January 1946 – onwards), Cochrane Central Register of Controlled Trials in the Cochrane Library (CENTRAL) and EMBASE (Ovid interface, January 1947- onwards). The following study registries will be searched: metaRegister of controlled trials (mRCT) and ClinicalTrials.gov. Online conference papers, abstracts, and presentations from the American College of rheumatology annual meeting (ACR, from 2012 – onwards) and European League against Rheumatism annual meeting (EULAR, from 2001 – onwards) will be searched. Page 6/14 Page 6/14 References will be screened to make a list of experts in the field. A citation index search of these experts will be performed on the ‘Web of Science’ platform and ‘researchgate.net’. Finally, a manual bibliography search of retrieved records will be performed to find additional references. We will search for retraction or errata statements that were published for every study we include. Search strategy Literature search strategies was developed using medical subject headings (MeSH) and text words related to GCA and antiplatelet therapy. There will be no language restriction for the search. A publication date filter from August 1st, 1990 - onwards will be used because official classification criteria for GCA were released in August 1990. This will allow correct identification of the population of interest. The search strategy was elaborated by the corresponding author and was peer-reviewed by all authors. A transcript of the MEDLINE search strategy is provided (Fig. 1). Search strategies for other information sources are provided in the data supplement (figure S1 to S5). We will provide the actual date when each search was performed during the review stage. Study records management, selection, and collection Covidence (covidence.org) will be used to upload literature search results, manage study records, select studies and for data extraction. Two co-authors (OT, YB) will work independently for the initial screening (title and abstract), selection of studies and data extraction. The two reviewers will have a practice-run with 10 records as a calibration exercise before beginning the official review process. Duplicate records will be counted and removed using Covidence. Multiple reports of the same study will be identified by juxtaposing author names, location, setting and date of the study. Multiple reports of the same study will not be discarded; they will be collated under one study identification. However, one record will be selected as the source (main report) of the study with a justification provided. Any disagreement between the two reviewers will be resolved through discussion. A third reviewer (CR) will be consulted if the disagreement persists. If required, additional information from the study authors will be requested to resolve remaining questions on eligibility. Data items Extracted data will include identification information (study id, record id), reason for excluding studies, characteristics of the study (author, year, country, design, duration, funding, conflict of interest), characteristics of participants (age, sex, ethnicity, disease subtype, baseline characteristics), details of the intervention (timing of initiation, type of antiplatelet medication, dose, other GCA therapy and immunosuppression), details of the comparator, outcomes (definitions, method of aggregation, measures of association, timing), and results (number of participants, exclusion, losses at follow-up, summary results, subgroup results). Furthermore, key conclusions, comments and references will be collected. Missing data will be recorded as such. Study authors will be contacted to retrieve missing data or resolve any uncertainties. Communications with study authors will be performed by email. A maximum of 3 attempts to reach the authors will be made in case of no response, with each attempt every 14 days. Risk of bias of individual studies For randomized studies, we will use version 2 of the Cochrane Collaboration tool for assessing the risk of bias (RoB 2).(22) For non-randomized intervention studies, the Risk Of Bias In Non-randomized Studies - of Interventions (ROBINS-I) tool will be used.(23) For observational studies, the Risk Of Bias In Non- randomized Studies - of Exposure (ROBINS-E) tool will be used.(24) The risk of bias will be assessed independently by two reviewers (OT, YB). Disagreements will be resolved by discussion, and if required, with arbitration from a third reviewer (JPM). Using the software RevMan web, we will summarise our findings and present them in a risk of bias table and figure. h Data synthesis If included studies are sufficiently homogeneous in terms of participants (similar proportions of new- onset or relapsing GCA), interventions (antiplatelet type), comparator, and study design (duration of follow-up), we will perform meta-analyses using a random-effects model. Our data will be binary: proportions of GCA-related ischemic complications, major bleeding event and deaths. We will use a Mantel-Haenszel method for quantitative synthesis, with odds ratio (OR) as a measure of association with 95% confidence intervals. A sensitivity analysis will be performed to evaluate the impact of studies with significant missing data (when there is ≥ 10% missing data for any outcome). Statistical heterogeneity will be tested using Chi2 test (significance level of 0.1) and I2 statistic. If there is a high level of statistical heterogeneity (p < 0.1 or I2 > 50%), we will analyze clinical heterogeneity by documenting the variability in participants, interventions, and outcomes in the included trials. We will also carefully analyze and compare study designs and settings to assess methodological heterogeneity. Furthermore, we will perform the following subgroup analyses to better understand the source of heterogeneity: 1) subgroup based on disease subtype (new onset vs. relapsing), 2) based on the timing of antiplatelet medication (prior to GCA vs. at GCA onset or relapse), 3) based on GCA therapy received (glucocorticoids alone vs. glucocorticoids with immunosuppression). We will also perform the following sensitivity analyses: 1) exclusion of non-randomized studies, 2) exclusion of studies with a high risk of bias. If quantitative analysis is not appropriate, a systematic narrative synthesis will be provided. Page 8/14 Confidence in cumulative evidence Two reviewers (JPM, CR) will independently evaluate the quality of evidence for all outcomes using the Grading of Recommendations, Assessment, Development and Evaluation working group methodology (GRADE).(26) Conclusions on GRADE for each outcome will be displayed in a ‘summary of findings’ table. An overall GRADE will be given to the body of all outcomes. Meta-biases assessment Publication bias will be evaluated with a funnel plot if at least 10 studies are included in the meta- analysis. We will use Egger’s test to assess potential publication bias via funnel plot asymmetry. For each included study, outcome reporting bias will be investigated by comparing reported outcomes against planned outcome measures “a priori” in the study protocol or trial registry. The risk of bias due to selective outcome reporting will be considered low if 1) the study protocol was published before the availability of study results, and 2) every “a priori” outcome in the protocol is reported in the study record (or if justification was provided for not reporting an outcome). If no study protocol is found, we will use the Outcome Reporting Bias in Trials (ORBIT) classification system to evaluate the risk of selective outcome reporting.(25) A sensitivity analysis to assess the impact of selective outcome reporting will be conducted if at least one study presents a high risk of bias due to selective outcome reporting. Discussion This systematic review will synthesize the available literature on the efficacy of antiplatelet therapy in GCA, with emphasis on a range of clinically important ischemic complications, including permanent visual loss, myocardial infarction, and strokes. These ischemic events are infrequently described in individual studies, with insufficient power to draw conclusions. The findings of this review are anticipated to assist advocacy groups and task-teams to develop practice guidelines, which will optimize the management in GCA and hopefully assist in preventing ischemic complications. This systematic review protocol has several strengths. This review will be conducted using an established methodology, with an advanced search strategy that includes grey literature. The included individual studies will be assessed thoroughly based on each risk of bias domain using the correct tool for the study type. By including non-randomized intervention studies and observational studies, we will increase the likelihood of finding eligible studies, as opposed to previous attempts to study this important research question. Nevertheless, we anticipate some difficulties in conducting this review. Ischemic and bleeding events in GCA studies are reported with variable consistency. The review team Page 9/14 will mitigate this by making a significant effort towards contacting study authors as necessary to obtain the required outcome information. will mitigate this by making a significant effort towards contacting study authors as necessary to obtain the required outcome information. The results of this systematic review will be reported in a peer-reviewed journal. Any amendments made to this protocol during the review will be reported in PROSPERO and in the final manuscript. Abbreviations ACR: American College of Rheumatology AHA: American Heart Association ASA: American Stroke Association CENTRAL: Cochrane Central Register of Controlled Trials. ECG: Electrocardiogram EULAR: European League Against Rheumatism GCA: Giant cell arteritis GRADE: Grading of Recommendations, Assessment, Development, and Evaluations MeSH: Medical Subject Headings mRCT: metaRegister of Controlled Trials OR: Odds ratio ORBIT: Outcome Reporting Bias in Trials PROSPERO: International Prospective Register of Systematic Reviews RCT: Randomised controlled trial RoB: Risk of bias ROBINS-I: Risk of Bias in Non-randomised Studies – of Interventions ROBINS-E: Risk of Bias in Non-randomised Studies – of Exposure Declarations Ethics approval and consent to participate. Abbreviations Abbreviations ACR: American College of Rheumatology AHA: American Heart Association ASA: American Stroke Association CENTRAL: Cochrane Central Register of Controlled Trials. ECG: Electrocardiogram EULAR: European League Against Rheumatism GCA: Giant cell arteritis GRADE: Grading of Recommendations, Assessment, Development, and Evaluations MeSH: Medical Subject Headings mRCT: metaRegister of Controlled Trials OR: Odds ratio ORBIT: Outcome Reporting Bias in Trials PROSPERO: International Prospective Register of Systematic Reviews RCT: Randomised controlled trial RoB: Risk of bias ROBINS-I: Risk of Bias in Non-randomised Studies – of Interventions ROBINS-E: Risk of Bias in Non-randomised Studies – of Exposure Declarations Ethics approval and consent to participate. ACR: American College of Rheumatology Declarations Ethics approval and consent to participate. Acknowledgements Mrs. Guylaine Marcotte (research coordinator), Mrs. Audrey Martineau (research assistant) and Dr Alexandra Mereniuk (collaborator). Mrs. Guylaine Marcotte (research coordinator), Mrs. Audrey Martineau (research assistant) and Dr Alexandra Mereniuk (collaborator). Authors’ contributions JPM and CR drafted the protocol and manuscript. All authors contributed to the development of the selection criteria, the risk of bias assessment strategy and data extraction criteria. JPM developed the search strategy. CR, JPM, CP and AM provided expertise on giant cell arteritis. All authors read, provided feedback, and approved the final manuscript. JPM is the guarantor. Competing interests The authors declare that they have no competing interests. No funding was required or obtained. No funding was required or obtained. Ethics approval and consent to participate. Page 10/14 This study does not contain any research conducted on human participants or animals that require institutional ethics review approval. Consent for publication Not applicable. Availability of data and materials Not applicable. References 1. Maz M, Chung SA, Abril A, Langford CA, Gorelik M, Guyatt G, et al. 2021 American College of Rheumatology/Vasculitis Foundation Guideline for the Management of Giant Cell Arteritis and Takayasu Arteritis. Arthritis Rheumatol. 2021;73(8):1349-65. 2. Andersen JB, Myklebust G, Haugeberg G, Pripp AH, Diamantopoulos AP. Incidence Trends and Mortality of Giant Cell Arteritis in Southern Norway. Arthritis Care Res (Hoboken). 2021;73(3):409- 14. 3. Dejaco C, Brouwer E, Mason JC, Buttgereit F, Matteson EL, Dasgupta B. Giant cell arteritis and polymyalgia rheumatica: current challenges and opportunities. Nat Rev Rheumatol. 2017;13(10):578-92. Page 11/14 4. Zarka F, Rheaume M, Belhocine M, Goulet M, Febrer G, Mansour AM, et al. Colour Doppler ultrasound and the giant cell arteritis probability score for the diagnosis of giant cell arteritis: a Canadian single- centre experience. Rheumatol Adv Pract. 2021;5(3):rkab083. 5. Hunder GG, Bloch DA, Michel BA, Stevens MB, Arend WP, Calabrese LH, et al. The American College of Rheumatology 1990 criteria for the classification of giant cell arteritis. Arthritis Rheum. 1990;33(8):1122-8. 6. Tomelleri A, Padoan R, Kavadichanda CG, Jose A, Singh K, Iorio L, et al. Validation of the 2022 American College of Rheumatology/EULAR classification criteria for Takayasu arteritis. Rheumatology (Oxford). 2023. 7. Szekeres D, Al Othman B. Current developments in the diagnosis and treatment of giant cell arteritis. Front Med (Lausanne). 2022;9:1066503. 8. Videman J, Skacelova M, Horak P, Skoumalova A, Hrabos D. Treatment of giant cell arteritis - current approach and new possibilities. Vnitr Lek. 2022;68(5):266-72. 9. Gomes de Pinho Q, Daumas A, Benyamine A, Bertolino J, Ebbo M, Schleinitz N, et al. Predictors of Relapses or Recurrences in Patients With Giant Cell Arteritis: A Medical Records Review Study. J Clin Rheumatol. 2023. 10. Kermani TA, Warrington KJ, Cuthbertson D, Carette S, Hoffman GS, Khalidi NA, et al. Disease Relapses among Patients with Giant Cell Arteritis: A Prospective, Longitudinal Cohort Study. J Rheumatol. 2015;42(7):1213-7. 11. Baalbaki H, Jalaledin D, Lachance C, Febrer G, Rheaume M, Makhzoum JP. Characterization of visual manifestations and identification of risk factors for permanent vision loss in patients with giant cell arteritis. Clin Rheumatol. 2021;40(8):3207-17. 12. Samson M, Jacquin A, Audia S, Daubail B, Devilliers H, Petrella T, et al. Stroke associated with giant cell arteritis: a population-based study. J Neurol Neurosurg Psychiatry. 2015;86(2):216-21. 13. Udayakumar PD, Chandran AK, Crowson CS, Warrington KJ, Matteson EL. Cardiovascular risk and acute coronary syndrome in giant cell arteritis: a population-based retrospective cohort study. References [GRADE: Evidence to Decision (EtD) frameworks - a systematic and transparent approach to making well informed healthcare choices. 2: Clinical guidelines]. Z Evid Fortbild Qual Gesundhwes. 2019;140:63-73. 26. Conrad S, Kaiser L, Kallenbach M, Meerpohl J, Morche J. [GRADE: Evidence to Decision (EtD) frameworks - a systematic and transparent approach to making well informed healthcare choices. 2: Clinical guidelines]. Z Evid Fortbild Qual Gesundhwes. 2019;140:63-73. References Arthritis Care Res (Hoboken). 2015;67(3):396-402. 14. Yousufuddin M, Young N. Aging and ischemic stroke. Aging (Albany NY). 2019;11(9):2542-4. 15. Coronel L, Rodriguez-Pardo J, Monjo I, de Miguel E. Prevalence and significance of ischemic cerebrovascular events in giant cell arteritis. Med Clin (Barc). 2021;157(2):53-7. 16. Martinez-Taboada VM, Lopez-Hoyos M, Narvaez J, Munoz-Cacho P. Effect of antiplatelet/anticoagulant therapy on severe ischemic complications in patients with giant cell arteritis: a cumulative meta-analysis. Autoimmun Rev. 2014;13(8):788-94. 17. Narvaez J, Bernad B, Gomez-Vaquero C, Garcia-Gomez C, Roig-Vilaseca D, Juanola X, et al. Impact of antiplatelet therapy in the development of severe ischemic complications and in the outcome of patients with giant cell arteritis. Clin Exp Rheumatol. 2008;26(3 Suppl 49):S57-62. 18. Weyand CM, Kaiser M, Yang H, Younge B, Goronzy JJ. Therapeutic effects of acetylsalicylic acid in giant cell arteritis. Arthritis Rheum. 2002;46(2):457-66. 18. Weyand CM, Kaiser M, Yang H, Younge B, Goronzy JJ. Therapeutic effects of acetylsalicylic acid in giant cell arteritis. Arthritis Rheum. 2002;46(2):457-66. Page 12/14 Page 12/14 19. Lee MS, Smith SD, Galor A, Hoffman GS. Antiplatelet and anticoagulant therapy in patients with giant cell arteritis. Arthritis Rheum. 2006;54(10):3306-9. 20. Bouget J, Balusson F, Viglino D, Roy PM, Lacut K, Pavageau L, et al. Major bleeding risk and mortality associated with antiplatelet drugs in real-world clinical practice. A prospective cohort study. PLoS ONE. 2020;15(8):e0237022. 21. Mollan SP, Sharrack N, Burdon MA, Denniston AK. Aspirin as adjunctive treatment for giant cell arteritis. Cochrane Database Syst Rev. 2014(8):CD010453. 22. Sterne JAC, Savovic J, Page MJ, Elbers RG, Blencowe NS, Boutron I, et al. RoB 2: a revised tool for assessing risk of bias in randomised trials. Bmj. 2019;366:l4898. 23. Sterne JA, Hernan MA, Reeves BC, Savovic J, Berkman ND, Viswanathan M, et al. ROBINS-I: a tool for assessing risk of bias in non-randomised studies of interventions. Bmj. 2016;355:i4919. 24. Bero L, Chartres N, Diong J, Fabbri A, Ghersi D, Lam J, et al. The risk of bias in observational studies of exposures (ROBINS-E) tool: concerns arising from application to observational studies of exposures. Syst Rev. 2018;7(1):242. 25. Kirkham JJ, Dwan KM, Altman DG, Gamble C, Dodd S, Smyth R, et al. The impact of outcome reporting bias in randomised controlled trials on a cohort of systematic reviews. Bmj. 2010;340:c365. 26. Conrad S, Kaiser L, Kallenbach M, Meerpohl J, Morche J. Figure 1 MEDLINE (OVID) search strategy Figures Page 13/14 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. DatasuppGCAPLT.docx DatasuppGCAPLT.docx PRISMAPchecklistGCAPLT.docx PRISMAPchecklistGCAPLT.docx PRISMAPchecklistGCAPLT.docx Page 14/14
https://openalex.org/W3097348585	https://www.nogr.org/jour/article/download/1455/1389	Russian	null	Cardiovascular comorbidity in non-alcoholic fatty liver disease	Èksperimentalʹnaâ i kliničeskaâ gastroènterologiâ	2,020	cc-by	7,398	 Corresponding author: Тимакова Арина Юрьевна, студент 4 курса лечебного факультета имакова Арина Юрьевна, студент 4 курса лечебного факультета Скирденко Юлия Петровна, ассистент кафедры факультетской терапии, профессиональных болезней, кандидат медицинский наук Скирденко Юлия Петровна, ассистент кафедры факультетской терапии, профессиональных болезней, кандидат медицинский наук Скирденко Юлия Петровна Yulia P. Skirdenko julija-loseva1@yandex.ru Ливзан Мария Анатольевна, ректор, заведующий кафедрой факультетской терапии, профессиональных болезней, профессор, д. м. н. Ливзан Мария Анатольевна, ректор, заведующий кафедрой факультетской терапии, профессиональных болезней, профессор, д. м. н. Кролевец Татьяна Сергеевна, ассистент кафедры факультетской терапии, профессиональных болезней, кандидат медицинский наук Кролевец Татьяна Сергеевна, ассистент кафедры факультетской терапии, профессиональных болезней, кандидат медицинский наук Николаев Николай Анатольевич, профессор кафедры факультетской терапии, профессиональных болезней, доцент, д. м. н. Нелидова Анастасия Владимировна, доцент кафедры пропедевтики внутренних болезней, доцент, кандидат медицинский наук , р ф р ф др ф у р , р ф , д ц , д Нелидова Анастасия Владимировна, доцент кафедры пропедевтики внутренних болезней, доцент, кандидат медицинский наук Arina Yu. Timakova, medical faculty, student Yulia P. Skirdenko, assistant of the department of faculty therapy, occupational disease, PhD Maria A. Livzan, D. Sci. (Med.), Professor, Rector, Head of the Department of Faculty Therapy, Occupational Diseases; ORCID: https://orcid.org/0000–0002–6581–7017, Scopus Author ID: 24341682600 Tatiana S. Krolevec, assistant of the department of faculty therapy, occupational disease, PhD Nikolay A. Nikolaev, professor of the department of faculty therapy, occupational disease, associate professor, MD Anastasia V. Nelidova, associate professor of the department of propaedeutics of internal diseases, associate professor, PhD Yulia P. Skirdenko, assistant of the department of faculty therapy, occupational disease, PhD ORCID: https://orcid.org/0000–0002–6581–7017, Scopus Author ID: 24341682600 Tatiana S. Krolevec, assistant of the department of faculty therapy, occupational disease, PhD Nikolay A. Nikolaev, professor of the department of faculty therapy, occupational disease, associate professor, MD Anastasia V Nelidova associate professor of the department of propaedeutics of internal diseases associate professor PhD обзор review обзор review DOI: 10.31146/1682-8658-ecg-182-10-88-95 Cardiovascular comorbidity in non-alcoholic fatty liver disease A. Yu. Timakova, Yu. P. Skirdenko, M. A. Livzan, T. S. Krolevets, N. A. Nikolaev, A. V. Nelidova eral State Funded Educational Institution for Higher Education Omsk State Medical University Ministry of Public Health, Russian Federat et, 12, Russia Для цитирования: Тимакова А. Ю., Скирденко Ю. П., Ливзан М. А., Кролевец Т. С., Николаев Н. А., Нелидова А. В. Кардиоваскулярная коморбидность при неал- когольной жировой болезни печени. Экспериментальная и клиническая гастроэнтерология. 2020;182(10): 88–95. DOI: 10.31146/1682-8658-ecg-182-10-88-95 For citation: Timakova A. Yu., Skirdenko Yu. P., Livzan M. A., Krolevets T. S., Nikolaev N. A., Nelidova A. V. Cardiovascular comorbidity in non-alcoholic fatty liver disease. Experimental and Clinical Gastroenterology. 2020;182(10): 88–95. (In Russ.) DOI: 10.31146/1682-8658-ecg-182-10-88-95 a A. Yu., Skirdenko Yu. P., Livzan M. A., Krolevets T. S., Nikolaev N. A., Nelidova A. V. Cardiovascular comorbidity in non-alcoholic fatty liv and Clinical Gastroenterology. 2020;182(10): 88–95. (In Russ.) DOI: 10.31146/1682-8658-ecg-182-10-88-95 For citation: Timakova A. Yu., Skirdenko Yu. P., Livzan M. A., Krolevets T. S., Nikolaev N. A., Nelidova A. V. Cardiovascular comorbidity in disease. Experimental and Clinical Gastroenterology. 2020;182(10): 88–95. (In Russ.) DOI: 10.31146/1682-8658-ecg-182-10-88-95  Corresponding author: Скирденко Юлия Петровна Yulia P. Skirdenko julija-loseva1@yandex.ru Резюме Актуальной проблемой современной медицины является поиск связей между неалкогольной жировой болезнью печени и сердечно-сосудистыми заболеваниями. Реальная клиническая практика нуждается в углублении знаний о взаимном влиянии этих патологий и разработке алгоритмов сопровождения таких коморбидных пациентов. В обзоре актуальной литературы показаны данные об увеличении кардиоваскулярных рисков и ускорении про- грессирования патологии печени при сочетании неалкогольной жировой болезни печени и сердечно-сосудистых заболеваний. В статье описаны звенья патогенеза неалкогольной жировой болезни печени, оказывающие влияние на развитие сердечно-сосудистых заболеваний (инсулинорезистентность, гипоадипонектинемия, гиперлептинемия, нарушения липидного обмена, активация воспаления и др.). Продемонстрирована роль воспаления, гормональных изменений и влияние кишечной микробиоты на течение обоих заболеваний. В обзоре представлены данные о влия- нии общих факторов риска (нарушение липидного обмена, нарушение углеводного обмена, ожирение и др.) на тече- ние обеих патологий. Приведены данные литературы о связи неалкогольной жировой болезни печени с развитием артериальной гипертензии (прежде всего за счет увеличения жесткости сосудистой стенки), ишемической болезнью 88 Кардиоваскулярная коморбидность при неалкогольной жировой болезни печени \| Cardiovascular comorbidity in non-alcoholic fatty liver dise сердца (прежде всего за счет эндотелиальной дисфункции и гиперлипидемии), нарушениями ритма сердца, сердеч ной недостаточностью, цереброваскулярными болезнями. Показано, что связь между неалкогольной жировой болезнью печени и сердечно-сосудистыми заболеваниями является двусторонней и кардиоваскулярные заболевания способны усугублять течение заболеваний печени. является двусторонней и кардиоваскулярные заболевания способны усугублять течение заболеваний печени. Важность дальнейшего изучения данного вопроса обусловлена взаимовлиянием неалкогольной жировой болез- ни печени и сердечно-сосудистых заболеваний не только на прогноз, но и на тактику ведения пациента. Важность дальнейшего изучения данного вопроса обусловлена взаимовлиянием неалкогольной жировой болез- ни печени и сердечно-сосудистых заболеваний не только на прогноз, но и на тактику ведения пациента. Ключевые слова: неалкогольная жировая болезнь печени, сердечно-сосудистые заболевания, артериальная гипер- тензия, сердечная недостаточность, нарушения ритма, ишемическая болезнь сердца Summary The actual problem of modern medicine is the search for links between non-alcoholic fatty liver disease and cardiovascu- lar diseases. The real clinical practice needs to deepen knowledge about the mutual inﬂ uence of these two diseases and develop algorithms to accompany these patients. A review of current literature shows data on increasing cardiovascular risks and accelerating progression of liver pathol- ogy in the combination of non-alcoholic fatty liver disease and cardiovascular disease. The article describes the links of pathogenesis of non-alcoholic fatty liver disease aﬀ ecting the development of cardiovascular diseases (insulin resistance, hypoadiponectinemia, hyperleptinemia, lipid metabolism disorders, activation of inﬂ ammation, etc.). The role of inﬂ amma- tion, hormonal changes and the inﬂ uence of intestinal microbiota on the course of both diseases has been demonstrated. The review presents data on the inﬂ uence of common risk factors (lipid metabolism disorder, carbohydrate metabolism disorder, obesity, etc.) on the course of both pathologies. The review presents the literature data on the connection of non-alcoholic fatty liver disease with the development of arterial hypertension (primarily due to an increase in vascular wall stiﬀ ness), coronary heart disease (primarily due to endothelial dysfunction and hyperlipidemia), heart rhythm disorders, heart failure, cerebrovascular diseases. It has been shown that the connection between non-alcoholic fatty liver disease and cardiovascular diseases is bilateral and cardiovascular diseases can aggravate the course of liver diseases. The importance of further study of this issue is due to the mutual inﬂ uence of non-alcoholic fatty liver disease and cardio- vascular disease not only on the prognosis, but also on the tactics of patient management. Keywords: non-alcoholic fatty liver disease, cardiovascular disease, arterial hypertension, heart failure, rhythm disorders, coronary heart disease Введение В настоящее время регистрируется высокая распро- страненность хронических мультифакториальных заболеваний, таких как атеросклероз, сахарный диабет, артериальная гипертензия, неалкогольная жировая болезнь печени и другие, а также их сочета- ния [1, 2]. Сосуществующие клинические состояния в 1970 году были охарактеризованы термином «ко- морбидность», предложенным американским ученым А. Файнштейном [3]. В современном понимании «ко- морбидность» – это одновременное существование двух и более заболеваний, связанных между собой патогенетическими и генетическими механизмами [4]. К таковым относятся кардиоваскулярные забо- левания при неалкогольной жировой болезни пече- ни. Актуальной задачей современной клинической медицины является поиск патогенетических и иных связей неалкогольной жировой болезни печени (НАЖБП) и сердечно-сосудистых заболеваний (ССЗ). НАЖБП рассматривается как системное мульти- факториальное хроническое заболевание печени, не связанное с употреблением алкоголя в гепатотоксич- ных дозах [5, 6]. НАЖБП является одним из наибо- лее частых заболеваний печени [7]. Так, у жителей европейских стран заболеваемость НАЖБП состав- ляет от 20 до 30%, в странах Азии несколько ниже – от 5 до 18% [8]. В России НАЖБП занимает одно из лидирующих мест в структуре заболеваний вну- тренних органов и встречается по разным данным у 29,3%-37,3% населения [5, 9, 10]. Известно, что при естественном течении патологии среди пациентов со стеатозом через 8–13 лет у 12–40% развивается неалкогольный стеатогепатит (НАСГ) [11]. По дан- ным исследования DIREG2 (2015 г.) встречаемость НАЖБП за 7 лет увеличилась на 10% у пациентов, обратившихся за амбулаторной медицинской помо- щью, а число больных с циррозом печени в исходе НАЖБП возросло до 5% [10]. Причем у 7% больных циррозом в течение последующих 10 лет повышен риск развития гепатоцеллюлярной карциномы [11]. В 2018 г. показано, что НАЖБП – вторая по значи- мости причина трансплантации печени в США, а по прогнозам, к 2030 году она выйдет на первое место [12]. В целом НАЖБП повышает уровень смертно- сти от любых причин [13]. лидирующих мест в структуре заболеваний вну- тренних органов и встречается по разным данным у 29,3%-37,3% населения [5, 9, 10]. Известно, что при естественном течении патологии среди пациентов со стеатозом через 8–13 лет у 12–40% развивается неалкогольный стеатогепатит (НАСГ) [11]. По дан- ным исследования DIREG2 (2015 г.) встречаемость НАЖБП за 7 лет увеличилась на 10% у пациентов, обратившихся за амбулаторной медицинской помо- щью, а число больных с циррозом печени в исходе НАЖБП возросло до 5% [10]. Причем у 7% больных циррозом в течение последующих 10 лет повышен риск развития гепатоцеллюлярной карциномы [11]. В 2018 г. Особенности патогенеза НАЖБП в контексте кардиоваскулярной коморбидности К й 2019 Atherosclerosis) [38] показало, что повышение цир- кулирующего интерлейкина-6 (ИЛ-6) при НАЖБП ассоциировано с заболеваниями сердца и сосудов, особенно с образованием бляшек в коронарных артериях. Также ИЛ-6 усугубляет течение самой НАЖБП, через стимуляцию ИЛ-17 и преобразова- ние сигнальных путей факторов роста бета (TGF-B), что приводит к активации воспаления и усиливает ИР, что ведет к прогрессированию НАЖБП [16]. Atherosclerosis) [38] показало, что повышение цир- кулирующего интерлейкина-6 (ИЛ-6) при НАЖБП ассоциировано с заболеваниями сердца и сосудов, особенно с образованием бляшек в коронарных артериях. Также ИЛ-6 усугубляет течение самой НАЖБП, через стимуляцию ИЛ-17 и преобразова- ние сигнальных путей факторов роста бета (TGF-B), что приводит к активации воспаления и усиливает ИР, что ведет к прогрессированию НАЖБП [16]. Китайские ученые в 2019 году показали, что кон- центрация десатуразы жирных кислот 2 (FADS2 – Fatty acid desaturase 2) в сыворотке крови играет важную роль в прогрессировании НАЖБП и па- тологии сердечно-сосудистой системы. FADS2 является ключевым ферментом в метаболизме полиненасыщенных жирных кислот (ПНЖК), которые выполняют противовоспалительную функцию, регулируют уровень липидов крови, а также уменьшают степень выраженности сте- атоза печени. Десатураза замедляет метаболизм ПНЖК, тем самым повышая уровень триглице- ридов в крови. Известно, что активность FADS2 у больных НАЖБП выше, чем у здоровых людей [39]. На данный момент активно изучается вклад полиморфизма гена, кодирующего синтез FADS2, однако однозначных результатов пока не получено. Китайские ученые в 2019 году показали, что кон- центрация десатуразы жирных кислот 2 (FADS2 – Fatty acid desaturase 2) в сыворотке крови играет важную роль в прогрессировании НАЖБП и па- тологии сердечно-сосудистой системы. FADS2 является ключевым ферментом в метаболизме полиненасыщенных жирных кислот (ПНЖК), которые выполняют противовоспалительную функцию, регулируют уровень липидов крови, а также уменьшают степень выраженности сте- атоза печени. Десатураза замедляет метаболизм ПНЖК, тем самым повышая уровень триглице- ридов в крови. Известно, что активность FADS2 у больных НАЖБП выше, чем у здоровых людей [39]. На данный момент активно изучается вклад полиморфизма гена, кодирующего синтез FADS2, однако однозначных результатов пока не получено. В 2012 году S. Coulon с коллегами определили, что у людей со стеатозом и стеатогепатитом более высокий уровень факторов роста сосудистого эн- дотелия, чем у здоровых. [19]. Высокая продукция протромботических факторов, в частности факто- ров VIII, IX, XI и XII, коррелирует с содержанием печеночного жира. Также, увеличение высвобо- ждения ингибитора активатора плазминогена-1 и повышение концентрации фибриногена при- водит к усилению тромбообразования и к повы- шению риска сердечно-сосудистых осложнений у пациентов с НАЖБП [8, 19]. Введение показано, что НАЖБП – вторая по значи- мости причина трансплантации печени в США, а по прогнозам, к 2030 году она выйдет на первое место [12]. В целом НАЖБП повышает уровень смертно- сти от любых причин [13]. В современной литературе имеются исследова- ния, результаты которых демонстрируют влияние НАЖБП на развитие сердечно-сосудистой патоло- гии [8, 14, 15, 16]. В проведенном G. Musso с соавт. метаанализе (2011 г.) показано, что риск развития ССЗ у пациентов с НАЖБП в 2 раза выше по срав- нению с контрольной группой [17]. По данным ис- следования, проведенного в США и включающего НАЖБП рассматривается как системное мульти- факториальное хроническое заболевание печени, не связанное с употреблением алкоголя в гепатотоксич- ных дозах [5, 6]. НАЖБП является одним из наибо- лее частых заболеваний печени [7]. Так, у жителей европейских стран заболеваемость НАЖБП состав- ляет от 20 до 30%, в странах Азии несколько ниже – от 5 до 18% [8]. В России НАЖБП занимает одно из 89 обзор \| review экспериментальная и клиническая гастроэнтерология \| выпуск 182 \| № 10 2020 более 11 тыс. человек, НАЖБП оказалась связана с повышенной распространенностью ССЗ независи- мо от других факторов риска [18]. Многочисленные данные показывают повышение кардиоваскулярных рисков и ускорение прогрессирования патологии печени при сочетании НАЖБП и ССЗ [19, 20, 21, 22], однако, существуют исследования, не подтвер- дившие ухудшение исходов [23, 24, 25]. Особенности патогенеза НАЖБП в контексте кардиоваскулярной коморбидности Atherosclerosis) [38] показало, что повышение цир- кулирующего интерлейкина-6 (ИЛ-6) при НАЖБП ассоциировано с заболеваниями сердца и сосудов, особенно с образованием бляшек в коронарных артериях. Также ИЛ-6 усугубляет течение самой НАЖБП, через стимуляцию ИЛ-17 и преобразова- ние сигнальных путей факторов роста бета (TGF-B), что приводит к активации воспаления и усиливает ИР, что ведет к прогрессированию НАЖБП [16]. Китайские ученые в 2019 году показали, что кон- центрация десатуразы жирных кислот 2 (FADS2 – Fatty acid desaturase 2) в сыворотке крови играет важную роль в прогрессировании НАЖБП и па- тологии сердечно-сосудистой системы. FADS2 является ключевым ферментом в метаболизме полиненасыщенных жирных кислот (ПНЖК), которые выполняют противовоспалительную функцию, регулируют уровень липидов крови, а также уменьшают степень выраженности сте- атоза печени. Десатураза замедляет метаболизм ПНЖК, тем самым повышая уровень триглице- ридов в крови. Известно, что активность FADS2 у больных НАЖБП выше, чем у здоровых людей [39]. На данный момент активно изучается вклад полиморфизма гена, кодирующего синтез FADS2, однако однозначных результатов пока не получено. По мере прогрессирования НАЖБП наблюдается ряд клинико-морфологических изменений, вклю- чающих в себя жировой гепатоз (стеатоз), стеато- гепатит, фиброз и цирроз [5, 27]. В основе НАЖБП лежит нарушение чувствительности рецепторов к инсулину, то есть инсулинорезистентность (ИР), что влечет за собой нарушение липидного, углево- дного и белкового обменов. Установлено, что на- рушение регуляции обмена липидов стимулирует повышенное выделение свободных жирных кислот (СЖК) адипоцитами, в результате чего триглице- риды (ТГ) откладываются в печени в виде вакуо- лей – развивается жировая инфильтрация печени (стеатоз) [5, 9, 28]. Также, ТГ активируют процессы перекисного окисления липидов, что приводит к разрушению митохондрий, лизосом и клеточной мембраны гепатоцитов. Дополнительное повре- ждающее воздействие на мембраны клеток оказы- вают продукты свободнорадикального окисления, в результате чего развивается хроническое воспа- ление и формируется НАСГ [5, 27, 29]. Имеются и другие механизмы перехода стеатоза в стеатоге- патит, например, повышение продукции фактора некроза опухолей –α (ФНО- α), активация цитохро- ма Р450, накопление активных форм кислорода (ок- сидативный стресс) с образованием избыточного количества высокотоксичных ксенобиотиков. Все эти факторы приводят к стимуляции коллагеноо- бразования и усугублению фиброза печени [9, 28]. Atherosclerosis) [38] показало, что повышение цир- кулирующего интерлейкина-6 (ИЛ-6) при НАЖБП ассоциировано с заболеваниями сердца и сосудов, особенно с образованием бляшек в коронарных артериях. Также ИЛ-6 усугубляет течение самой НАЖБП, через стимуляцию ИЛ-17 и преобразова- ние сигнальных путей факторов роста бета (TGF-B), что приводит к активации воспаления и усиливает ИР, что ведет к прогрессированию НАЖБП [16]. НАЖБП и атеросклероз НАЖБП связана со многими маркерами атероскле- роза (толщина комплекса «интима-медиа» (КИМ), кальцификация, увеличение ригидности коронар- ных артерий, снижение лодыжечно-плечевого ин- декса (ЛПИ), скорость распространения пульсовой волны) [42, 45, 46]. Фрамингемское исследование на 3529 пациентов, прошедших компьютерную томографию, показало наличие связи НАЖБП с отложением кальция в коронарных артериях даже после корректировки факторов метаболического риска, таких как ожирение и гиперлипидемия [19]. дальнейших исследований для уточнения гендер- ных различий и выявления их механизмов [23]. Тем не менее известно, что в сравнении с пациентами без поражения печени, пациенты с НАЖБП имеют более высокую летальность от острого коронарного синдрома (3,1% против 21,4%) [20, 43]. НАЖБП связана со многими маркерами атероскле- роза (толщина комплекса «интима-медиа» (КИМ), кальцификация, увеличение ригидности коронар- ных артерий, снижение лодыжечно-плечевого ин- декса (ЛПИ), скорость распространения пульсовой волны) [42, 45, 46]. Фрамингемское исследование на 3529 пациентов, прошедших компьютерную томографию, показало наличие связи НАЖБП с отложением кальция в коронарных артериях даже после корректировки факторов метаболического риска, таких как ожирение и гиперлипидемия [19]. В метаанализе, проведенном в 2016 году Pais R., Giral P., Khan J. F., с участием 25 837 больных НАЖБП был выявлен более высокий риск разви- тия ИБС по сравнению с группой контроля [47]. Подобное исследование проводилось и в Японии, где после пяти лет наблюдения обнаружено, что из 1637 человек с диагностированной НАЖБП, у 1221 из них имелись проявления ИБС [48]. Имеются данные, что НАЖБП часто коррелиру- ет с атеросклерозом сонных артерий [45, 49]. В ис- следовании с участием 1021 пациента в возрасте 30–79 лет значения толщины КИМ у пациентов с НАЖБП были значительно выше, чем у группы сравнения [45], сопоставимые результаты полу- чил и Th akur M. L. с соавторами [50]. Увеличение толщины КИМ чаще всего выявляют у пациентов на стадии стеатоза печени [45]. Риск развития ка- ротидного атеросклероза у больных НАЖБП ока- зался выше средне популяционного в 1,85 раза [45], что заставило Abid Rasool с соавт рекомендовать всем пациентам с НАЖБП проходить скрининг с определением толщины КИМ [49]. В метаанализе, проведенном в 2016 году Pais R., Giral P., Khan J. F., с участием 25 837 больных НАЖБП был выявлен более высокий риск разви- тия ИБС по сравнению с группой контроля [47]. Подобное исследование проводилось и в Японии, где после пяти лет наблюдения обнаружено, что из 1637 человек с диагностированной НАЖБП, у 1221 из них имелись проявления ИБС [48]. Особенности патогенеза НАЖБП в контексте кардиоваскулярной коморбидности у у у У пациентов с НАЖБП регистрируется умень- шение уровня адипонектина и повышение уровня лептина [30, 31]. Адипонектин – это гормон, выра- батываемый клетками жировой ткани, обладающий противовоспалительным (за счет снижения про- дукции ФНО-α и интерлейкина-6), кардиопротек- тивным [30, 32, 33] и антистеатотическим (за счет усиления окисления свободных жирных кислот) действиями [34]. Лептин – это пептидный гормон, се- кретируемый, в основном, адипоцитами, выделение которого прямо пропорционально жировой массе и обеспечивает эффект «анти-ожирение» за счет ак- тивации гипоталамических клеток [32, 33]. Имеются предположения, что лептин сам по себе участвует в патогенезе НАЖБП, способствуя развитию ИР за счет активации дефосфорилирования субстрата ин- сулинового рецептора и уменьшения глюконеогенеза [34, 35], а провоспалительное действие, способствует прогрессированию НАЖБП [35, 36]. Исследования на животных показали, что НАЖБП является мультисистемным заболевани- ем с поражением не только печени, но и органов сердечно-сосудистой системы. Так, при моделиро- вании НАЖБП на крысах, были выявлены атеро- склеротические изменения стенок сосудов [14, 40]. В другом же эксперименте НАЖБП была ассоци- ирована с развитием эндотелиальной дисфункции у кроликов [14]. Изменение липидного профиля является одним из ведущих механизмов развития ССЗ при НАЖБП. Как говорилось раннее, изменение функционально- го состояния печени при НАЖБП влияет на основ- ные обменные процессы, в том числе усиливает ИР и липолиз, что способствует повышению уровня триглицеридов, липопротеинов низкой и очень низ- кой плотности, и снижению уровня липопротеинов высокой плотности [8, 15]. Известно, что НАЖБП НАЖБП усугубляет ИР, что приводит к выра- ботке большего количества провоспалительных, прокоагулянтных и профиброгенных медиаторов и способствует развитию структурных нарушений в миокарде и эндотелии. Однако, роль биомарке- ров воспаления при НАЖБП и ССЗ изучена недо- статочно [14, 15, 37]. Одно из крупнейших иссле- дований по этому вопросу (Multi-Ethnic Study of 90 рдиоваскулярная коморбидность при неалкогольной жировой болезни печени \| Cardiovascular comorbidity in non-alcoholic fatty liver disease ты на течение различных заболеваний. Так, было выявлено, что изменения кишечной микробиоты играют важную роль у пациентов с НАЖБП и ише- мическими болезнями сердца (ИБС). Показано, что количество Collinsella и Parabacteroides, оказываю- щих протективное действие в отношении обмена веществ, у пациентов с ИБС и поражением печени было ниже, чем у пациентов контрольной груп- пы. Так же обнаружено, что количество Sutterella stercoricanis, ассоциирующееся с ожирением и пора- жением печени, у пациентов с НАЖБП было выше, чем у здоровых людей. Имеются данные и о низком содержании Ruminococcus albus у пациентов с ИБС, но значение этих данных остается неизвестным [43]. Существуют предположения о возможном патогене- тическом механизме взаимовлияния микробиоты и ССЗ. Особенности патогенеза НАЖБП в контексте кардиоваскулярной коморбидности Данные влияния связывают с кишечным микробным метаболизмом холина до накопления триметиламина, который превращается в печени хо- зяина до флавинмонооксигененазы 3 и триметила- мин-N-оксида (ТМАО), что связывают с повышен- ным риском сердечно-сосудистых заболеваний [44]. сопровождается образованием эктопического жира, причем не только в печени, но и в эпикарде и ми- окарде. В норме эпикардиальный жир поглощает свободные жирные кислоты из кровеносного русла, которые могут быть использованы кардиомиоцита- ми в качестве дополнительного источника энергии [31]. При избыточном их накоплении наблюдается повышение риска развития ССЗ [41], в частности фибрилляции предсердий [8, 14]. У пациентов с НАЖБП и ССЗ существуют из- вестные общие факторы риска, например, эндо- телиальная дисфункция, оксидативный стресс, воспаление, дислипидемия, сахарный диабет, ожи- рение, метаболический синдром и др. [30], а также выделяют «новые» факторы риска, такие как уве- личение толщины эпикардиального жира и утол- щение комплекса интима-медиа, развитие эндоте- лиальной дисфункции, увеличение С-реактивного белка (СРБ), гиперурикемия, гипоадипонектине- мия и гиповитаминоз D [11, 18, 31, 42]. В последнее время уделяется значительное вни- мание влиянию особенностей кишечной микробио- НАЖБП и атеросклероз По результатам коронароангиографии ате- росклероз коронарных артерий был обнаружен у 84,6% пациентов с НАЖБП, тогда как у пациентов без НАЖБП атеросклеротические бляшки выяв- лялись в 64,1% случаев [48]. Часть исследований показывает, что НАЖБП связана с наибольшей распространенностью нестабильных бляшек в ко- ронарных артериях [13, 31]. Однако по другим дан- ным, связь ИБС и НАЖБП оказалась несуществен- ной после корректировки большинства факторов риска, а именно гиперлипидемии, ожирения или избытка массы тела, диабета, курения и повышен- ного артериального давления [24]. Так, обследова- ние 471849 больных, выполненное в 2019 году, не выявило связи между НАЖБП и ССЗ [23]. Данный анализ показал, что при НАЖБП повышается риск смертности от всех причин, в особенности от пече- ночных осложнений, а не от сердечно-сосудистой патологии. Также было показано повышение ри- ска смертности у женщин с НАЖБП, что требует У пациентов с НАЖБП выявлен высокий риск развития цереброваскулярных заболеваний (ЦВЗ) [19, 51]. Abdeldyem, S.M. с соавт. [19, 25] провели исследование 200 пациентов с ишемическим инсуль- том, оказалось, что НАЖБП ассоциирована с плохой оценкой по шкале NIHSS (National Institutes of Health Stroke Scale) и более высоким риском инвалидиза- ции. Сходные данные получены в ретроспективном исследовании [25] 306 пациентов с острым наруше- нием мозгового кровообращения, среди которых пациенты с НАЖБП имели более тяжелые невро- логические последствия. Но в то же время, другое исследование пациентов, перенесших инсульт не выявило различий в группах с НАЖБП и без [52]. Помимо поражения крупных артерий, при НАЖБП увеличивается риск развития и микро- сосудистых осложнений, что может привести к раз- витию ретинопатии, нейропатии, а также к нару- шению функции почек [13]. 91 обзор \| review экспериментальная и клиническая гастроэнтерология \| выпуск 182 \| № 10 2020 НАЖБП и нарушения ритма сердца Наличие НАЖБП тесно связано с увеличением интервала QT, что в свою очередь приводит к раз- витию желудочковых аритмий и повышению риска внезапной сердечной смерти [14, 26, 31], однако механизмы влияния на интервал QT не известны. Также, имеются данные, что пациенты с блокадой правой ножки пучка Гиса более подвержены раз- витию НАЖБП, но более подробно этот вопрос не изучен [18, 26]. НАЖБП и артериальная гипертензия (АГ) НАЖБП – независимый предиктор повышения жесткости артериальной стенки, в том числе и у лиц, не страдающих диабетом, АГ или ожи- рением [27, 45, 54]. Исследование 8632 пациентов старше 40 лет, продемонстрировало повышение лодыжечно-плечевого индекса у больных НАЖБП независимо от наличия факторов риска ССЗ [45]. Также, в недавних работах имеются данные о том, что НАЖБП увеличивает риск повышения ригид- ности артерий у женщин в постменопаузальном периоде [8]. У больных с неалкогольным поражением печени АГ встречается в 49,5% случаев, в то время как у лю- дей со здоровой печенью частота составляет 38,5% [55, 56]. Известно, что у больных с НАЖБП чаще встречается повышение систолического артери- ального давления, нарушение суточного профиля АД, повышение пульсового давления [27, 51, 57]. НАЖБП и хроническая сердечная недостаточность (ХСН) НАЖБП усугубляет течение ХСН [11, 55, 58]. В свою очередь, пациенты с сердечной недостаточностью имеют более высокую (до 77%) частоту развития неалкогольного поражения печени, чем без ХСН [11]. В Пекине было проведено исследование 102 пациентов с сердечной недостаточностью, в котором у 36% была выявлена НАЖБП, причем пациенты с НАЖБП были моложе, имели более высокий ин- декс массы тела и индекс массы левого желудочка (ЛЖ) [22, 59]. Другие исследования продемонстри- ровали, что НАЖБП ассоциируется с худшей толе- рантностью к физической нагрузке и более выра- женным ремоделированием сердца [55]. НАЖБП также независимо коррелирует со степенью фиброза левого желудочка и, возможно, усугубляет прогрес- сирование ХСН [59]. Однако, по данному вопросу необходимы дальнейшие исследования для выяв- ления механизмов взаимовлияния НАЖБП и ХСН. (ЛЖ) [22, 59]. Другие исследования продемонстри- ровали, что НАЖБП ассоциируется с худшей толе- рантностью к физической нагрузке и более выра- женным ремоделированием сердца [55]. НАЖБП также независимо коррелирует со степенью фиброза левого желудочка и, возможно, усугубляет прогрес- сирование ХСН [59]. Однако, по данному вопросу необходимы дальнейшие исследования для выяв- ления механизмов взаимовлияния НАЖБП и ХСН. Влияние ССЗ на НАЖБП Не только НАЖБП способна влиять на развитие ССЗ, но и наоборот, некоторые кардиоваскуляр- ные патологии усугубляют течение НАЖБП. Так, Jiantao Ma. и соавт. показали, что при наличии ар- териальной гипертензии повышается риск разви- тия НАЖБП даже после поправки на индекс массы тела и количество жировой ткани [60]. Нарушение периферического кровообращения при АГ, атеро- склеротическое поражение сосудов, формирование венозного застоя при хронической правожелудоч- ковой недостаточности приводят к еще более зна- чимому повреждению гепатоцитов, что не может не отразиться на состоянии гепатобилиарной системы. Таким образом, «порочный круг» замыкается [61]. НАЖБП и нарушения ритма сердца Существуют данные о связи НАЖБП и развитием нарушений ритма сердца [14, 42, 53]. Было отмечено, что у пациентов с НАЖБП фибрилляция предсер- дий (ФП) встречается в 2,5 раза чаще, чем у лиц без патологии печени. [47, 53]. Käräjämäki A. J. и соавт. приводят эпидемиологические доказательства того, что НАЖБП является независимым фактором ри- ска развития ФП [53]. Targher G. С соавт. выявили, что в течение десятилетнего периода наблюдения у пациентов с НАЖБП частота возникновения ФП больше, чем у пациентов со здоровой печенью [14]. В другом исследовании Sinner M. F., с соавторами проанализировали 3744 пациента и обнаружил что повышенный уровень аспартат-трансамина- зы (АСТ) и аланин- трансаминазы (АЛТ) связан с повышенным риском возникновения ФП [14]. Механизм возникновения ФП при НАЖБП до конца не выяснен, но существуют предположения, что накопление жира в гепатоцитах и окислитель- ный стресс приводит к увеличению синтеза про- воспалительных цитокинов, что является факто- ром риска развития ФП [47]. По другим данным, вегетативная дисфункция, развивающейся на фоне НАЖБП, нарушает работу симпатической нервной системы и провоцирует возникновение ФП [47, 53]. Наличие НАЖБП тесно связано с увеличением интервала QT, что в свою очередь приводит к раз- витию желудочковых аритмий и повышению риска внезапной сердечной смерти [14, 26, 31], однако механизмы влияния на интервал QT не известны. Также, имеются данные, что пациенты с блокадой правой ножки пучка Гиса более подвержены раз- витию НАЖБП, но более подробно этот вопрос не изучен [18, 26]. конца не выяснен, но существуют предположения, что накопление жира в гепатоцитах и окислитель- ный стресс приводит к увеличению синтеза про- воспалительных цитокинов, что является факто- ром риска развития ФП [47]. По другим данным, вегетативная дисфункция, развивающейся на фоне НАЖБП, нарушает работу симпатической нервной системы и провоцирует возникновение ФП [47, 53]. Наличие НАЖБП тесно связано с увеличением интервала QT, что в свою очередь приводит к раз- витию желудочковых аритмий и повышению риска внезапной сердечной смерти [14, 26, 31], однако механизмы влияния на интервал QT не известны. Также, имеются данные, что пациенты с блокадой правой ножки пучка Гиса более подвержены раз- витию НАЖБП, но более подробно этот вопрос не изучен [18, 26]. конца не выяснен, но существуют предположения, что накопление жира в гепатоцитах и окислитель- ный стресс приводит к увеличению синтеза про- воспалительных цитокинов, что является факто- ром риска развития ФП [47]. По другим данным, вегетативная дисфункция, развивающейся на фоне НАЖБП, нарушает работу симпатической нервной системы и провоцирует возникновение ФП [47, 53]. Литература  References 1. Наумова Л.А., Осипова О. Н. Коморбидность: механиз- мы патогенеза, клиническое значение. Современные проблемы науки и образования. – 2016, 5, стр. 57–64 Naumova L. A., Osipova O. N. Comorbidity: mechanisms of pathogenesis, clinical signifi cance. Modern problems of science and education. 2016. No. 5, pp. 57–64. 7. Parise E. R. Nonalcoholic fatty liver disease (NAFLD), more than a liver disease NASH. Arquivos de Gastroenterologia. 2019;56(3)243–245. doi: 10.1590/ s0004–2803.201900000–45 8. Ахмедов В. А. Взаимосвязь сердечно-сосудистых ос- ложнений и неалкогольной жировой болезни печени. РМЖ, 2018. –1(2), стр. 86–88 Akhmedov V. A. Th e relationship of cardiovascular com- plications and non-alcoholic fatty liver disease. RMZh. 2018;1 (2):86–88. 2. Самородская И.В., Болотова Е. В. Терминологические и демографические аспекты коморбидности. Успехи Геронтологии. –2016, 29, 3, стр. 471 I. V. Samorodskaya, E. V. Bolotova. Terminological and demographic aspects of comorbidity. Advances in Gerontology. 2016;29(3):471. 2. Самородская И.В., Болотова Е. В. Терминологические и демографические аспекты коморбидности. Успехи Геронтологии. –2016, 29, 3, стр. 471 р р I. V. Samorodskaya, E. V. Bolotova. Terminological and demographic aspects of comorbidity. Advances in Gerontology. 2016;29(3):471. 9. Лазебник Л. Б., Радченко В. Г., Голованова Е. В. и др. 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РЖГГК- 2015. – 6, стр. 31–41 Ivashkin V. T. and others. Prevalence of non-alcoholic fatty liver disease in outpatient patients in the Russian Federation: results of the DIREG 2 study. RZHGGK-2015, 6, pp. 31–41. 10. Ивашкин В. Т. и др. Распространенность неалко- гольной жировой болезни печени у пациентов ам- булаторно-поликлинической практики в Российской Федерации: результаты исследования DIREG 2. РЖГГК- 2015. – 6, стр. 31–41 10. 4. Оганов Р.Г., Симаненков В. И., Бакулин И. Г., Ба ку ли- на Н. В. и др. Заключение фактором риска для развития сердечно-сосуди- стой патологии или лишь сопутствующим забо- леванием, остается открытым. Таким образом, данные современной литературы свидетельствуют о наличии патогенетической связи между НАЖБП и ССЗ, однако, некото- рые исследования, такую связь не подтвержда- ют. Поэтому, вопрос о том, является ли НАЖБП В связи с высокой распространенностью, необ- ходимо признать значимость ранней диагностики 92 Кардиоваскулярная коморбидность при неалкогольной жировой болезни печени \| Cardiovascular comorbidity in non-alcoholic fatty liver disease рдиоваскулярная коморбидность при неалкогольной жировой болезни печени \| Cardiovascular comorbidity in non-alcoholic fatty liver disease заболеваемости и смертности от болезней системы кровообращения. И наоборот, при наблюдении пациента с сердечно-сосудистой патологией, сле- дует помнить о НАЖБП, которая может усугубить течение кардиоваскулярных заболеваний. Таким образом, подтверждение связей между НАЖБП и ССЗ имеет важное клиническое значение и тре- бует дальнейшего изучения. и совершенствования лечебно-профилактической помощи пациентам с НАЖБП. Следует уточнить механизмы влияния НАЖБП на развитие и про- грессирование болезней системы кровообраще- ния. При подтверждении таких связей следует рассматривать включение углубленного кардиова- скулярного скрининга в программу курации боль- ных НАЖБП, как возможный путь к уменьшению Источник финансирования: Грант Президента РФ для государственной поддержки ведущих научных школ (НШ-2558.2020.7) (соглашение № 075–15–2020–036 от 17 марта 2020 года) «Разработка технологии здоровьесбереже- ния коморбидного больного гастроэнтерологического профиля на основе контроля приверженности». Литература  References doi: 10.15403/jgld-344 Akhmedov V. A., Mamedova N. F., Gaus O. V. Non-alcoholic fatty liver disease and comorbidity. Gastroenterology of St. Petersburg. 2019;3(4):47–52. 27. Athyros V. G., Tziomalos K., Katsiki N., Doumas M. et al. Cardiovascular risk across the histological spectrum and the clinical manifestations of non-alcoholic fatty liver disease: An update.. World Journal of Gastroenterology, 2015;21(22): 6820–6834. doi: 10.3748/wjg.v21.i22.6820 14. Mangi M A, Rehman H, Minhas A M, et al. 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https://openalex.org/W4200573677	https://bmcgeriatr.biomedcentral.com/counter/pdf/10.1186/s12877-021-02644-6	English	null	Older adults who receive homecare are at increased risk of readmission and mortality following a short ED admission: a nationally register-based cohort study	BMC geriatrics	2,021	cc-by	7,244	Open Access Abstract Background: Older adults admitted to an emergency department (ED) who are dependent on homecare may be especially challenged with respect to readmission and mortality. This study aimed to assess whether receiving homecare prior admission was associated with readmission or mortality within 30 days of a short ED admission and to explore whether the amount of homecare received was associated with an increased risk of readmission or mortality. Methods: This nationwide register-based cohort study included patients aged 65 or above who were admitted to an ED at any Danish hospital from 1 December 2016 to 30 November 2017 and discharged within 48 h. Data were extracted from national registers through Statistics Denmark. Homecare was categorized into groups; patients without homecare and three groups according to the amount of homecare received per week. Logistic regression analyses were used to explore the association between the four homecare groups and outcomes, readmissions and mortality. Results: In total, 80,517 patients (51% female, median age 75 years) were included in the study. Overall, 64,886 patients without homecare, 15,631 (19%) patients received homecare (64% female, median age 83 years), of which 4938 patients received homecare ≤30 min, 4033 received > 30 min to ≤120 min and 6660 received > 120 min per week. The risk of readmission and mortality increased concurrently with the minutes of homecare received: Patients receiving homecare > 120 min per week had the highest odds ratios (ORs) for readmission within 30 days (OR 1.8 95% CI: 1.7–1.9) and mortality within 30 days (OR 4.5 95% CI: 4.1–4.9) compared with patients without homecare. Conclusion: Receiving homecare was associated with an increased risk of readmission and death following a short ED admission. Collaboration between the ED and primary health care sector in relation to rehabilitation and end- of-life care is essential to improve quality of care for older adults who receive homecare, particularly those receiving homecare > 2 h a week, because of their increased risk of readmission and mortality. Keywords: Primary health care, Older adults, Death, Acute illness, Acute hospitalisation, Multimorbidity, Comorbidities, Emergency medicine Mette Elkjær1,2,3* , Donna Lykke Wolff2,4, Jette Primdahl2,5,6, Christian Backer Mogensen1,2, Mikkel Brabrand2,7,8 and Bibi Gram2,3 Mette Elkjær1,2,3* , Donna Lykke Wolff2,4, Jette Primdahl2,5,6, Christian Backer Mogensen1,2, Mikkel Brabrand2,7,8 and Bibi Gram2,3 © 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. Background Correspondence: mette.elkjaer@rsyd.dk 1 Department of Emergency Medicine, University hospital of Southern Denmark, Kresten Philipsens Vej 15, 6200 Aabenraa, Denmark Full list of author information is available at the end of the article Correspondence: mette.elkjaer@rsyd.dk 1 Department of Emergency Medicine, University hospital of Southern Denmark, Kresten Philipsens Vej 15, 6200 Aabenraa, Denmark Full list of author information is available at the end of the article Correspondence: mette.elkjaer@rsyd.dk 1 Department of Emergency Medicine, University hospital of Southern Denmark, Kresten Philipsens Vej 15, 6200 Aabenraa, Denmark Full list of author information is available at the end of the article Admission at emergency department (ED) is especially challenging for older adults aged 65 or above owing to a high prevalence of multimorbidity [1–3]. One-fifth of Older adults who receive homecare are at increased risk of readmission and mortality following a short ED admission: a nationally register-based cohort study Mette Elkjær1,2,3 , Donna Lykke Wolff2,4, Jette Primdahl2,5,6, Christian Backer Mogensen1,2, Mikkel Brabrand2,7,8 and Bibi Gram2,3 Elkjær et al. BMC Geriatrics (2021) 21:696 https://doi.org/10.1186/s12877-021-02644-6 © 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. Elkjær et al. BMC Geriatrics (2021) 21:696 Elkjær et al. BMC Geriatrics (2021) 21:696 Elkjær et al. BMC Geriatrics (2021) 21:696 Page 2 of 9 all acutely admitted older adults are readmitted within 30 days, leading to a significant financial burden for society [4]. Moreover, an acute admission is associated with a critical increase in risk of mortality [5]. Among acutely admitted older adults aged 65 or above who are discharged, 20% will die within 1 year of discharge [5]. Although considerable research has been conducted on older adults who are acutely admitted, there is a lack of agreement on how to identify patients at risk of readmis- sion and mortality. receive practical help, the majority of patients receiving > 30 to ≤120 min per week receive some assistance with personal care in addition to practical help and the major- ity of the patients receiving > 120 min of homecare are dependent on daily assistance for ADL. Thus, we hypoth- esise that the amount of help received reflects the degree of dependency on external help. Examination of whether older adults receive homecare at the time of admission to the ED may be a simple way to identify patients at high risk of readmission or mortality. Thus, this study aimed to assess whether receiving home- care was associated with readmission or mortality within 30 days in older adults, after a short ED admission, and to explore whether the amount of homecare received was related to an increased risk of readmission or mortality. A 2015 study from Belgium reported that older adult patients 75 years or above who were discharged from an ED had a higher readmission rate compared with other hospitalised patients [3]. ED care involves high activ- ity, and the accelerated treatment processes and rapid discharges might affect the readmission rate. A Danish study concluded that 49% of readmissions were prevent- able among older adults 75 years or above and these were more likely to occur soon after hospital discharge [6]. In addition, a study from the Netherlands found that (adults median aged 71) the most common reason for prevent- able readmission was a lack of coordination within the health care system [7]. Identifying patients at risk for readmission would allow health professionals to apply target interventions to prevent readmissions among the patients with the greatest needs. Study design and setting A retrospective register-based cohort study was con- ducted. National data on acutely admitted patients were extracted from Danish registers to construct a database on readmission rates, mortality and associated variables. The Danish health care system is tax funded for all 5.8 million residents, and Danish patients do not bear any additional financial costs for hospital care and treatment or for homecare.h The Danish EDs are organised with hospital beds for patients in need of a short admission and where the discharge is planned directly from the ED. A short ED admission is nationally defined as an admission less than 48 h. Acutely admitted older adults are often extraordinar- ily fragile with multiple comorbidities, functional limita- tions and cognitive impairments [1, 2, 8]. Municipalities offer cost-free homecare to residents in Denmark for those who find activities of daily living (ADL) chal- lenging because of functional limitations or cognitive impairments [9]. The probability of receiving homecare increases with age, and two-thirds of those who receive homecare are aged 65 or above [10]. In 2015, around 12% of older adults aged 65 or above received homecare in Denmark [9]. Homecare to support ADL consists of “personal care”, which includes assistance with personal hygiene, dress- ing, morning routines and mealtimes, and “practical help”, which includes assistance with cleaning, laundry and shopping. The type and amount of homecare allo- cated to a resident is based on an individual assess- ment, with the specific aim of restoring, maintaining and improving mental and physical functionality [9]. Indi- vidual needs are continually adjusted and are planned by a specialist. Data on the type (personal/practical) and amount of homecare are shared electronically between the municipality of residence and local and regional hos- pitals at admission and discharge. Around 35% of medical patients in a Danish ED aged 65 or above, receive homecare [11]. A population-based cohort study from Denmark included 3775 medical patients from a single municipality and reported that receiving homecare before an acute admission was a strong predictor of subsequent readmission and mortal- ity [11]. However, it is not known whether these results are transferable to an ED population and whether the risk of readmission and mortality is associated with the amount of homecare received. Exposure variableh The study population was categorised into one of the fol- lowing four groups according to the proportion of home- care received: 1) patients without homecare, 2) patients receiving homecare ≤30 min per week, 3) patients receiv- ing homecare between > 30 to ≤120 min per week and 4) patients receiving homecare > 120 min per week.i The Danish Registration of Elderly contains informa- tion about services supplied by the municipalities to resi- dents. Information about health care use and homecare services among acutely admitted older adult patients was extracted [15].h Homecare is defined as personal and practical help received and recorded in minutes per week. To assess the total time of homecare, we merged time allocated to practical help and time allocated to personal help. The homecare variable was assessed a month before the index admission and was calculated as a mean (minutes) of homecare received per week throughout the entire month. Patients receiving homecare for less than 1 min per week were categorised as patients without homecare. The Danish National Prescription Registry (NPR) con- tains individual-level data on prescriptions dispensed from pharmacies [16]. Information about the prescrip- tion of medication for mental health Anatomical Thera- peutic Chemical (ATC) codes (N05–N07) was extracted and used to describe mental comorbidity.h The Income Statistics Register provides data on the income composition of the entire Danish population. The patients’ disposable income was extracted from the Income Statistics Register [17]. The Danish Registration of Elderly receives monthly data from the municipalities. Unfortunately, data from some municipalities are inadequate and there may be variations over the year that are not recorded. If the municipalities do not approve the data as valid, Statis- tics Denmark uses the data delivered the month previ- ously. Statistics Denmark provides information on those months for which data might not be valid. Using this information, we performed a sensitivity analysis (see Appendix B). Data sources Data were extracted from the following registries in the Statistics Denmark [12].h We hypothesise that the risk of readmission and mor- tality among older adults receiving homecare is associ- ated with their ADL difficulties, which is reflected in the amount (in time) of homecare they receive per week. We categorise homecare based on the assumption that the majority of patients receiving ≤30 min per week only The Danish Civil Registration System (CRS) contains individual-level information on all persons residing in Denmark [13]. The unique 10-digit identification num- ber assigned to all Danish citizens in the CRS at birth or immigration enables linking data from different data Elkjær et al. BMC Geriatrics (2021) 21:696 Page 3 of 9 sources. We extracted information about marital status, age, sex and the date of death among the study popula- tion from this database. as the primary outcome as we do not consider them to be outpatient appointments. Secondary outcomes were readmission within 7 days and mortality within 30, 180 and 360 days. Primary and secondary outcome variables were assessed from the day of discharge from the index admission. The Danish National Patient Registry (DNPR) pos- sesses longitudinal data on inpatient and outpatient con- tacts, as well as discharge diagnosis codes according to the International Classification of Diseases, Revision 10 (ICD10) [14]. The DNPR was used to define the study population and obtain information about readmission and comorbidity. Study population From 1 December 2016 to 30 November 2017, patients aged 65 years or above with an admission to any Dan- ish ED were included in the study population. The study population was extracted from the DNPR. In the DNPR, patients are registered as inpatients or outpatients (patient type) and as admission type, acute or planned. As a proxy for ED admissions, we included all acute inpa- tients (admissions) with a length of stay below 48 h in the study population. Covariables Explanatory variables were selected based on their pos- sible influence on readmission and mortality. We used information about the patients’ age, sex and marital sta- tus (unmarried/married) recorded on the date of their index admission. Disposable income was included as a socio-economic factor. Disposable income is defined as the amount of money a person has available to spend and save after accounting for income tax. We calculated the median yearly disposable income over the past 5 years up to the index admission. The updated version of the Charlson Comorbidity Index (CCI) [18] was used to calculate and describe the number of patients’ chronic diseases using ICD-10 diagnoses and included both A (primary) and B (secondary) diagnoses. All registered diagnoses from 5 years before the index admission were included. Since the CCI contains little to no information about mental disorders, we used information on medi- cal prescriptions for mental disorders at any time within Since each medical specialty, department or hospital contact is coded as an individual contact in the DNPR, contacts of less than 3 h apart were merged into a single contact. An additional file shows this in more detail (see Additional file 1). We excluded patients registered as an acute outpatient visit only, patients living in residential care and patients without Danish residency. The patient’s first contact at the hospital in the study period was regis- tered as the index admission. The follow-up periods for the study population were 30 days regarding readmission and 360 days regarding mortality. Statistical methods Descriptive statistics were used to report demographic and clinical characteristics for the following categories: all patients, patients without homecare and patients with homecare. Categorical data were reported as frequen- cies and continuous variables as median and interquartile range (IQR). The distribution of data was assessed with Q-Q plots. Differences between groups were tested with the Wilcoxon rank sum test or Pearson’s chi-square test, depending on the distribution of the data. The associations between received homecare and out- comes (readmission rate on day 7 and 30 and mortal- ity on day 30, 180 and 360) were tested using logistic regression analyses and presented as crude ORs with 95% confidence intervals (CIs). Furthermore, the regres- sion analyses were preformed adjusting for age, Charlson Comorbidity Index and income (as continuous variables) and sex, marital status and treatment for mental disor- der (as categorical variables) and presented as adjusted ORs with 95% CIs. Finally, a margins plot calculating the probability for mortality and readmission within the homecare categories from the adjusted logistic regres- sion analysis was created. Patients who died before read- mission were excluded from the analyses calculating the risk of readmission. Further, we repeated all the analy- ses excluding the months with inadequate data from the municipalities in sensitivity analyses. All analyses were performed using Stata version 16.1 (StataCorp, College Station, TX, USA). P-values of < 0.05 were considered statistically significant. Fig. 1 Flowchart of study inclusions and exclusions Fig. 1 [0–2] vs. 0 [0–1]) compared with patients who did not receive homecare (Table 1). Further, patients receiv- ing homecare experienced a significant increased rate of readmission and mortality after an acute admission (Table 1). Outcome variablesh The primary outcome was readmission, which was defined as an acute contact, occurring within 30 days of the day of previous discharge. We included acute outpa- tient contacts above 6 h equal to inpatients (admissions) Elkjær et al. BMC Geriatrics (2021) 21:696 Elkjær et al. BMC Geriatrics (2021) 21:696 Elkjær et al. BMC Geriatrics (2021) 21:696 Page 4 of 9 the year before the index admission as a proxy for men- tal disorder (yes/no). Different types of medication for mental disorders included antipsychotics, anxiolytics, hypnotics and sedatives, antidepressants, and medica- tion for dementia. Psychostimulants were rarely used (< 0.03%) and were therefore not included. Type of medi- cation was used to describe the cohort and to compare patients receiving homecare with patients not receiving homecare. Type of medication was not included in the analyses. the year before the index admission as a proxy for men- tal disorder (yes/no). Different types of medication for mental disorders included antipsychotics, anxiolytics, hypnotics and sedatives, antidepressants, and medica- tion for dementia. Psychostimulants were rarely used (< 0.03%) and were therefore not included. Type of medi- cation was used to describe the cohort and to compare patients receiving homecare with patients not receiving homecare. Type of medication was not included in the analyses. Fig. 1 Flowchart of study inclusions and exclusions Outcomesh The distribution of the amount of homecare was as fol- lows: 4938 patients (32%) received ≤30 min (~ practical help), 4033 patients (26%) received ≥31 and ≤ 120 min (~ personal care in addition to practical help) and 6660 patients (42%) received ≥121 min per week (~ dependent on daily assistance for ADL). Participants no homecare Aged (year) Median (IQR) 75 (70–82) 74 (70–80) 83 (76–88) < 0.001 Marital status (married) 43,498 (54%) 39,725 (62%) 3773 (24%) < 0.001 Sex (female) 40,882 (51%) 30,918 (48%) 9964 (64%) < 0.001 In treatment for mental disorders 27,517 (34%) 19,243 (30%) 8274 (53%) < 0.001 In treatment with * Antipsychotics 1696 (2%) 1078 (2%) 618 (4%) < 0.001 Anxiolytics 4311 (5%) 3125 (5%) 1186 (8%) < 0.001 Hypnotics & sedative 9069 (11%) 6740 (10%) 2329 (15%) < 0.001 Antidepressants 6706 (8%) 4558 (7%) 2148 (14%) < 0.001 Medicine for dementia 1272 (2%) 628 (1%) 644 (4%) < 0.001 Income after taxes (Euro) median (IQR) 22,162 (17,756–29,300) 22,323 (17,513–30,435) 21,801 (18,710–25,796) < 0.001 Charlson Comorbidity Index median (IQR) 0 (0–2) 0 (0–1) 1 (0–2) < 0.001 0 48,548 (60%) 41,358 (64%) 7190 (46%) 1 10,066 (12%) 7414 (11%) 2652 (17%) 2 13,562 (17%) 10,418 (16%) 3144 (20%) > 3 8341 (11%) 5696 (9%) 2645 (17%) Mortality < 30 days 3010 (4%) 1790 (3%) 1220 (8%) < 0.001 < 180 days 7022 (9%) 4320 (7%) 2702 (17%) < 0.001 < 360 days 10,191 (13%) 6.325 (10%) 3866 (25%) < 0.001 Readmission < 7 days 5371 (7%) 4094 (6%) 1277 (8%) < 0.001 < 30 days 10,895 (14%) 8127 (13%) 2768 (18%) < 0.001 Table 1 Sociodemographic characteristics of the study population Note: IQR is interquartile range and n is numbers of patients. The difference between groups was tested with the Wilcoxon rank sum test Pearson’s chi-squared test. Patients can appear in more than one category Table 2 Association between homecare categories and readmission Notes: OR is the odds ratio and CI is confidence intervals. The logistic regression analyses were adjusted for age, sex, marital status, income, Charlson Comorbidity Index and treatment for mental disorder. Participants In total, 80,517 patients were included as the study popu- lation (Fig. 1). Among the study population, 5371 patients (7%) were readmitted within 7 days and 10,895 patients (14%) were readmitted within 30 days. Table 2 shows the association between amount of homecare received and risk of read- mission. The risk of readmission within 30 days increased with the amount of time for which patients received homecare; patients receiving homecare for > 120 min Among the study population, 15,631 patients (19%) received homecare. Patients receiving homecare before the index admission were older (median [IQR]: age 83 [76–88] vs. 74 [70–80]) years, fewer were married (24% vs. 62%), the majority were female (64% vs. 48%), a larger proportion received treatment for mental disorders (53% vs. 30%), and they had a higher CCI score (median [IQR]: Elkjær et al. BMC Geriatrics (2021) 21:696 Page 5 of 9 Table 1 Sociodemographic characteristics of the study population Note: IQR is interquartile range and n is numbers of patients. The difference between groups was tested with the Wilcoxon rank sum test (unequal distribution) or Pearson’s chi-squared test. Patients can appear in more than one category Variables All patients N = 80,517 Patients with no homecare n = 64,886 Patients receiving homecare n = 15,631 P-value Homecare vs. Participants Significance levels with a p-value of < 0.05 are marked with * and significance levels with a p-value of < 0.001 are marked with Variables Patients with no homecare Patients receiving homecare ≤ 30 min/week Patients receiving homecare > 30 to ≤120 min/week Patients receiving homecare > 120 min/week Readmission < 7 days Crude OR (95% CI) Adjusted OR (95% CI) 1 (ref.) 1.26 (1.13–1.40) 1.15 (1.02–1.29)* 1.26 (1.12–1.42) 1.13 (1.00–1.29) 1.53 (1.40–1.68) 1.30 (1.17–1.43) Readmission < 30 days Crude OR (95% CI) Adjusted OR (95% CI) 1 (ref.) 1.41 (1.30–1.52) 1.22 (1.12–1.31) 1.46 (1.34–1.59) 1.24 (1.13–1.35) 1.82 (1.70–1.94) 1.43 (1.33–1.54) ** CI is confidence intervals. The logistic regression analyses were adjusted for age, sex, marital status, income, Charlson Comorbidity l disorder. Significance levels with a p-value of < 0.05 are marked with * and significance levels with a p-value of < 0.001 are marked Notes: OR is the odds ratio and CI is confidence intervals. The logistic regression analyses were adjusted for age, sex, marital status, income, Charlson Comorbidity Index and treatment for mental disorder. Significance levels with a p-value of < 0.05 are marked with * and significance levels with a p-value of < 0.001 are marked with** mental disorders, patients receiving homecare > 120 min a week still had significant increased odds for readmis- sion – OR 1.43 (95% CI: 1.33–‍1.54) – compared with patients without homecare (Table 2). The increased risk a week had the highest odds for readmission – OR 1.82 (95% CI: 1.70–1.94) – compared with patients without homecare in the crude analysis (Table 2). When adjusting for age, sex, marital status, income, CCI and treatment for Elkjær et al. BMC Geriatrics (2021) 21:696 Elkjær et al. BMC Geriatrics (2021) 21:696 Page 6 of 9 Page 6 of 9 The sensitivity analyses excluding the months with inadequate data from the municipalities revealed no sig- nificance for the results regarding readmission and mor- tality. An additional file shows this results in detail (see Additional file 2). of readmission within 30 days among patients receiving homecare for ≤30 min and between > 30 and ≤ 120 min was similar to that found in the crude analysis. In the study population, 3010 (4%) patients died within 30 days, 7022 (9%) within 180 days and 10,191 (13%) within 360 days. Participants Table 3 shows the association between amount of homecare received and mortality. The crude risk for 30-days mortality increased related to the amount of time for which patients received homecare (Table 3). The patients receiving homecare for > 120 min per week had the highest OR for 30-days mortality – OR 4.51 (95% CI: 4.12–4.93) – when comparing the three homecare categories (Table 3). Discussion Overall, our study showed that older adults aged 65 or above receiving homecare within the month prior to an ED admission were at a significantly higher risk of read- mission and a significantly higher risk of dying compared with older adults not receiving homecare. Although we adjusted for explanatory variables such as comorbidity, sex and age, receiving homecare was a strong predictor of readmission and early death. Older adults who were dependent on daily assistance for ADL (receiving home- care for > 2 h) were at the highest risk among the home- care categories. When adjusting for age, sex, marital status, income, CCI and treatment for mental disorders, patients receiving homecare for > 120 min per week still had an increased OR of 2.25 (95% CI: 2.01–2.50) for 30-days mortality compared with patients without homecare (Table 3). Among our study population, 19% received homecare, which was fewer than found in other studies [1, 11]; how- ever, our study population differs because it consists of both medical and surgical complaints and solely com- prises short ED admissions of < 48 h. Receiving homecare was associated with an increased probability of readmission and mortality and patient’s receiving homecare > 120 min per week had the high- est probability within 30 days from the day of discharge (readmission: 21% (95% CI: 20.1–22.1) and mortality: 11% (95% CI: 10.6–12.1)). For comparison, older adults without homecare had a probability of readmission 13% (95% CI: 12.5–13.1) and of mortality 3% (95% CI: 2.6–2.9) within 30 days. Mortality and homecare We found that receiving homecare was a significant pre- dictor of the risk of dying within 30 days of discharge. Moreover, older adults receiving homecare for more than 2 h a week had a nearly fourfold risk (OR 4.5) and prob- ability (11% vs 3%), compared with patients who did not receive homecare. Similarly, Tanderup et al. reported that Danish medi- cal patients receiving homecare had an increased risk of mortality within 30 days of an acute admission, with a HR of 2.8, compared with patients not receiving homecare [11]. In addition, we found different risks between the homecare groups. Older adults receiving homecare for > 120 min a week had a significantly higher risk of mortal- ity compared with adults receiving homecare for ≤30 min a week (OR 4.5 vs. OR 1.7). Thus, our study contributes Readmission and homecare BMC Geriatrics (2021) 21:696 Page 7 of 9 Fig. 2 Probability for readmission and mortality within 30 days for the categories of homecare. Note: Results show probability (%) with 95% CI for readmission and mortality within 30 days calculated from the adjusted logistic regression analyses Fig. 2 Probability for readmission and mortality within 30 days for the categories of homecare. Note: Results show probability (%) with 95% CI for readmission and mortality within 30 days calculated from the adjusted logistic regression analyses important results to the existing knowledge since older adults dependent on daily assistance for ADL (i.e. receiv- ing homecare for > 2 h) have a markedly increased risk of dying after an ED admission compared to patients with- out homecare. (≥ 75 years) reported no significant association between readmission and homecare [3]. However, the study from Belgium was relatively small, with only 97 patients [3]. Our study clearly demonstrates that older adult Dan- ish residents receiving homecare are at increased risk of readmission following an ED admission. We adjusted for age, comorbidities, income, sex, marital status and treat- ment for mental disorders. However, there might be some overlooked confounding variables not included in the analyses affecting to which extent homecare is an inde- pendent predictor of an increased risk of readmission. A Spanish cohort study identified male sex, comor- bidity, hospital admission and degree of pressure ulcers as significant predictors of mortality among older adults receiving homecare [19]. Male sex, age and comorbid- ity were also identified as independent predictors of one-year mortality in the Danish population of medical patients admitted ≥65 years [11]. We adjusted for age, comorbidities, income, sex, marital status and treatment for mental disorders, and when comparing the crude and the adjusted analyses, the results indicated that some of the selected explanatory variables influenced the results. However, further research is required to elu- cidate the influence of the covariables (e.g. age, sex and comorbidity). Readmission and homecare We found that receiving homecare was a significant predictor of readmission within 30 days of discharge. Older adults receiving homecare > 2 h per week had an increased risk (OR 1.8) corresponding to nearly doubled probability, compared to older adults without homecare (21% vs 13%). When adjusting for age, sex, marital status, income, CCI and treatment for mental disorders, patients receiv- ing homecare for > 120 min per week had a probability for readmission within 30 days of 17% (95% CI: 16.3–18.2) and 5% (95% CI: 4.3–5.3) for mortality compared to a probability of readmission of 13% (95% CI: 12.5–13) and 2% (95% CI: 2.1–2.3) of mortality within 30 days among older adults without homecare (Fig. 2). Tanderup et al. also reported that receiving homecare was a strong predictor of readmission among Danish medical patients (≥ 65 years) compared with medical patients without homecare (HR for competing risk 1.8) [11]. In contrast, a study from Belgium of ED patients Table 3 Association between homecare categories and mortality Notes: OR is the odds ratio and CI is confidence intervals. The logistic regression analyses were adjusted for age, sex, marital status, income, Charlson Comorbidity Index and treatment for mental disorder. Significance levels with a p-value of < 0.05 are marked with * and significance levels with a p-value of < 0.001 are marked with Variables Patients with no homecare Patients receiving homecare ≤ 30 min/week Patients receiving homecare > 30 to ≤120 min/week Patients receiving homecare > 120 min/week Mortality < 30 days Crude OR (95% CI) Adjusted OR (95% CI) 1 (ref.) 1.72 (1.50–1.99) 1.08 (0.92–1.26) 2.18 (1.90–2.50)** 1.26 (1.07–1.47)* 4.51 (4.12–4.93) 2.25 (2.01–2.50) Mortality < 180 days Crude OR (95% CI) Adjusted OR (95% CI) 1 (ref.) 1.71 (1.56–1.88) 1.08 (0.97–1.20) 2.23 (2.03–2.45) 1.31 (1.18–1.46) 4.48 (4.20–4.77) 2.28 (2.11–2.46) Mortality < 360 days Crude OR (95% CI) Adjusted OR (95% CI) 1 (ref.) 1.81 (1.67–1.96) 1.14 (1.05–1.25) * 2.34 (2.16–2.54) 1.39 (1.27–1.52) 4.71 (4.45–4.99) 2.45 (2.28–2.62) Table 3 Association between homecare categories and mortality Notes: OR is the odds ratio and CI is confidence intervals. The logistic regression analyses were adjusted for age, sex, marital status, income, Charlson Comorbidity Index and treatment for mental disorder. Significance levels with a p-value of < 0.05 are marked with * and significance levels with a p-value of < 0.001 are marked with** Elkjær et al. Acknowledgments g The authors thank Andreas Kristian Pedersen, Hospital Sønderjylland, Univer- sity Hospital of Southern Denmark, for statistical support, and Caroline Mar- garet Moss, Hospital Sønderjylland, University Hospital of Southern Denmark, for providing language help. Further, we thank OPEN (Odense Patient data Explorative Network), Region of Southern Denmark, for assisting with prepar- ing the application for the data and with data management. Our study had some limitations. As described, there is no specific registration of ED patients and we thus had to develop a proxy for ED admissions. This cod- ing may have imposed a risk of misclassification bias. However, this data management method is well known and used in other register studies [22, 23]. We included both primary and secondary diagnoses calculating patients CCI but did not differentiate between acute and chronic conditions. Furthermore, lifestyle factors Abbreviations d ED: Emergency department; ADL: Activities of daily living; DNPR: Danish National Patient Registry; NPR: National Prescription Registry; CRS: Danish Civil Registration System; CCI: Charlsons Comorbidity Index; IQR: Interquartile range; CI: Confidence interval; OR: Odds ratio; HR: Hazard ratio. Clinical implications Knowledge about the risk profile of patients receiving homecare can be used to organise and coordinate ED care and preventative treatment. To be able to identify patients at high risk of readmission and death will allow health professionals to implement supportive interven- tions to ease transfer from the ED to primary health care and prevent unnecessary readmission and early death. Elkjær et al. BMC Geriatrics (2021) 21:696 Elkjær et al. BMC Geriatrics (2021) 21:696 Page 8 of 9 Page 8 of 9 Early follow-up visits at home after hospital discharge can reduce preventable readmissions among geriatric patients [6]. Combining several rehabilitative interven- tions before discharge (individual planning, geriatric assessment and patient education) with follow-up after discharge may further reduce readmissions [20]. In Denmark, information about whether patients receive homecare or not is easy to access from the electronic communication between the municipality and local hos- pitals, and it is relatively reliable and requires no addi- tional training for use. such as smoking and overweight can be confounding factors, but was not possible to included using registry data. Further, we used data on marital status (married/ unmarried); thus, we could not tell whether the older adults lived with adult children or cohabitants. How- ever, the study population was aged 65 or above, so we assumed that relatively few persons lived together with- out being married according Danish culture. Finally, the Danish municipalities had months where data transfer was inadequate due to change of registrations system; however, we completed sensitivity analyses excluding these months, which confirmed the robustness of our results. A study from Canada found that older adults receiving homecare experienced at least one care transition (moves between care settings such as a home nursing, home and hospital) during the last 30 days before death, and one- fifth experienced two or more hospitalisations [21]. If this knowledge is combined with our results demonstrating a nearly fourfold increased risk of dying within 30 days of discharge among older adults receiving homecare, the ED admission may be considered a time-limited window of opportunity to offer help to fragile patients. Strengths and limitations Our study has important strengths. This is the first study to investigate readmission rates after a short ED admission comparing older adult patients related to the amount of homecare received, in a large national cohort. We used the DNPR to identify our study population and linked the patients between the registries using the unique 10-digit identification number. Registry data have high completeness and validity [12–17], and to supple- ment the CCI, we added data from the Danish NPR to describe the patients’ mental health to obtain a complete picture of the patients’ total comorbidities. Conclusion h Receiving homecare prior admission was associated with an increased risk of readmission and death follow- ing admission to an ED for older adults aged 65 or older. Older adults who received homecare for more than 2 h a week were at the highest risk compared with those who received less or no homecare. Collaboration between the ED and primary health care facility in relation to reha- bilitation and end-of-life care is essential to improve the quality of care for older adults who receive homecare, particularly those receiving homecare for more than 2 h a week, because of their increased risk of readmission and mortality. f We suggest increased attention given to older adults dependent on daily assistance for ADL (receiving home- care for > 2 h). Care interventions could include evalu- ation of their needs and preferences for rehabilitative interventions or end-of-life care. Whether older adults receive homecare or not and the amount of time allo- cated to homecare can also be used to develop a plan for future treatment and care and ensure that collaboration between the ED and primary health care sector focuses on preventative interventions. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12877-021-02644-6. Additional file 1. Additional file 2. Funding h visits for geriatric patients discharged from hospital. Eur Geriatr Med. 2018;9(5):613–21. g The Novo Nordisk Foundation and Hospital Sønderjylland, University Hospital of Southern Denmark, supported the study financially but were not involved in any aspect of study design, data collection, data analysis or interpretation of the data. 7. Fluitman KS, van Galen LS, Merten H, Rombach SM, Brabrand M, Cooksley T, et al. Exploring the preventable causes of unplanned readmissions using root cause analysis: coordination of care is the weakest link. Eur J Intern Med. 2016;30:18–24. Consent for publication Not applicable. 13. Schmidt M, Pedersen L, Sorensen HT. The Danish civil registration system as a tool in epidemiology. Eur J Epidemiol. 2014;29(8):541–9. 14. Schmidt M, Schmidt SA, Sandegaard JL, Ehrenstein V, Pedersen L, Sorensen HT. The Danish National Patient Registry: a review of content, data quality, and research potential. Clin Epidemiol. 2015;7:449–90. Availability of data and materialsi 8. Gray LC, Peel NM, Costa AP, Burkett E, Dey AB, Jonsson PV, et al. Profiles of older patients in the emergency department: findings from the interRAI multinational emergency department study. Ann Emerg Med. 2013;62(5):467–74. The data that support the findings of this study are available from Statistics Denmark. However, data were used under licence for the current study and are not publicly available. 9. Ministry of Health. Healthcare in Denmark: an overview. Copenhagen: Ministry of Health; 2017. ISBN: 978-87-7601-365-3. Declarations 10. Wilson DM, Truman CD, Hewitt JA, Els C. Are chronically ill patients high users of homecare services in Canada? Am J Manag Care. 2015;21(10):e552–9. Competing interests The authors declare that they have no competing interests. y 15. Danish Registration of Elderly [Web Page]. Statistics Denmark 2020 [cited 2020 2108]. Available from: https://www.dst.dk/da/Statistik/dokumentat ion/Times/aeldredokumentation. Accessed 29 Mar 2021. Author details 1 Department of Emergency Medicine, University hospital of Southern Denmark, Kresten Philipsens Vej 15, 6200 Aabenraa, Denmark. 2 Department of Regional Health Research, University of Southern Denmark, Odense, Denmark. 3 Research Unit of Health Sciences, Hospital of South West Jut- land, Esbjerg, University hospital of Southern Denmark, Esbjerg, Denmark. 4 Department of Internal Medicine, University hospital of Southern Denmark, Aabenraa, Denmark. 5 Sygehus Sønderjylland, University hospital of Southern Denmark, Aabenraa, Denmark. 6 Danish Hospital for Rheumatic Diseases, University hospital of Southern Denmark, Sønderborg, Denmark. 7 Department of Emergency Medicine, University hospital of Southern Denmark, Esbjerg, Denmark. 8 Department of Emergency Medicine, Odense University Hospital, Odense, Denmark. 16. Pottegard A, Schmidt SAJ, Wallach-Kildemoes H, Sorensen HT, Hallas J, Schmidt M. Data resource profile: The Danish National Prescription Regis- try. Int J Epidemiol. 2017;46(3):798–f. y p 17. Baadsgaard M, Quitzau J. Danish registers on personal income and trans- fer payments. Scand J Public Health. 2011;39(7 Suppl):103–5. 18. Quan H, Li B, Couris CM, Fushimi K, Graham P, Hider P, et al. Updating and validating the Charlson comorbidity index and score for risk adjustment in hospital discharge abstracts using data from 6 countries. Am J Epide- miol. 2011;173(6):676–82. 19. Badia JG, Santos AB, Segura JCC, Terén CA, González LC, Ramírez EL, et al. Predictors of mortality among elderly dependent home care patients. BMC Health Serv Res. 2013;13(1):316. Received: 14 April 2021 Accepted: 16 November 2021 Received: 14 April 2021 Accepted: 16 November 2021 20. Åhsberg E. Discharge from hospital – a national survey of transition to out-patient care. Scand J Caring Sci. 2019;33(2):329–35. 21. 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The Danish database for acute and emergency hospital contacts. Clin Epidemiol. 2016;8:469–74. y 23. The Danish database for acute and emergency hospital contacts. Clin Epidemiol. 2016;8:469–74. p 2. Kennelly SP, Drumm B, Coughlan T, Collins R, O’Neill D, Romero-Ortuno R. Characteristics and outcomes of older persons attending the emergency department: a retrospective cohort study. QJM. 2014;107(12):977–87. 2. Kennelly SP, Drumm B, Coughlan T, Collins R, O’Neill D, Romero-Ortuno R. Characteristics and outcomes of older persons attending the emergency department: a retrospective cohort study. QJM. 2014;107(12):977–87. Authors’ contributions BG, MB, JP and ME designed the study. BG, MB and JP supervised the research project. DLW and ME performed the preparation of data for analysis. ME ana- lysed the data. BG, MB, JP, CBM, DLW and ME contributed to data interpreta- tion and the manuscript. All authors provided substantial contribution revising the manuscript critically for important intellectual content and approved the version to be published. Page 9 of 9 Elkjær et al. BMC Geriatrics (2021) 21:696 Elkjær et al. BMC Geriatrics (2021) 21:696 Ethics approval and consent to participate The study was reported to the Danish Data Protection Agency (file no. 2008-58-0035). In accordance with Danish law, register studies are exempted from gaining informed consent from participants. The need for formal ethi- cal approval was waived by the Regional Scientific Ethical Committee for the Region of Southern Denmark (file no. 20182000–68) because the study did not involve biomedical interventions. Reporting followed the STROBE guidelines. 11. Tanderup A, Ryg J, Rosholm JU, Lassen AT. Association between the level of municipality healthcare services and outcome among acutely older patients in the emergency department: a Danish population-based cohort study. BMJ Open. 2019;9(4):e026881. 12. Thygesen LC, Daasnes C, Thaulow I, Bronnum-Hansen H. Introduction to Danish (nationwide) registers on health and social issues: structure, access, legislation, and archiving. Scand J Public Health. 2011;39(7 Suppl):12–6. Publisher’s Note 3. Deschodt M, Devriendt E, Sabbe M, Knockaert D, Deboutte P, Boonen S, et al. Characteristics of older adults admitted to the emergency depart- ment (ED) and their risk factors for ED readmission based on compre- hensive geriatric assessment: a prospective cohort study. BMC Geriatr. 2015;15:54. 3. Deschodt M, Devriendt E, Sabbe M, Knockaert D, Deboutte P, Boonen S, et al. Characteristics of older adults admitted to the emergency depart- ment (ED) and their risk factors for ED readmission based on compre- hensive geriatric assessment: a prospective cohort study. BMC Geriatr. 2015;15:54. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. 4. Klinge M, Aasbrenn M, Öztürk B, Christiansen CF, Suetta C, Pressel E, et al. Readmission of older acutely admitted medical patients after short- term admissions in Denmark: a nationwide cohort study. BMC Geriatr. 2020;20(1):203. 4. Klinge M, Aasbrenn M, Öztürk B, Christiansen CF, Suetta C, Pressel E, et al. Readmission of older acutely admitted medical patients after short- term admissions in Denmark: a nationwide cohort study. BMC Geriatr. 2020;20(1):203. 5. Fløjstrup M, Henriksen DP, Brabrand M. An acute hospital admission greatly increases one year mortality – getting sick and ending up in hos- pital is bad for you: a multicentre retrospective cohort study. Eur J Intern Med. 2017;45:5–7. 6. Pedersen LH, Brynningsen P, Foss CH, Gregersen M, Barat I, Damsgaard EM. Avoidable readmissions seem to be reduced by early follow-up 6. Pedersen LH, Brynningsen P, Foss CH, Gregersen M, Barat I, Damsgaard EM. Avoidable readmissions seem to be reduced by early follow-up
https://openalex.org/W2978702772	https://revistas.ucm.es/index.php/CLAC/article/download/65657/4564456551868	es		Estudio contrastivo sobre los adverbios de actos de habla en español y sus paralelos en chino: una perspectiva de semántica de eventos	Círculo de lingüística aplicada a la comunicación	2,019	cc-by	15,797	ARTÍCULOS Círculo de Lingüística Aplicada a la Comunicación ISSN: 1576-4737 http://dx.doi.org/10.5209/CLAC.65657 Estudio contrastivo sobre los adverbios de actos de habla en español y sus paralelos en chino: una perspectiva de semántica de eventos Chen Chen 陈晨1 Recibido: 28 de mayo de 2019 / Aceptado: 17 de junio de 2019 Resumen. El presente estudio, basado en la semántica de eventos, profundiza en la diferencia de los recursos lingüísticos utilizados en español y chino para expresar las semánticas conceptuales de eventos de actos de habla con orientación hacia emisor o receptor, y observa la evolución diacrónica de los adverbios de actos de habla española y sus paralelos en chino, con el fin de descubrir la motivación de la diferencia inter-lingüística de las dos lenguas en contraste. El desarrollo diacrónico de los adverbios de actos de habla en español y sus paralelos en chino muestra un patrón de evolución similar, pero las dos expresiones en las dos lenguas, en contraste, se encuentran en diferentes etapas de lexicalización. La razón de la diferencia del grado de lexicalización podría ser, que los dos idiomas son morfológicamente diferentes. El español, rico en aspectos morfológicos, es más capaz de combinar varias semánticas de eventos mediante recursos léxicos; los adverbios se encargan de la proyección de eventos de actos de habla a la estructura superficial, pues, otras constituyentes semánticas conceptuales no necesitan obligatoriamente presentarse, y especialmente, la entidad semántica de los verbos enunciados se puede codificar implícitamente. Mientras el chino, como es una lengua que carece de aspectos morfológicos, tiene que recurrir a los recursos sintácticos para codificar el mismo concepto de evento, las constituyentes semánticas y, sobre todo, los verbos enunciados tienen que ser proyectados explícitamente en la estructura superficial. Nuestro trabajo también pone de manifiesto la viabilidad y la efectividad de la estructura conceptual compartida por las dos lenguas en contraste, que sirve como tertium comparationis en el estudio inter-lingüístico y abre una nueva perspectiva del análisis contrastivo entre los dos idiomas tipológicamente diferentes como el español y el chino. Palabras clave: adverbios de actos de habla, evento de semánticas, semántica conceptual, verbo enunciativo, lexicalización [en] A contrastive study on the Speech-act Adverbs in Spanish and its parallels in Chinese: An Event Semantics perspective Abstract. The present study, based on the semantics of events, delves into the difference of the linguistic resources used in Spanish and Chinese to express the conceptual semantics of events of speech acts oriented towards transmitter or receiver, and observes the diachronic evolution of adverbs of Spanish-speaking acts and their parallels in Chinese, in order to discover the motivation of the interlinguistic difference of the two languages in contrast. The diachronic development of adverbs of speech acts in Spanish and their parallels in Chinese shows a similar pattern of evolution, but the two expressions in the two languages, in contrast, are in different stages of lexicalization. The reason for the difference in the degree of lexicalization could be that the two languages are morphologically different. Spanish, rich in morphological aspects, is more capable of combining several semantics of events through lexical resources; Adverbs are responsible for the projection of events of speech acts to the _____________ 1 Xiangtan University 湘潭大学 (China)/Instituto Confucio de la Universidad de León (Spain). Correo electrónico: cche@unileon.es/ chenchenleon@163.com CLAC 79: 217-256 217 218 Chen, C 陈晨. CLAC 79 2019: 217-256 surface structure, because other conceptual semantic constituents do not necessarily need to be presented, and especially, the semantic entity of the verbs enunciated can be encoded implicitly. While Chinese, as it is a language that lacks morphological aspects and therefore it has to resort to syntactic resources to encode the same concept of event, the semantic constituents and, above all, the enunciated verbs have to be explicitly projected in the surface structure. Our work also highlights the feasibility and effectiveness of the conceptual structure shared by the two contrasting languages, which serves as tertium comparationis in inter-linguistic study and opens a new perspective of contrastive analysis between the two typologically different languages such as the Spanish and Chinese. Keywords: adverbs of speech acts, semantic event, conceptual semantics, verb of speaking, lexicalization [ch] 西班牙语“言说行为副词”与其在汉语中对应表达的对比研究: 基于事件语义学的视角提要:本文基于事件语义学的视角，以西班牙语“言说行为副词”及其在汉语中的对应表达为研究对象, 分析两者在语言表达方式上的差异，从历时角度考察了西汉语中言说行为表达的词汇化和语法化过程，探究西汉语中言说行为表达方式差异的内在成因。西班牙语“言说行为副词”及其在汉语中的对应表达的历时演化路径相似，但词汇化程度不一致，其主要成因在于汉西两种语言的形态类型不同: 西班牙语的形态变化相对丰富，“言说行为副词”词汇化程度较高，具备融合言说事件语义信息成分的能力，将言说行为事件投射到句法表层结构，其他语义概念信息在语言表层则不必显性编码。与西班牙语相比，汉语形态手段不发达，言说行为表达手段的词汇化程度较低，词汇融合言说事件语义能力较弱。因此，汉语更倾向于将言说事件中各语义元素组合起来表达，其他语义概念信息尤其是言说动词在语言表层必须显性编码。另外，本文还强调了概念语义结构在跨语言对比研究中的有效性和可行性，将西汉语中共享的概念语义结构作为语际对比的基础，能为西班牙语和汉语这两种不同语言类型的对比分析开辟新的研究视角。关键词: 言说行为副词，事件语义学，概念语义，言说动词，词汇化 Cómo citar: Chen, Chen 陈晨 (2019). Estudio contrastivo sobre los adverbios de actos de habla en español y sus paralelos en chino: una perspectiva de semántica de eventos. Círculo de Lingüística Aplicada a la Comunicación 79, 217-256, http://dx.doi.org/10.5209/CLAC.65657 Índice. 1. Introducción. 2. Tertium comparationis y perspectiva del análisis. 2.1 Tertium comparationis: Estructura conceptual. 2.2 Perspectiva del análisis: Teoría semántica de eventos. 2.3 Semántica de eventos de los actos de habla en español. 3. Los recursos lingüísticos asumidos como los paralelos de los AAHs españoles en chino. 3.1 Las consultas de las posibles EAHs en el corpus CCL. 3.2 Clasificación de las EAHs en chino. 4. Análisis y observaciones. 4.1 Las correspondencias entre los AAHs y las EAHs no son bilaterales. 4.2 Dos tipos de recursos lingüísticos: recursos léxicos versus recursos sintácticos. 5. Evolución de los AAHs y las EAHs en las dos lenguas en contraste. 5.1 Evolución de los AAHs en español. 5.2 Evolución de las EAHs en chino. 6. Conclusiones. Agradecimientos. Bibliografía. Anexo 1. Anexo 2. 1. Introducción En español los adverbios oracionales francamente, sinceramente, honestamente, honradamente pueden ser interpretados como adverbios de actos de habla (en adelante, AAHs). Los adverbios oracionales que nos ocupamos en el presente trabajo, que han recibido denominaciones muy diversas en la bibliografía: adverbios orientados hacia el hablante Jackendoff (1972), Dik, Hengeveld, Vester y Vet (1990), Ernst (2002), adverbios parentéticos, Bartsch (1976); adverbios performativos, Borillo (1976), Egea (1979); modalizadores de la enunciación, Nef y Nølke (1982) ; Chen, C. 陈晨 CLAC 79 2019: 217-256 219 adverbios pragmáticos, Bellert(1977), Conte (1988) ; adverbios disjuntos de estilo, Greenbaum (1969), Quirk y otros (1985), Schreiber (1972), Koktová (1986) ; cualificadores del decir, Kovacci (1986) ; adverbios de enunciación, Conte (1987), Ducrot (1980), Berrendonner (1987), Fuentes (1991), González (1997) ; adverbios de acto lingüístico, Lonzi (1991); adverbios ilocutivos, Wilson y Sperber(1993), Nøjgaard (1992, 1995, 1995) ; satélites ilocutivos, Dik (1989, 1997a, 1997b) ; adverbios de manera del decir, Rodríguez Ramalle (2003) ; adverbios orientados hacia el emisor o el receptor, Kovacci (1999) , Torner Castell (2005). En el presente trabajo nosotros tomamos la denominación de Lonzi (1995), Cinque (1999) y San (2012): adverbios de actos de habla. Generalmente, los AAHs se orientan hacia el hablante/emisor del enunciado en contextos declarativos, ocupan posiciones periféricas con respecto a la predicación oracional, su semántica conceptual (Jackendoff 1997) se puede parafrasear como: indicar una evaluación del hablante/emisor sobre su propia actitud en cuanto enunciador, la actitud del hablante/emisor ante lo que dice. Se trata de un grupo relativamente exiguo, que en español cuenta tan sólo con cuatro unidades que acabamos de mencionar, y están derivados de los siguientes cuatro adjetivos: franco, sincero, honesto, honrado. El uso que de este grupo de adverbios son esperables, en función del significado que poseen los adjetivos con que se forman. En primer lugar, todos ellos presentan lecturas oracionales en las que se orientan hacia el hablante/emisor: (1) Con que fuimos viendo todos los cuadros, y yo, francamente, no encontraba ninguno en que pudieras estar tú. (Hidalgo Manuel, Azucena, que juega al tenis, 1988) (2) Yo, sinceramente, creo que ya ha llegado el momento de que se largue. (VV.AA. El club de la Comedia Presenta Ventajas de ser incompetente y otros monólogos de humor,2001) (3) Si tengo que elegir, prefiero cazar vampiros, honestamente, aunque todavía siento el sofoco que me provocaban los corsés. (Fotogramas nº 1921, 11/2003) (4) Y creo, honradamente, que los dos diarios tienen su parte de razón. (El Mundo, 25/05/1996) Paralelamente a este uso, los AAHs también pueden orientarse hacia el oyente/receptor en contextos interrogativos o imperativos. Esto ocurre cuando, en un turno de habla, un hablante pide o espera una respuesta del oyente/receptor y le solicita que en ésta tenga la actitud denotada por el adverbio: (5) {Francamente / honestamente / honradamente / sinceramente}, ¿crees que esto es así? (Sergi 2005) (6) {Francamente / honestamente / honradamente / sinceramente}, dime qué opinas. (Sergi 2005) En concreto, al orientarse hacia el hablante/emisor, los AAHs ponen de relieve que este se compromete a cumplir la cualidad denotada por su base adjetiva a la hora de proferir el enunciado. Si se orientan hacia el oyente/receptor, el hablante/emisor exige a su interlocutor que cumpla la misma condición al responder (San,2012). De hecho, los adverbios francamente, sinceramente, honestamente, honradamente no solo aparecen como modificadores de actos de habla, sino también pueden actuar como adverbios de manera orientados al sujeto. Véase los ejemplos de (7) a (8). Chen, C 陈晨. CLAC 79 2019: 217-256 220 (7) María respondió francamente que todo había sido mentira. (Rodríguez 2003) (8) María honestamente devolvió todo el dinero. (Rodríguez 2003) No es casual que existe la relación entre los adverbios de manera orientados al sujeto y los AAHs orientados hacia el responsable de la enunciación, ya que las mismas bases adjetivas franco, sincero, honesto, honrado pueden intervenir en la formación de ambos adverbios, predicándose a los individuos en relación a la acción que realizan y, por lo tanto, estos pueden llegar a expresar propiedades en las acciones que realiza un individuo. Cabe citar, los adverbios brevemente, sencillamente, escuetamente, que caracterizan la forma en que expresa la proposición mediante su uso metalingüístico (Bellert 1977), y que pueden considerarse como otro tipo de AAHs, pero no forman parte del objetivo del presente estudio. En el presente trabajo delimitamos estrictamente el empleo del término orientación en el sentido que lo mencionábamos antes y resumimos las clasificaciones y características de los AAHs en español en la tabla 1: Los AAHs en español Grupos de adverbios Contexto en lo que aparecen Semántica conceptual A. Orientación hacia el emisor/hablante Los contextos declarativos Indican una evaluación del hablante/emisor sobre su propia actitud en cuanto enunciador, la actitud del hablante/emisor ante lo que dice. B. Orientación hacia el receptor/oyente Los contextos interrogativos o imperativos El hablante/emisor pide o espera una respuesta del oyente/receptor y le solicita que en ésta tenga la actitud denotada por el adverbio. Tabla 1: Clasificaciones y características de los AAHs en español En cuanto al chino, nos llaman la atención varios fenómenos lingüísticos. En primer lugar, curiosamente, no encontramos los denominados adverbios de actos de habla (Zhang 2000). Las expresiones subrayadas en los ejemplos (9) a (13) no son correctas en chino: (9) 坦率地 (Franco DE) ，……。 (10) 坦白(地) (Franco DE) ，……。 (11) 真诚地 (Sincero DE) ，……。 (12) 诚实地 (Honesto DE) ，……。 (13) 老实(地) (Honrado/Honesto/Sincero/Franco DE) ，……。 Generalmente, los elementos adjetivos 坦率, 坦白, 真诚, 诚实, 老实 no se usan independientes, sino que se suelen combinar con los verbos enunciativos como 说 o 讲 (decir o contar), formándose una estructura ¨Adverbio + [ 地 ] + verbo enunciativo ¨. En tal estructura, puede aparecer la partícula 地 (DE); pero es evidente que las partículas estructurales ¨的¨ y ¨地¨(DE) tienen unas peculiaridades sintácticas que hacen que se siga discutiendo sobre si se trata ¨的¨ y ¨地¨ de la Chen, C. 陈晨 CLAC 79 2019: 217-256 221 misma partícula o se trata ¨的¨ como marcador de modificador adjetival y ¨地¨ de marcador de modificador adverbial (Lu 1963, 1983; Lyu 1981, 2002; Zhang 2010, 2012; Chen 2016; Yang 2017). Aceptamos la explicación que propone Lyu (2002) para la elección de una misma partícula y no de dos partículas distintas, la aclaración es la siguiente: La distinción de 的／地 no apareció hasta la época del Movimiento del Cuatro de Mayo cuando se introdujeron muchas obras literarias extranjeras. Los traductores descubrieron que en idiomas como inglés y francés la distinción entre el adjetivo y el adverbio se representaba en el sufijo e intentaron mostrar en el texto traducido al chino la distinción con 的 y 地 (Lyu, 1981). En realidad, 的 y 地 son una misma palabra, y no se pueden utilizar para calificar las categorías gramaticales de palabra en función de las dos formas de escritura. Si por una parte se dice que se escribe 的 porque es adjetivo, mientras se escribe 地 porque es adverbio, pero a su vez, se dice que es adjetivo porque se escribe 的 mientras es adverbio porque se escribe 地, caeremos en una petición de principio (Lyu, 2002). Además, según los datos de la frecuencia de uso de las dos palabras 的 y 地 en el corpus de CCL, también se indica que en la lengua moderna china no es compatible con la idea de que las dos palabras son completamente diferentes (Zhang 2000,2012). Hay que sumar a este factor, el hecho de que 的 y 地 son una misma palabra, es muy importante, según Yang (2017), poder calificar la categoría gramatical de los elementos coordinados de DE. Pues, en dicha construcción verbal ¨Adverbio + [DE] + verbo enunciativo ¨, las palabras 坦率, 坦白, 真诚, 诚实, 老实 son originalmente adjetivos que se convierten en adverbios al indicarnos la actitud del hablante/emisor ante lo que dice, o hacia lo que se requiere al oyente/receptor en el turno siguiente. Este es el caso de los llamados ¨adjetivos adverbializados¨. En español, estos adjetivos ‘adverbializados’ pueden ser reemplazados por adverbios terminados en –mente, han sido dominados por ¨adverbios adjetivales¨. Para saber más sobre su definición se recomienda la lectura del trabajo de Kovacci (1999). Por lo tanto, a nuestro juicio, será razonable que modifiquemos la denominación de dicha construcción por ¨Adjetivo adverbializado + [DE] + verbo enunciativo ¨ que se refleja claramente las funciones gramaticales de los constituyentes de los adjetivos adverbializados y estatus sintáctica de la partícula estructural DE en ella. Véase los ejemplos de (14) a (15). (14) 坦率地讲，我认为这完全是偶合。 (CCL de chino moderno) Glosa: Franco DE decir, yo creer esto en absoluto es coincidencia. Trad.: Francamente, creo que esto es pura coincidencia. (15) 老实说，你想不想出去看看？ (CCL de chino moderno) Glosa: Honesto [DE] decir, ¿tú querer no querer salir y ver? Trad.: Honestamente/Francamente, ¿si quieres o no salir a ver mundo? Como se observa en los datos españoles (1) y (6) y los datos chinos (14) y (15), se nota otra diferencia, destacando los comportamientos sintácticos de los enunciados: en las oraciones chinas se presentan sintácticamente los verbos enunciativos, 222 Chen, C 陈晨. CLAC 79 2019: 217-256 mientras que, en español, no aparecen. Dejaremos, sin embargo, este tema aquí, puesto que de los verbos enunciativos vamos a ocuparnos en el apartado 3. Con todo esto, hemos ido viendo como surgen algunas dudas o preguntas sobre los fenómenos lingüísticos de las dos lenguas que se nos muestran arriba, por ello, nos preguntamos lo siguiente: a) La comparación total o parcial entre dos lenguas distintas exige la existencia de un terreno común que la haga posible. Es lo que se conoce como tertium comparationis: the common ground on which two languages can be compared to be able to establish (dis)similarities” (Ebelin 1998). Por tanto, ¿Cuál sería el tertium comparationis de los AAHs en español y las EAHs en chino? Dado que, aunque las dos expresiones en las dos lenguas son semánticamente equivalentes, son sintácticamente diferentes. b) Si en chino no se dispone de los AAHs, ¿cuáles son las posibles expresiones de actos de habla (en adelante, las EAHs) del chino? Es decir, creemos que los recursos lingüísticos típicos para la expresión de determinadas semánticas conceptuales de actos de habla son distintos en las dos lenguas analizadas, ¿Cuáles son los recursos más representativos en chino? c) ¿Por qué en las EAHs se representan los verbos enunciativos, pero en español no se representan sintácticamente? En otros términos, ¿Por qué la entidad semántica de los verbos enunciados se puede codificar implícitamente en español y mientras, los verbos como ''说/讲/告诉(decir/hablar/contar)'' se deben codificar explícitamente en chino? ¿Por qué la lengua española tiene capacidad de proyectar la semántica conceptual del evento de actos de habla mediante recursos léxicos, mientras que la lengua china dependería más de recursos sintácticos? d) ¿Tiene que ver esta diferencia intre-lingüística con la tipología morfológica? ¿Han alcanzado estos recursos lingüísticos el mismo grado de lexicalización en las dos lenguas en contraste? Con el propósito de responder a tales interrogantes, primero hay que determinar cuál es el tertium comparationis (en adelante, TC) del presente análisis contrastivo acerca de los distintos recursos lingüísticos en español y chino; para ello recurriremos a la hipótesis de la Semántica Conceptual (en adelante, SC) desarrollada por Jackendoff (1983, 1990, 2002, 2007 y 2010) y Pinker (1989) y Pustejovsky (1995), que se centra en el estudio de la interfaz entra la semántica y sintáctica. Ellos proponen la existencia de una Estructura Conceptual (en adelante, EC), que es innata y común a todas las lenguas. Tomaremos como el TC la EC para desarrollar la comparación entre dos lenguas analizadas. Para describir e interpretar directamente de la EC de eventos de actos de habla en las dos lenguas analizadas, acudiremos a la teoría semántica de eventos propuesta por Davidson (1967) y desarrollada por Parsons (1990). Para proceder al análisis de la distribución sincrónica y la evolución diacronía de los AAHs y las EAHs, hemos acudido a tres corpus masivos: el Corpus de Referencia del Español Actual (CREA) y Corpus de Referencia Diacrónico del Español (CORDE) y Corpus del Centro de Investigación de la Lingüística China de la Universidad de Pekín (CCL/北京大学中国语言学研究中心语料库). El Corpus de CCL consta del corpus de chino moderno, el corpus de chino clásico y el Chen, C. 陈晨 CLAC 79 2019: 217-256 223 corpus bilingüe chino-inglés. En el presente trabajo, consultaremos al corpus de chino moderno y el clásico. Cabe mencionar, que el trabajo con el corpus requiere un ¨factor humano¨, ya que las búsquedas de tales corpus, deben revisarse manualmente, tanto por los errores en el etiquetado, que se producen de forma automática, como para evitar que haya secuencias que no se corresponden con las que buscamos (se detallará más adelante, en el apartado 3). Aparte de los corpus, los ejemplos de obras de otros autores también nos servirán de orientación útil para nuestro análisis. El trabajo está organizado del siguiente modo. En el apartado 2 se introduce el TC y la perspectiva del análisis del presente trabajo, con el fin de construir la forma lógica de la EC de eventos de actos habla en español. En el apartado 3, basándose en las representaciones obtenidas en el anterior apartado, se trata de deducir las posibles expresiones de actos de habla en chino mediante la consulta avanzada o compleja en el corpus CCL. En el apartado 4, se analizan las correspondencias entre los AAHs y las EAHs y las diferencias de recursos lingüísticos utilizados en las dos lenguas en contraste. En el apartado 5, se observa la evolución diacrónica de los AAHs y las EAHs. Y finalmente en el último apartado se presentan las conclusiones del estudio. 2. Tertium comparationis y perspectiva del análisis 2.1. El TC: Estructura conceptual Tekin (2012) tiende a interpretar el TC como características universales, como se puede leer en la siguiente cita: “...was kann dann als t.c. angesetzt werden? Was ist allen Sprachen gemeinsam, oder besser gefragt: Was die Vereinigungsmenge von Sprachen? ” (Tekin 2012, énfasis: ÖT) ¿...qué podría entonces aplicarse como el tertium comparationis? ¿Qué tienen todas las lenguas en común, o mejor aún: ¿cuál sería el conjunto en común de las lenguas?” (Tekin 2012, énfasis: ÖT, traducción: ARGT) Determinar cuál es el TC de un análisis contrastivo es uno de los principales quebraderos de cabeza de este campo. Todo estudio contrastivo tiene que abordarse desde algún modelo teórico. El estructuralismo toma como el TC la correspondencia formal, en otras palabras, la estructura superficial en términos de Chomsky (1957). Sin embargo, como argumenta Krzeszowski (1984), si solo se tiene en cuenta el criterio formal, no sería posible realizar la comparación de un recurso lingüístico de una lengua que no tiene su equivalente (formal) en otra lengua. El generativismo propone como el TC la equivalencia en el nivel de la estructura profunda, ¨equivalent constructions have identical deep structures even if on the surface they are markedly different. ¨ (Krzeszowski 1971). Pero la estructura profunda recupera solo el significado proposicional que solo es una parte del significado de una expresión lingüística determinada (Halliday 1973, 1975), y no proporciona un equivalente semántico adecuado que pueda usarse como el TC. Para determinar cuál es el TC del presente análisis contrastivo acerca de los distintos recursos lingüísticos en español y chino, recurriremos a la hipótesis de SC, que es una de las propuestas semánticas más importantes de la gramática de los 224 Chen, C 陈晨. CLAC 79 2019: 217-256 últimos años, donde plantea el léxico como un componente independiente, con sus propios principios, que interactúa con la sintaxis. Es decir, la teoría SC radica en estudiar la forma en que los conceptos, pensamientos o ideas se expresan en la sintaxis del lenguaje natural, explora las relaciones existentes entre la semántica léxica y la sintaxis desde un punto de vista semántico-céntrico, nos es especialmente útil porque permite establecer qué aspectos estructurales de significado comparten las unidades léxicas semánticamente relacionadas (Lieber 1992, Plag 1999, Gràcia et al, 2000). Los problemas que plantea la creatividad del lenguaje y su adquisición, sobre los que se fundamenta la teoría sintáctica generativista, se reflejan de igual manera en el dominio de la semántica conceptual. Jackendoff (1983, 1987, 1990) justifica la existencia de una representación mental, denominada estructura conceptual (en adelante, EC), que ejemplifica los significados de las oraciones y sirve como base formal para la inferencia y la conexión con el conocimiento, así como de la percepción mundial, que sería innata y común a todas las lenguas y que sirve como la “sintaxis del pensamiento” (Jackendoff 2010: 7). La hipótesis central de la SC es que, el inventario de conceptos léxicos, potencialmente infinito, se construye a partir de una base innata de conceptos posibles, que han de ser codificados por una mente finita. Esto nos lleva forzosamente a la hipótesis de que la base innata de conceptos, consiste en un conjunto de principios generativos: un conjunto de elementos primitivos y principios combinatorios que determinan el conjunto de conceptos léxicos. Figura 1. Relación inherente de Primitivos conceptuales, Semánticas conceptuales, Estructura conceptual y Estructura sintáctica, tomado y adoptado de Jackendoff (2010) A la vista de Figura 1, comprendemos rápidamente que la EC estaría en correspondencia con unas ciertas reglas de formación, por un lado, con otros tipos de representación mental (sistemas visual, auditorio, motor, etc.) por el medio de los principios de proyección (mapping), y por otro, con el componente sintáctico del lenguaje. Cabe preguntarse, ¿cómo se forma la EC y dónde situamos la EC? Jackendoff (1983, 1990) postula que los primitivos conceptuales que forman parte del pensamiento, son iguales en todas las lenguas, por lo que cabe considerarlos una especie de principios de clasificación del mundo, categorías ontológicas que no son estrictamente lingüísticas. Para este autor, existe en el análisis semántico un conjunto de categorías, que él denomina las “partes del habla conceptuales”, tales como Cosa, Evento, Estado, Acción, Lugar, Camino, Chen, C. 陈晨 CLAC 79 2019: 217-256 225 Propiedad y Cantidad. Dichos primitivos conceptuales pueden formar las ECs mediante unas ciertas reglas de formación, las cuales corresponden a las estructuras sintácticas. Cada constituyente sintáctico (exceptuando expletivos vacíos, como it y there) se proyecta en uno conceptual, pues muchos constituyentes conceptuales se encuentran contenidos dentro de las piezas léxicas. Véase el ejemplo (16): (16) Juan corrió hacia la casa. (Jackendoff 1991) En esta oración, Juan y casa corresponden al constituyente Cosa, el sintagma prepositivo hacia la casa indica un Camino, y la oración completa es un Evento. El componente Camino no aparece en correr, pero si en adentrarse, que además iría sujeto a un sintagma prepositivo indicativo de Lugar. Así, Jackendoff propone que las ECs son previas a la sintaxis. Las reglas de correspondencia deben ir de una EC universal a una sintaxis particular de una lengua, es decir, la sintaxis resulta de la proyección de las ECs. Concretamente, resumimos abajo las ideas básicas de la hipótesis SC que están relacionadas con el presente trabajo: a) Los primitivos conceptuales son innatos y tienen características universales en el cerebro humano, forman parte del pensamiento y de las semánticas conceptuales. b) Mediante unas ciertas reglas de formación, las semánticas conceptuales conectan con las ECs, que serán compartidas tanto por la lengua española como por la lengua china. c) Las ECs compartidas son innatas y comunes en las dos lenguas en contraste y éstas sirven como “sintaxis del pensamiento”, corresponden a la estructura sintáctica a través de los principios de proyección. d) La EC es una estructura sintáctica más rica y está proyectada selectivamente en la estructura sintáctica superficial. Observando este último punto, nos damos en cuenta que tanto en el nivel intralingüístico como en el inter-lingüístico la correspondencia entre el significado y la forma es one to many (Chesterman1998). En cuanto al nivel intralingüístico, hay que saber que una misma lengua cuenta con múltiples posibilidades para expresar y realizar la proyección de un determinado semántico conceptual, siendo unas más típicas o más predominantes que otras. Por ejemplo, para realizar la proyección de un semántico conceptual de acto de habla en español, a su vez aparte de los AAHs, también se acuden a otros recursos lingüísticos: a) Adverbios: francamente, sinceramente, honestamente, honradamente; b) Otras expresiones adverbiales equivalentes: con (toda) franqueza, con sinceridad, en confianza, etc.; c) Cláusula: (Te) digo francamente / sinceramente / honestamente / honradamente/, (Yo) Soy franco/sincero/ honesto/honrado al decirte, etc.; Dado que el presente trabajo está enfocado en la investigación de los AAHs, no se va a detallar acerca del análisis de otras expresiones de actos de habla en español. (Véase el anexo 1.) 226 Chen, C 陈晨. CLAC 79 2019: 217-256 Por otro lado, al tratarlo a nivel inter-lingüístico, y dado que, en las dos lenguas en contraste, tanto el español como el chino, los AAHs y las EAHs que nos ocupan en el presente trabajo poseen las mismas semánticas conceptuales y comparten la misma EC, pero difieren en los recursos lingüísticos concretos para realizar la proyección en las estructuras sintácticas superficiales; tomaremos como el TC la equivalencia en el nivel de la EC, y a partir de allí, analizaremos las diferencias y semejanzas de las expresiones de actos de habla en ambas lenguas. 2.2. Perspectiva del análisis: Teoría semántica de eventos Según Qin y Wang (2012), Kong (2014), la semántica de eventos es una forma directa y efectiva de describir e interpretar la EC de la lengua, su forma lógica tiene características universales e intre-lingüísticas, que pueden mostrar de manera lógica y profunda la concordancia de los constituyentes semánticos conceptuales de la EC y la correspondencia entre los constituyentes semánticos conceptuales de la EC, así como también, los constituyentes sintácticos de la estructura superficial , tal y como se observará más adelante. El término evento fue por primera vez utilizado en el campo de la filosofía. Davidson (1967) es considerado como el primer autor en proponerlo en el campo del análisis de la semántica de eventos (en adelante SE). En su clásico trabajo The Logical Form of Action Sentences, Davidson (1967) propuso la existencia de una variable de evento o argumento davidsoniano presente en la estructura argumental de ciertos verbos. El término evento, que se considera como una entidad lingüística y un argumento adicional(x) de los otros predicados (agente, paciente, tema…), como se recoge en la cita a continuación: The basic idea is that verbs of action-verbs that say ´what someone did´-should be construed as containing a place, for singular terms or variables, that they do not appear to. For example, we would normally suppose that ´Shem kicked Shaun´ consists in two names and a two-place predicate. I suggest, though, that we think of ´ kicked´ as a three-place predicate. This third argument is the event (Davidson, 1967: 118). Así, la representación de ES de la forma lógica de la oración ¨Pedro pateó a Juan (Shem kicked Shaun) ¨ será: (17) (∃x) (Pateó a (pedro, Juan x)) Davidson trata el evento como un elemento que contribuye a la forma lógica de una oración, en el ejemplo (17), concretamente, como un argumento adicional (x) paralelo a los otros dos argumentos (Pedro, Juan). De acuerdo con este análisis, los predicados poseen un argumento extra dentro de su estructura argumental reservado para el evento. Así, la entrada léxica de un verbo como morir será (+V, -N, <1, e>), donde se representa la posición de evento. La representación de la forma lógica de la oración en la que se proyecte este predicado como Cesar murió será: (18) (∃e) [morir(e) & Objeto (e, x)] La interpretación (18) es ¨existe un e, tal que se denota un evento de morir, en el que x es el objeto de e y x es César¨. En esta representación, el argumento Chen, C. 陈晨 CLAC 79 2019: 217-256 227 seleccionado por el predicado principal se relaciona con el verbo indirectamente, a través del vínculo que se establece entre el evento y los participantes que intervienen en él. El análisis de Davidson se basa en la idea de que la información eventiva, está representada en la estructura argumental de los verbos que denotan acción mediante una posición de argumento e implica que hay que introducir ¨los eventos como entidades acerca de las cuales se puede decir un número indefinido de cosas¨ (Davidson 1967: 119-120). Dicho análisis es dominado por Análisis Davidsoniano y ha sido profundizado por Parsons. Además de incluir un argumento de evento, Parsons (1990) también asigna papel temático a los argumentos en función de ese argumento evento. En el trabajo de Parsons (1990), el autor introduce 6 tipos de papeles temáticos que un Sintagma Nominal (SN) puede desempeñar en inglés, que son: Agente, Tema, Meta, Benefactivo, Instrumento, y Experimentes. Según Parsons, la forma lógica de ES de la oración como: (19) Brutus apuñaló a César por la espalda con un cuchillo. Se puede interpretar como: (20) (∃e) (Apuñalar (e) & Pasado (e) & Agente (e, Brutus) & Tema (e, César) & por la espalda (e) & con un cuchillo (e)). La interpretación (20) es ¨existe un evento de apuñalar en el que Brutus es el agente, César el paciente, se realizó por la espalda y con un cuchillo¨. El análisis de la estructura de los predicados de Parson ha sido dominado por el ¨Análisis Neo-Davidsoniano¨ y una de las ventajas de dicho análisis reside en que nos permite explicar las implicaciones que dan lugar los adverbios. Por ejemplo, La forma lógica de ES de la oración ¨Brutus mató a Cécar violentamente¨ se expresaría de la siguiente manera: (21) (∃e) (Apuñalar (e) & Pasado (e) & Agente (e, Brutus) & Tema (e, César) & Adj (e, violento)). En líneas generales, Higginbotham (1985) y (1989) sigue al Análisis Davidsoniano y Análisis Neo-Davidsoniano. En lo que respecta a la modificación adverbial, según Higginbotham (1989:476), está puede integrarse en el proceso de identificación temática, uno de los cuatro modos de descarga temática que propone él mismo. Véase un ejemplo de los adverbios modificadores del predicado. (22) María se resbaló fatalmente. (Cresswell 1985) Higginbotham (1989:476) propone que el adverbio fatalmente posee una red temática propia con dos posiciones <1,2>, heredada del adjetivo base, fatal, en la que posición 1 es un evento que se identifica con la posición E de evento del predicado principal; mientras que la posición 2 se refiere al sujeto de la predicación María y se identifica con la posición temática 1 del predicado principal: (23) [V´ [fatalmente (1, 2)] [V´ resbaló (1, E]] El resultado de estas relaciones de identificación es una interpretación representada por la ES siguiente: (24) a. ∃e [resbaló (María, e) & fatal (e, María)] 228 Chen, C 陈晨. CLAC 79 2019: 217-256 La interpretación de (24) es ¨ existe un evento (e) de resbalarse María, tal que fue fatal para María¨. Los adverbios oracionales han sido analizados generalmente como operadores (Thomason y Stalnaker, 1973; Cresswell, 1985; Espinal, 1985 y 1987; Parsons, 1990:8). Según Higginbotham (1988), la representación de una oración debe identificarse con la variable de evento (e), de una variable proposicional (P), y de otra variable del acto de habla (E), restringidas a su vez por los operadores de evento, proposicionales y de actos de habla o ilocutivos, respectivamente. De acuerdo con esta línea, a continuación, analizamos la semántica de eventos de actos de habla en español, y a partir de allí, pretenderemos deducir las posibles expresiones de actos de habla en chino a través la consulta avanzada o compleja en el corpus CCL. 2.3 Semántica de eventos de los actos de habla en español Según Kovacci (1986), la semántica conceptual de oración (25) admite la paráfrasis como (26): (25) Francamente /honestamente / honradamente / sinceramente, creo que está bien. (26) (Yo) Soy franco / sincero / honesto / honrado al decirte que creo que está bien. Sin embargo, en el caso de eventos de actos de habla de orientación hacia al oyente, su semántica conceptual sería un poco compleja de parafrasearse. Según el punto de la vista de la hipótesis performativa, los AAHs en el ejemplo (27) deberían estar modificando a un verbo enunciativo del tipo "preguntar". De ser esto cierto, se puede parafrasear como "Te pregunto francamente qué piensas" y "Te pregunto sinceramente cómo se te ocurrió hacer eso". Sin embargo, es obvio que, en estos enunciados, los adverbios en cuestión, se encuentran orientados hacia el oyente, el hablante espera de su interlocutor una respuesta franca y una respuesta sincera. Por tanto, en la semántica conceptual de la oración (27) sería adecuado y razonable parafrasear como vemos en el ejemplo (28). (27) Francamente /honestamente / honradamente / sinceramente, ¿crees que está bien? (28) (Tú) Sé franco / sincero / honesto / honrado al contestarme si crees que está bien. Cabe, no obstante, la posibilidad de acudir a la descomposición léxica del verbo enunciado "preguntar" e interpretarlo como " pedir + decir"(Schreiber, 1972). La figura 2 representa tentativamente la estructura semotáctica (Kovacci 1986) de (25) - (28). El nodo superior se puede interpretar como una coordinación o una relación paratáctica o una proyección realizada. Se ven dos casos que en el primero, el condicionamiento en (A) permite generar (26) / (28), y en el segundo, el condicionamiento en (B) determina la estructura de (25) / (27). Chen, C. 陈晨 CLAC 79 2019: 217-256 229 Figura 2: Tomado y adaptado de Kovacci (1994) En el marco de la teoría de la SC, el semema s/franc/ se puede interpretar como primitivo conceptual, pues, en dicho primer caso, se realiza con el lexema adjetivo L /franco/, mientras en el segundo, con el lexema adverbio L/francamente/. Basándonos en el análisis arriba expuesto, ya podemos construir las formas lógicas de los dos grupos de eventos de actos de habla con orientación al hablante/oyente y leer sus interpretaciones. Primero, analizamos el grupo de evento de actos de habla de orientación al hablante. Tomaremos nuevamente los ejemplos (25) como (29): (29) Francamente /honestamente / honradamente / sinceramente, creo que está bien. La oración (29) se representaría de la siguiente manera: (30) (∃e) [E(e) & Agente (e, yo) & Tema (e, P)]& Franco / Sincero /Honesto / Honrada (e, yo) Se puede leer: existe un evento del acto de habla(E), cuyo agente es yo, el contenido es la proposición(P): creo que está bien, y cuando el agente(yo) realiza la proposición, su característica es Franco / Sincero /Honesto / Honrada. Pues, la forma lógica de la semántica de eventos de actos de habla de orientación hacia al hablante, se puede interpretar de la siguiente manera: (31) (∃e) [E(e) & Agente (e, emisor-hablante) & Tema (e, P)]& ADJ (e, emisor-hablante) Ahora, vamos a analizar el grupo de evento de actos de habla de orientación hacia al oyente. Retomaremos el ejemplo (27) como (32): (32) {Francamente / honestamente / honradamente / sinceramente}, ¿crees que esto es así? (Sergi, 2005) Dado que el emisor/hablante pide o espera una respuesta del receptor/oyente, la proposición está implicada por un operador imperativo. Por la tanto, la forma lógica de la estructura de eventos de actos de habla de orientación hacia al oyente puede ser representada de la siguiente manera: (33) IMP(∃e) [E(e) [E´(e) & Agente (e, receptor-oyente) & Tema (e, P)] & ADJ (e, receptor-oyente)]. 230 Chen, C 陈晨. CLAC 79 2019: 217-256 Pues, en términos más técnicos, la oración (33) debe ser reducible semánticamente a una interpretación como: existe un evento del acto de habla(E) que consiste en un sub-evento(E´)-un emisor/hablante que pide o espera una respuesta del receptor/oyente-, siendo el contenido de la información la proposición(P): ¿crees que está bien?, y cuando el agente (receptor) realiza la proposición, su característica se requiere ser franco, sincero. honesto, honrado. 3. Los recursos lingüísticos asumidos como los paralelos de los AAHs españoles en chino A continuación, basándonos en las representaciones obtenidas en la sección 2.3, pretenderemos deducir las posibles expresiones de actos de habla en chino a través de la consulta avanzada en el corpus CCL, es decir, para saber cuáles son los recursos lingüísticos expresivos de dichas determinadas semánticas conceptuales en chino. 3.1 La consulta de las posibles EAHs en el corpus CCL Para poder usar la consulta avanzada en el corpus CCL, en primer lugar, hay que determinar las expresiones lógicas que consisten en grupos por caracteres relacionados y puntuaciones entre sí mediante los operadores lógicos (SPACE, \|, $, #, +, -, ~, !, : ) y del operador de distancia. Gracias a las formas lógicas del EC obtenidas en el anterior apartado, podemos delimitar los caracteres relacionados que podrían formar las expresiones lógicas adecuadas de eventos de actos de habla en chino. Figura 3. La correspondencia entre los recursos lingüístico y la EC de actos de habla de orientación al hablante/emisor Figura 4. La correspondencia entre los recursos lingüísticos y la EC de actos de habla de orientación al oyente/receptor Chen, C. 陈晨 CLAC 79 2019: 217-256 231 Como se observa en la figura 3 y 4, podemos coger cinco grupos de los caracteres relacionados de las expresiones lógicas: a) Los pronombres ¨我(yo)¨/¨你(tú)¨ que corresponden a AGENTE; b) Los adjetivos ¨老实/坦率/坦白/诚实/诚恳/真诚/诚恳¨ (franco, sincero. honesto, honrado) que corresponden a ADJ. c) Los verbos enunciativos ¨说/讲/告诉¨. Tal y como recoge C. Fuente (1987), en realidad en todo enunciado existe una estructura implícita con un verbo enunciativo, la diferencia tipológica entre las dos lenguas es que, en algunas lenguas como el español los verbos enunciativos no representan sintácticamente (Garrido 1999), pero en otras lenguas como el chino si aparecen obligatoriamente (Zhang 2002). d) El verbo ¨ 请¨ o los prenombres ¨ 给/ 跟 (con)¨ corresponden a IMP, los denominamos como ¨marcadores imperativos¨(IMP). e) Los sustantivos ¨实话/老实话/实在的/真的/真话/真心话¨ (las palabras honestas/las palabras honestas y humildes/la verdad/las palabras verdaderas/las palabras del corazón/las palabras sinceras del corazón) que conllevan los adjetivos o morfemas adjetivos ¨实/老实/实在/真 (franco, sincero. honesto, honrado) ¨. Veamos algunos ejemplos ilustrativos de la manera de formular las primeras consultas y las segundas consultas: a) Expresión lógica A: (ADJETIVO\| … \|ADJETIVO) $1(VERBO\| … \| VERBO) Por ejemplo: (老实\|坦率\|诚恳\|诚实\|坦白\|真诚\|直率) $1(说\|讲\|告诉). Lectura: los adjetivos y los verbos deben estar presentes en el documento, y los adjetivos deben de aparecer a una distancia medida en carácter no superior a 1 del verbo, debido a que en esa construcción existe la posibilidad de aparecer la partícula estructura DE. b) Expresión lógica B: (VERBO \| … \| VERBO) $2(SUSTANTIVO\| … \|SUSTANTIVO) Por ejemplo: (说\|讲\|告诉) $1(实话\|老实话\|实在的\|真的\|真话\|真心话). Lectura: el verbo y el sustantivo deben estar presentes en el documento, y los verbos deben de aparecer a una distancia medida en caracteres no superior a 1 de los sustantivos, debido a que tales sustantivos puedan llevar un clasificador nominal ¨句(frase)¨ antepuesto. c) Expresión lógica C: (IMP \| … \| IMP) $2(VERBO\| … \|VERBO) $2(SUSTANTIVO\| … \|SUSTANTIVO) Lectura: los marcadores imperativos, los verbos y los sustantivos deben estar presentes en el documento, así como los marcadores imperativos, deben de aparecer a una distancia medida en carácter no superior a 2 de los verbos, debido a que en esa construcción existe la posibilidad de aparecer los pronombres, por ejemplo: 我 (yo), 你 (tú), 您 (usted), 我们 (nosotros), 你们 (vosotros). Chen, C 陈晨. CLAC 79 2019: 217-256 232 Por ejemplo, (请\|跟\|给) $2(说\|讲\|) $2(实话\|老实话\|实在的\|真的\|真话\|真心话) d) Expresión lógica D: Expresión lógica A/B/C…$0(，\|！) Lectura: Basándonos en los resultados de las primeras consultas, realizamos las segundas consultas. Los caracteres conseguidos por las primeras consultas deben estar antepuestos de ¨ ，¡¨o ¨ ！¨ sin distancia. Así ya nos salen las expresiones que ocupen posiciones periféricas con respecto a la predicación oracional. Las primeras consultas en el corpus CCL Las segundas consultas en el corpus CCL Expresión lógica D: A/B/C…$0(，\|！) Expresión lógica A: (ADJETIVO\|…\|ADJETIVO\|$\|VERBO \|…\|VERBO) Por ejemplo: (老实\|坦率\|诚恳\|诚实\|坦白\|真诚\|直率) $1(说\|讲\|告诉). (franco \|sincero \|honesto\| honrado) $1(decir \|hablar \| contar). Los ejemplos registrados: 老实说, 老实讲, 坦率地说, 坦率的说, 坦率地讲, 坦率的讲, 坦白地说, 坦白的说, 坦白地讲, 坦白的讲, 诚实地说, 诚实的说, 诚实讲, 真诚地说, 真诚的说, 真诚地讲, 真诚的讲, 诚恳地说, 诚恳的说, 诚恳地讲, 诚恳的讲 Expresión lógica B: (VERBO\|…\|VERBO) $2(SUSTANTIVO\|…\|SUSTANTIVO) Por ejemplo: (说\|讲\|告诉) $1(实话\|老实话\|实在的\|真的\|真话\|真心话). (decir \|hablar \| contar) $1(las palabras honestas \| las palabras honestas y humildes \| la verdad) Los ejemplos registrados: 说实话, 讲实话, 说老实话, 讲老实话, 说实在的, 讲实在的, 说真的, 讲真的, 说真话, 讲真话, 说真心话, 讲真心话, 实话实说, 坦诚相告, 实话告诉你/您, 实话告诉我 Las expresiones representativas de actos de habla en el corpus CCL Expresiones lingüísticas Casos Expresiones lingüísticas 老实说，[P] 993 老实说/讲，[P] 老实讲，[P] 88 说实话，[P] 2098 讲实话，[P] 184 说老实话，[P] 398 讲老实话，[P] 30 说实在的，[P] 625 说实在的，[P] 实话实说，[P] 114 实话实说，[P] 坦诚相告，[P] 17 坦诚相告，[P] 坦率 DE 说， [P] 429 坦率 DE 说/讲， [P] 坦率 DE 讲， [P] 79 坦白（ DE ）说，[P] 343 坦白（ DE ）讲，[P] 36 诚实（ DE （说，[P] 31 诚实（ DE ）讲，[P] 2 真诚 DE 说， [P] 159 诚恳 DE 说， 288 说/讲实话，[P] 说/讲老实话，[P] 坦白（ DE ）说 / 讲，[P] 诚实 DE 说/讲， [P] Chen, C. 陈晨 CLAC 79 2019: 217-256 Expresión lógica C: (IMP\|…\|IMP\|)$2(VERBO\|…\|VERBO) $2(SUSTANTIVO\|…\|SUSTANTIVO) Por ejemplo: (请\|跟\|给) $2(说\|讲\|告诉) $2(实话\|老实话\|实在的\|真的\|真话\|真心话) (IMP\|IMP\|IMP) $2(decir hablar\| contar) $2(las palabras honestas \| las palabras honestas y humildes \| la verdad) Los ejemplos registrados: (我)坦率地告诉你, 我坦白(DE)告诉你(吧), (我) 实话告诉你/您(吧), (你/您)实话告诉我, (我)跟你/您说实话(吧), (你)给我说实话, (你)跟我说实话, (我)老实告诉你(吧)/您, (你)老实告诉我, (我)老实跟你/您说(吧), (你)老实跟我说 48 expresiones lingüísticas, 9996 casos 233 [P] 说真的，[P] 766 说/讲真的，[P] 讲真的，[P] 2 说真话，[P] 595 讲真话，[P] 161 说真心话，[P] 51 说真心话，[P] (我)坦率地告诉你, [P] 1 我坦率地告诉你， [P] (我)坦白(DE)告诉你(吧), [P] 10 我坦白(DE)告诉你 (吧), [P] (我)实话告诉你 /您(吧)，[P] 91 (我)实话告诉你/您 (吧)，[P] ( 你/您) 实话告诉我，[P] 5 (你/您)实话告诉我，[P] ( 我) 跟你/您说实话(吧)，[P] 35 (我)跟你/您说实话 (吧)，[P] (你)给我说实话，[P] 15 (你)给我说实话， [P] (你)跟我说实话，[P] 26 (你)跟我说实话， [P] (我)老实告诉你 /您(吧)，[P] 115 (我)老实告诉你/您 (吧)，[P] (你)老实告诉我，[P] 33 (你)老实告诉我， [P] ( 我) 老实跟你/ 您说(吧)，[P] 19 (我)老实跟你/您说 (吧)，[P] (你)老实跟我说，[P] 2 (你)老实跟我说， [P] 33 expresiones lingüísticas 7841 23 expresiones lingüísticas 说真话，[P] Tabla 2. Los ejemplos registrados de las posibles EAHs en el corpus CCL Como se observa en la tabla 2, a la izquierda vemos que, mediante las expresiones lógicas A, B y C, nos salen 45 tipos de expresiones lingüísticas a las primeras consultas, en toda son 9996 casos. En el medio, se puede ver que las expresiones lógicas D nos han permitido recopilar los 23 tipos de expresiones lingüísticas que ocupan posiciones periféricas con respecto a la predicación oracional, y sus ejemplos registrados individuales, que en total son 7841 casos. Sin embargo, como mencionábamos en el apartado 1, el trabajo con corpus requiere un ¨factor humano¨, las búsquedas de tales corpus deben revisarse 234 Chen, C 陈晨. CLAC 79 2019: 217-256 manualmente. Una vez guardados todos los ejemplos de las segundas consultas, leemos uno por uno y eliminamos los que no son válidos para el presente trabajo. Después de la evaluación, como vemos a la derecha en la tabla, nos quedan 23 tipos de expresiones lingüísticas, que en total son 4186 casos. 3.2 Clasificación de las EAHs en chino Una vez analizados los registros del corpus, se ha observado que, los 23 tipos de las EAHs en chino, sintácticamente, se pueden dividir en 4 grupos (Véase la tabla 3. y el anexo 1.): 1) Adjetivo adverbializado + [DE] + verbo enunciativo (34)老实说，在你心目中，我是个怎样的皇帝？(CCL de chino moderno) Glosa: Honesto hablar, ¿en tus ojos, yo soy cómo el emperador? Trad.: Honestamente, en tu opinión, ¿qué tal soy cómo emperador? (35)坦率地讲，我也有过同样的想法。(CCL de chino moderno) Glosa: Franco DE decir, yo también he tenido la misma idea. Trad.: Francamente, yo he tenido la misma idea que tú. (36)坦白地说，你最不满意我的是什么? (CCL de chino moderno) Glosa: Franco DE hablar, ¿tú lo más no satisfecha a mí es qué? Trad.: Francamente, ¿qué es lo que te moleta de mí? (37)诚实地讲，我现在还想再成就一番事业。(CCL de chino moderno) Glosa: Honesto DE decir, yo ahora todavía quiero otra vez lograr un gran carrera. Trad.: Honestamente, aún no he perdido la ilusión por hacer la carrera. 2) Verbo enunciativo + NP (38)说实话，你是想看老吕的笑话吧。(CCL de chino moderno) Glosa: Dime las palabras honestas/Sé sincero, tú eres querer reírse del Sr. Lyu. Trad.: Sinceramente, ¿es tu intención burlarte de Sr. Lyu? (39)说实在的，一点不想不可能，想多了也不现实。(CCL de chino moderno) Glosa: Digo lo que es real, un poco no querer no es posible, pensar más tampoco es real. Trad.: Sinceramente, no puedo dejar de pensar en eso, pero si pienso demasiado tampoco es conveniente. (40)说真的，你怎么放这样的片？不怕没人看吗？(CCL de chino moderno) Glosa: Digo la verdad, ¿tú cómo puedes poner este tipo de películas, no temes que nadie la vea? Trad.: Sinceramente, no entiendo cómo puedes escoger este tipo de películas. ¿No piensas que igual no viene nadie? 3) Locución verbal Chen, C. 陈晨 CLAC 79 2019: 217-256 235 (41)实话实说，谁寻你开心了？(CCL de chino moderno) Glosa: Hablas las palabras verdaderas, ¿quién te busca para ser feliz? Trad.: Honestamente, ¿Quién te corteja? (42)小兄弟，你我不是外人，务请坦诚相告，你是不是想回冰火岛去 Glosa: Hermanito, tú y yo no somos forasteros, por favor sé honesto al decirme, ¿quieres volver a la isla Icefire? Trad.: Hermanito, tú y yo nos conocemos bien, honestamente, ¿quieres volver a la isla Icefire? 4) Cláusula (43)您实话告诉我，现在到底在怀疑谁啊？(CCL de chino moderno) Glosa: Usted dígame las palabras honestas, ¿ahora al final sospechar a quién? Trad.: Sinceramente, ¿De quién sospecha ahora? (44)老实告诉你，我认为它们令人作呕。(CCL de chino moderno) Glosa: Honestamente y humildemente te digo a ti, yo creo que ellos hacen que las personas vomiten. Trad.: Honestamente, me parecen repugnantes. (45)赵鞅，你老实告诉我，你对我有没有意思？(CCL de chino moderno) Glosa: Zhao Yang, tú dime honestamente y humildemente, ¿tú a mí tener no tener significados?Trad.: Zhao Yang, honestamente, ¿significo algo para ti? (46)不过，亲爱的，我老实跟您说，确实难受极了。(CCL de chino moderno) Glosa: Pero, querido, yo honestamente y humildemente le digo, de verdad me siento muy mal. Trad.: Pero querido, honestamente, me siento muy mal. Recursos lingüísticos Las expresiones representativas de actos de habla en chino Casos en el corpus CCL Recursos léxicos -- -- Recursos sintácticos 20 tipos de expresiones lingüísticas Casos en el corpus CCL (4186) Adjetivo adverbializado + Verbo enunciativo 老实说/讲, [P] 870(21%) 坦率地说/讲,[P] 246 坦白（DE）说/讲,[P] 189 诚实地说/讲,[P] 6 说/讲实话,[P] 1039(25%) 说/讲老实话,[P] 267 1311 2550 Chen, C 陈晨. CLAC 79 2019: 217-256 236 说实在的,[P] 569 说/讲真的,[P] 588 说真话,[P] 60 说真心话,[P] 27 Locución verbal 实话实说, [P] 57 坦诚相告, [P] 1 Cláusula (我)坦率地告诉你, [P] 1 1 (我)坦白(DE)告诉你(吧), [P] 10 10 (我)实话告诉你/您(吧),[P] 87 87 (你/您)实话告诉我,[P] 2 2 (我)跟你/您说实话(吧),[P] 27 27 (你)给我说实话,[P] 9 9 (你)跟我说实话,[P] 18 18 (我)老实告诉你/您(吧),[P] 76 76 (你)老实告诉我,[P] 23 23 (我)老实跟你/您说(吧),[P] 12 12 (你)老实跟我说,[P] 2 2 Verbo enunciativo + NP 58 Tabla 3. Los 4 grupos de las EAHs en chino Entre los 4 grupos de las EAHs del chino, en realidad, las estructuras de ¨Adjetivo adverbializado + Verbo enunciativo ¨, ¨ Verbo enunciativo + NP ¨, ¨ Locución verbal ¨ sintácticamente, pertenecen a la misma construcción verbal, que en total son 3918 ejemplos registrados, ocupando 94% en el corpus CCL. Por tanto, podemos decir, que en chino, las construcciones verbales son los principales recursos lingüísticos para proyectar la semántica conceptual de eventos de actos de habla a la estructura superficial. Entre todas las variantes lingüísticas mostradas en la tabla 5, nos llaman la atención que todas las cláusulas presentan 267 ejemplos registrados, ocupando solamente 6% en el corpus CCL, mientras las expresiones más condensadas como ¨ 老实说/讲¨ presentan 870 ejemplos registrados y ¨说/讲实话¨ 1039 ejemplos registrados, ocupando 21% y 25 % en el mismo corpus, respectivamente. Los datos de la distribución de tales variantes sincrónicas nos hacen pensar en dos aspectos. Primero, la distribución de variaciones sincrónicas puede reflejar la evolución de la lengua, en la que el principio de economía (economy principle) juega un papel bastante importante. Según Hopper y Traugott (2003: 71-72), las expresiones lingüísticas experimentan la lexicalización motivada por el principio de la economía. Para aprovechar la ¨maximización de la economía¨ (maximization of economy), los hablantes tienden a reducir la señal del habla (speech signal) para lograr la máxima eficiencia de comunicación (communicating efficiency), lo que implica una simplicidad de la señal (signal simplicity). Pues, impulsados por el Chen, C. 陈晨 CLAC 79 2019: 217-256 237 principio de economía, las expresiones lingüísticas se desarrollarían en formas más condensadas. Segundo, la distribución de las variaciones sincrónicas, también pueden interpretar la relación entre la frecuencia de uso y la lexicalización de las unidades lengüisucias. Elvira (2006, 2007) señala: Como en otro tipo de cambios lingüísticos, la frecuencia de uso es el factor que está presente en la mayoría, si no en todos los casos de lexicalización. El uso reiterado de una expresión favorece su acomodo y arraigo en la memoria y su retención como un conjunto global e integrado más que como un todo analizable. Según los ejemplos extraídos del corpus CCL, la estructura Verbo enunciativo + NP¨说/讲实话¨ y Adjetivo adverbializado + Verbo enunciativo ¨老实说/讲¨ son las dos expresiones más representadas entre los 23 tipos de EAHs del chino, pudiendo ser las unidades lingüísticas que ya estén en el proceso de lexicalización, o ya estén más lexicalizadas que las demás expresiones de actos de habla en chino. 4. Análisis y observaciones 4.1 Las correspondencias entre los AAHs y las EAHs no son bilaterales Aunque todos los AAHs del español encuentran sus paralelos en el chino, las correspondencias no son bilaterales, en muchos casos, se observan correlaciones sintácticas entre un mismo adverbio español y varios posibles equivalentes del chino. Sin ánimo de ser exhaustivos, tomamos el adverbio honestamente como un ejemplo para manifestar estas correspondencias: Semántica conceptual: Soy honesto al decirte que… ¨ / ¨Sé honesto al decirme que…¨ Forma lógica de estructura conceptual: (∃e) [E(e) & Agente (e, yo) & Tema (e, P)]& Honesto (e, yo) IMP(∃e) [E(e) [E´(e) & Agente (e, yo) & Tema (e, P)] & Honesto (e, yo)] Adverbio en español Construcción verbal en chino Honestamente A. Adjetivo adverbializado + Verbo enunciativo 老实说/讲 B. Verbo enunciativo + NP 说/讲实话 C. Locución verbal 实话实说 D. Cláusula 我老实告诉你/您 Posibles paralelos del chino 你老实告诉我 Tabla 4: El AAH Honestamente y sus paralelos del chino Gracias a las correlaciones mostradas en la tabla 4 se ha podido comprobar nuevamente que, como habíamos mencionado en el apartado 2, la correspondencia entre el significado y forma es ¨one to many¨, tanto en el nivel intralingüístico como en el inter-lingüístico. 238 Chen, C 陈晨. CLAC 79 2019: 217-256 En cuanto al nivel intralingüístico, al igual que el español (Véase el anexo 1.), el chino también cuenta con múltiples posibilidades de expresiones para realizar la proyección de ¨ Honesto (e, yo)]¨, como se puede observar en la tabla 5, que son los 4 grupos de variantes sincrónicas lingüísticas: A. Adjetivo adverbializado + Verbo enunciativo¨ 老实说 / 讲 ¨, B. Verbo enunciativo + NP¨ 说 / 讲实话 ¨, C. Locución verbal¨实话实说¨, D. Cláusula¨我老实告诉你/您¨ y ¨你老实告诉我¨. Se sabe que, la coexistencia de variantes sincrónica lingüísticas no solo sirven como un fósil viviente donde se puede observar el proceso de la evolución diacrónica de la historia lingüística, sino que también puede ser un indicio de un cambio lingüístico (Berta 1999, Zhang 2003), ya que todo el cambio es el resultado de una variación, aunque no toda variación lingüística termina produciendo cambios. En cuanto al nivel inter-lingüístico, según la teoría SC, tanto el adverbio de actos de habla honestamente como sus paralelos chinos ¨老实说/讲, 说/讲实话, 实话实说 , 我老实告诉你 / 您 , 你老实告诉我 ¨ poseen la misma semántica conceptual: ¨Soy honesto al decirte que… ¨ o ¨Sé honesto al decirme que…¨ y comparten la misma estructura conceptual: ¨ (∃e) [E(e) & Agente (e, yo) & Tema (e, P)]& Honesto (e, yo) ¨ o ¨IMP(∃e) [E(e) [E´(e) & Agente (e, yo) & Tema (e, P)] & Honesto (e, yo)]¨, sin embargo, difieren en los recursos lingüísticos, que en español es la forma léxica (adverbio), en chino la forma sintáctica (construcciones verbales y cláusulas). 4.2 Dos tipos de recursos lingüísticos: recursos léxicos versus recursos sintácticos Hasta ahora, ya podemos afirmar que, para la expresión de determinadas semánticas conceptuales de actos de habla, en español se pude recurrir a recursos léxicos, y mientras en chino, a recursos sintácticos. A continuación, tomamos como ejemplo, el adverbio honestamente y su paralelo en chino, con el fin de demostrar la diferencia al realizar la proyección de la semántica conceptual de eventos de actos de habla a la estructura superficial en ambas lenguas. Véase la tabla 5 y 6 en la página siguiente. Tanto en la oración ¨Honestamente, creo que está bien. ¨como en la oración ¨Honestamente, ¿crees que está bien? ¨, la información de eventos de habla se codifica mediante su descomposición en el (sub)evento de actos de habla. Es decir, la oración completa es un Evento, y dicho evento está compuesto por unos constituyentes que son Agente (yo o tú), Predicación (ser honesto), Paciente (tú o yo), y Proposicional. Sin embargo, cuando se realiza la proyección de la semántica conceptual a la estructura superficial en español, solamente se presenta el adverbio honestamente. En otras palabras, a nivel de la estructura superficial, el adverbio honestamente combina los constituyentes ¨Agente¨, ¨Predicación¨ y ¨Paciente¨. En cuanto a las EAHs, bien sea una estructura de ¨ Adjetivo adverbializado + Verbo enunciativo ¨, o bien sea una locución, o sea una cláusula, no son capaces de combinar los constituyentes ¨Agente¨, ¨Predicación¨ y ¨Paciente¨. Para que lo vean más claro, convertimos las tablas 5 y 6 a la figura 5, en la que nos indica directamente la relación entre los constituyentes semánticos y la estructura superficial. Chen, C. 陈晨 CLAC 79 2019: 217-256 239 Honestamente, creo que está bien. 老实说(/说实话/实话实说)，我认为挺好的。 Semántica conceptual (Yo) Soy honesto al decirte que creo que está bien. Presentación de forma lógica de estructura conceptual (∃e) [E(e) & Agente (e, yo) & Tema (e, P)]& honesto (e, yo) Constituyentes de eventos de actos de habla Agent e (YO) Estructura superficial en español Estructura superficial en chino Predicación SER HONESTO DECIR Paciente Proposicional TÚ(TE) P creo que está bien. [Honestamente] （WO） [LAOSHI] [SHUO/JIAN G/GAOSU] （NI）我认为挺好的。 Tabla 5. La realización de proyección de semántica conceptual de actos de habla de orientación al emisor/hablante Honestamente, ¿crees que está bien? 老实说(/说实话/实话实说)，你觉得还行吗？ Semántica conceptual (Tú) Sé honesto al contestarme si crees que está bien. Presentación de forma lógica de estructura conceptual IMP(∃e) [E(e) [E´(e) & Agente (e, tú) & Tema (e, P)] & honesto (e, tú] Constituyentes de eventos de actos de habla IMP Agente (TÚ) Estructura superficial en español Estructura superficial en chino Predicación SER HONEST O DECIR Paciente Proposicional YO(ME) P ¿creo que está bien? [Honestamente] （NI） [LAOSHI] [SHUO/JI ANG/GA OSU] （WO）你觉得还行吗？ Tabla 6. La realización de proyección de semántica conceptual de actos de habla de orientación al receptor/oyente 240 Chen, C 陈晨. CLAC 79 2019: 217-256 Tanto en la oración ¨Honestamente, creo que está bien. ¨como en la oración ¨Honestamente, ¿crees que está bien? ¨, la información de eventos de habla se codifica mediante su descomposición en el (sub)evento de actos de habla. Es decir, la oración completa es un Evento, y dicho evento está compuesto por unos constituyentes que son Agente (yo o tú), Predicación (ser honesto), Paciente (tú o yo), y Proposicional. Sin embargo, cuando se realiza la proyección de la semántica conceptual a la estructura superficial en español, solamente se presenta el adverbio honestamente. En otras palabras, a nivel de la estructura superficial, el adverbio honestamente combina los constituyentes ¨Agente¨, ¨Predicación¨ y ¨Paciente¨. En cuanto a las EAHs, bien sea una estructura de ¨Adjetivo adverbializado + Verbo enunciativo¨, o bien sea una locución, o sea una cláusula, no son capaces de combinar los constituyentes ¨Agente¨, ¨Predicación¨ y ¨Paciente¨. Para que lo vean más claro, convertimos las tablas 5 y 6 a la figura 5, en la que nos indica directamente la relación entre los constituyentes semánticos y la estructura superficial. Figura 5. La relación entre los constituyentes semánticos y la estructura superficial Lo expuesto arriba permite observar el proceso de composición de los constituyentes semánticos de eventos de actos de habla en español. Así como el adverbio honestamente se encarga de la proyección de dicho evento en la estructura superficial, otros constituyentes semánticos, no necesitan obligatoriamente presentarse explícitamente, y, sobre todo, comparado con sus paralelos chinos, el verbo no tiene que aparecer sintácticamente. Según afirma Salvador (1996), los adverbios no afectan directamente al ¨enunciado dictal¨, establecen con él una relación indirecta a través del verbo oculto¨. Garrido (1999) también señala, ¨las expresiones adverbiales aluden al proceso de decir, pero no lo representan sintácticamente, es decir, no modifican a un verbo que supuestamente representara el acto de decir, preguntar, etc. No hay este verbo porque no hace falta. ¨ Mientras en chino, es todo lo contrario, en todos los casos, tanto en las cláusulas ¨我老实告诉你/你老实告诉我¨ que se considera que no están nada lexicalizadas, como en la estructura de ¨Adjetivo adverbializado + Verbo enunciativo ¨老实说¨ que se considera que está más lexicalizada (Li 2012), los verbos están siempre explícitamente proyectados en la estructura superficial. Cabe decir, que en cuanto al nivel inter-lingüístico, los adverbios de actos de habla en español están más lexicalizados que sus paralelos chinos. Según Banczerowski (1980), los adverbios son expresiones sintéticas, las construcciones verbales son expresiones analíticas, y generalmente, se considera que la expresión sintética es una expresión más lexicalizada, mientras que, la expresión analítica está menos o nada lexicalizada. Como es sabido, en la tipología morfológica, la lengua china se considera una lengua aislante y analítica, que carece de aspectos Chen, C. 陈晨 CLAC 79 2019: 217-256 241 morfológicos, mientras que la lengua española constituye un ejemplo representativo de lengua sintética y flexiva. Por tanto, suponemos que tiene que ver la diferencia inter-lingüística en cuestión con la tipología morfológica, las variaciones tipológicas nos pueden ayudar a explicar la diferencia o similitud del grado de lexicalización y gramaticalización (Kong, Qin 2017). 5. Evolución de los AAHs y las EAHs en las dos lenguas en contraste 5.1 Evolución de los AAHs en español Como señala Grandgent (1928) que la construcción endocéntrica adverbial latina de ¨adjetivo+ mente ¨ se usó primero ¨para denotar un estado de ánimo, espíritu, o disposición, como forti mente, obstinata mente, jocunda mente, firma mente. ¨ sin embargo, ¨pasó a emplearse en un sentido más general: pari mente, mala mente. ¨ Más tarde, tal vez ya el período del latín vulgar, -mente se usó con algunos adjetivos que podían formar un adverbio de modo, como sola mente, longa mente. ¨ Según la RAE (1973): ¨Los adverbios en –mente proceden del sustantivo feminino lation mensmentis… Delibitada la significación del sustantivo, los dos términos llegaron a formar una unidad léxica con valor de adverbio de modo en la mayor parte de la Romania. ¨ En cuanto a los adverbios en –mente que nos ocupamos en el presente trabajo, podemos encontrar muchos casos con valor de adverbio de modo en el CORDE. Véase los ejemplos de (47) a (50). (47) Otrossí, damos e otorgamos a los de barrio de Francos, por merçet que les fazemos, preuilegio de calle de Francos que vendan e compren francamente e libremientre en sus casas sus pannos et sus merchandías en gros et a detal e a varas, et toda cosa que quieran comprar et vender en sus casas que lo puedan fazer, et que ayan hy pelligeros et alfayates assí commo en Toledo, e que puedan tener camios en sus casas. (Privilegio que dio el rey don Fernando a Sevilla cuando la ganó, 1251) (48) Y estos son los principales capítulos de la confederación y paz entre Su Majestad y el Duque Guillermo, los cuales se hicieron a siete días de septiembre en el Real sobre Venlot, los cuales se profirieron á guardar sinceramente y sin engaño alguno. (Crónica del Emperador Carlos V, 1550) (49) Podemos dezir que justiçia es convenible disposiçión en cada una de las cosas derechamente judgante; e así, justiçia es dante a cada uno lo suyo e su derecho e bevir honestamente e non fazer daño a otro. (Catecismo, 1325) (50) Esta concupiçençia se trasforma en figura de Escanio, queriendo ser vista recta intinçión, e fingiéndolo, así como cuando uno pone su amor en las riquezas e dize que lo non faze con cobdiçia sinon por mantenerse honradamente. (Traducción y glosas de la Eneida, 1427) El primer uso de los adverbios francamente, sinceramente, honestamente, honradamente como adverbios de modo en el CORDE se remonta a los 1251s. Una búsqueda del corpus (desde 1251s - 1602s) muestra que dichos adverbios denotan 242 Chen, C 陈晨. CLAC 79 2019: 217-256 una forma de acción. Durante este período, los adverbios se usaron solo como un modificador del predicado, una cláusula interna adverbial. Además, como adverbio de modo, los adverbios francamente, sinceramente, honestamente, honradamente también se usan ocasionalmente juntos con los verbos enunciativos, formándose la expresión ¨Agente + adverbio + verbo enunciativo¨ o ¨Agente + verbo enunciativo + adverbio¨, como ¨fablar francamente / contar sinceramente / honestamente fabla / honradamente hablaron¨ en (51) - (54). (51) ¿cómo fue ello sostenido que descendiese el señorío en personas privadas? que usurpasen assí grant poder que davan en la corte fablar francamente, e de fuera de la corte que ellos tiren del todo la palabra" (que quier dezir, que non puede ninguno fablar con ellos). (Taducción de las Décadas de Tito Livio, 1400) (52) Quien honestamente fabla entre estraños non deve fablar desonestamente entre los suyos. (Floresta de philósophos,1430) (53) Serenissimo principe: honradamente hablaron mis dos compañeros: cada qual segund su juyzio. (Exemplario contra los engaños y peligros del mundo, 1493) (54) Es narración contar sinceramente algún suceso bueno, con cuyo ejemplo anima y esfuerza a los oyentes a emprender otro semejante, o malo, para que sea escarmiento. (Cisne de Apolo, 1602) Luego, en la década de 1729, la combinación ¨Agente + adverbio + verbo enunciativo¨ o ¨Agente + verbo enunciativo + adverbio¨ comenzó a moverse hacia la periferia izquierda de la oración, ocupando posiciones periféricas con respecto a la proposición que ahora se ha convertido en la cláusula subordinada. En tal posición sintáctica, esta combinación asumió la función de actos de habla, es decir, pidiendo la respuesta del receptor/oyente cuando se usa con una oración interrogativa (58-59), o expresando la manera de habla del emisor/hablante cuando se usa con una oración declarativa (55-57). Este cambio implica un nuevo análisis jerárquico de la combinación, lo que lleva a una nueva estructura con relaciones de alcance modificadas, que es un proceso típico de gramaticalización. En la cláusula de actos de habla, los adverbios francamente, sinceramente, honestamente, honradamente son todavía adverbios de modo que modifican el verbo enunciativo. Sin embargo, los verbos enunciativos ¨hablar, decir¨ en (55-59) ya no se usan con la tercera persona, sino solo con la primera y segunda persona, ya que solo aparecen en su forma básica sin cambios morfológicos con tiempo y aspecto. Esto indica que los verbos enunciativos han sufrido una descategorización, otra característica típica de la gramaticalización (Brinton, Traugott 2005). Además, la cláusula de actos de habla ha obtenido una función pragmática. En este período, algunas expresiones como ¨te hablo sinceramente / os diré sinceramente / francamente le digo / dime sinceramente / decidme francamente¨ se utilizaron con frecuencia para mostrar la actitud positiva del emisor/hablante en la comunicación. (55) Y para que veas que te hablo sinceramente, haré aquí la justicia que debo a uno de ellos. (Teatro crítico universal,1729) (56) os diré sinceramente, que yo no conozco entre vuestros vassallos otro más capaz, que cierto labrador mozo, que aún no está casado. (Teatro crítico universal,1729) Chen, C. 陈晨 CLAC 79 2019: 217-256 243 (57) Pero francamente le digo a V. mrd., que no admito sus reglas, porque no las juzgo convenientes, por más que la común aceptación las haya hecho plausibles. (Cartas eruditas y curiosas, 1742) (58) ¿Gustarías más de llevar un vestido rico que otro roto? Dime sinceramente tu sentir. (Eusebio, 1786) (59) Decidme francamente, ¿por qué os interesa este desgraciado? (Voz de la naturaleza, 1787) En este período, los adverbios francamente, sinceramente, honestamente, honradamente todavía se usaban como adverbios de modo, aunque se usaban en una cláusula de actos de habla, pero encontramos que los adverbios también se usaban como un elemento independiente, véase el ejemplo (60). Lo que indica una tendencia hacia una relación más flexible entre el adverbio y el verbo que modifica. (60) Por lo que confieso, y confesaré siempre, mi obligación y deuda, y estimaré que vuestra merced, sinceramente, y como verdadero amigo, me informé de las resultas, dándome exacta cuenta del bueno o malo concepto que de esa obra hacen nuestros compañeros de la misma Academia, para quienes me tomé el trabajo de hacer esas colecciones. (Carta a Rodríguez Campomanes, 1759) A partir de la década de 1801, los adverbios francamente, sinceramente, honestamente, honradamente se encontraron utilizando independientemente para realizar el acto de habla, es decir, los verbos enunciativos ya no aparcaren sintácticamente en la estructura superficial. Véase los ejemplos de (61) a (62). (61) Y francamente, ¿qué se puede saber sobre tal cosa? ¿Con qué autoridad fijarlo? La imaginacion es la única guia. (Cartas y disertaciones sobre la lengua vascongada, 1801) (62) Me alegro, porque en la ópera... francamente, me duermo. (Traducción de Partir a tiempo, de Scribe, 1835) Como analizábamos anteriormente en el apartado 2, los adverbios francamente, sinceramente, honestamente, honradamente son una parte del evento de actos de habla que cuenta con el emisor/hablante, el receptor/oyente, y el verbo enunciativo, pues podemos decir que los adverbios de actos de habla surgen de la lexicalización de las cláusulas semánticamente saturadas del acto de habla. Según esta línea, el adverbio de acto de habla ¨francamente¨ en (61) es un adverbio lexicalizado de la expresión ¨dime francamente¨; y ¨francamente¨ en (62) es un adverbio lexicalizado de la expresión ¨te digo francamente¨. Los constituyentes semánticos del emisor/hablante, del receptor/oyente, y del verbo enunciativo si están disponibles en la estructura conceptual, pero no están codificados explícitamente en la estructura superficial. En otros términos, Los constituyentes del evento de actos de habla (el emisor/hablante, el receptor/oyente, el verbo enunciativo) se han ido combinando gradualmente en una forma léxica de los adverbios ¨francamente / sinceramente / honestamente / honradamente¨, que se han convertido en adverbios de actos de habla, los cuales han ganado la fuerza ilocutiva, y ocupan posiciones periféricas con respecto a la predicación oracional. 244 Chen, C 陈晨. CLAC 79 2019: 217-256 5.2 Evolución de las EAHs en chino Aunque se suele considerar que las construcciones verbales son menos lexicalizadas, las investigaciones también apoyan la afirmación de que la estructura ¨Adverbio + verbo enunciativo ¨ha estado en el proceso de lexicalización (Dong 2003). Por ejemplo, la estructura Adjetivo adverbializado + DE + Verbo enunciativo¨老实说/讲¨ (honesto hablar/decir), ¨坦率/坦白 DE 说/讲¨ (franco DE hablar/decir), en algunos contextos, han perdido su composicionalidad semántica y los verbos enunciativos como ¨ 说 / 讲 ¨ (decir/hablar) han sufrido una desclasificación, que se refleja en su uso no finito y la pérdida de su capacidad de colocación junto con la tercera persona. A continuación, tomamos la estructura Verbo enunciativo + NP ¨说实话¨ (Decir la verdad/ francamente/ sinceramente/ honestamente) ¨ como un ejemplo para ilustrar la evolución de las EAHs en chino. La búsqueda de corpus muestra que el primer uso de ¨说实话¨ se remonta a la Dinastía Song (antiguo mandarín). En ese momento, ¨说实话¨ era sintácticamente una frase situada internamente en una cláusula, que se puede descomponer en verbo ¨说¨ (hablar/decir) y su objeto ¨实话¨ (palabras verdaderas). El significado de la frase es compositivo. El verbo ¨说¨ (hablar/decir) podría combinarse con un sujeto de primera persona, segunda persona o tercera persona, y la frase puede ser negada, por lo tanto, es una proposición semántica. (63) 若是脱空诳诞, 不说实话 , 虽有两人相对说话, 如无物也。 (CCL del chino clásico, Dinastía Song) Glosa: Si hablan sin sentido, no hablan palabras verdaderas, su conversación no tiene sentido. Trad.: Si no hablan la verdad, su conversación no tiene sentido. Si las palabras carecen de peso, se las lleva el viento. (64) 我为你带了二年重孝，受了千辛万苦，今日还不说实话，待怎么？ (CCL del chino clásico, Dinastía Ming) Glosa: Yo para ti llevo dos años de luto, y he sufrido muchas dificultades. Hoy todavía no dices la verdad. ¿Qué estás esperando? Trad.: Llevo dos años de luto y sufrimiento por ti, y aún no dices la verdad. ¿A qué estás esperando? El corpus muestra que después de la dinastía Song, en las novelas de la dinastía Qing, ¨ 说实话¨ comenzó a aparecer en las oraciones imperativas, expresando órdenes, peticiones y también prohibiciones. Por lo general, la mayoría de este tipo de expresiones se estructuran después de oraciones interrogativas, indicando que el emisor/hablante pide una respuesta del receptor/oyente y le solicita una actitud. (65) 阮英、郑翠屏、郑梦熊来到贼人面前问道: “你这店住下的五个人哪里去了？快说实话！若不实说，我就打你。” (CCL del chino clásico, Dinastía Qing) Glosa: Qi Ying, Zheng Cuiping y Zheng Mengxiong se acercaron al ladrón y le preguntaron: "¿Dónde están las cinco personas que se alojaban en este hotel? Díme la verdad. Si no me dices la verdad, te golpearé". Chen, C. 陈晨 CLAC 79 2019: 217-256 245 Trad.: Qi Ying, Zheng Cuiping y Zheng Mengxiong se acercaron al ladrón y le preguntaron: "¿Dónde están las cinco personas que se alojaban en este hotel? Díme la verdad. Si no, te golpearé". (66) 问说: “巡抚吴德往哪里去了你等急速快说实话！如要不然，我定然结果你等性命！。” (CCL del chino clásico, Dinastía Qing) Glosa: Preguntó: "¿Adónde va el gobernador Wu De? ¡Tú de prisa decir la verdad! Si no, ¡te voy a matar! Trad.: Preguntó: "¿Adónde va el gobernador Wu De? Dime rápidamente la verdad, si no quieres que te mate. Como se puede observar en los ejemplos (65) y (66), el significado de la expresión ¨说实话¨. sigue siendo compositivo, es decir, en esa época, todavía está combinado por el verbo ¨说¨ (hablar/decir) y su objeto ¨实话¨ (palabras verdaderas), es el contrario de la frase ¨说假话 (decir las mentiras) ¨. Tambien nos damos en cuenta, que a partir de la dinastía Qing (mandarín medio), ya aparecen algunos casos que tienen el uso inicial e independiente de la expresión ¨说实话¨. Véase el ejemplo (67) y (68). (67) 吴福说: “大太爷，饶命。我说实话，你说的秃子，他是我们:庄主爷，叫吴元豹。” (CCL del chino clásico, Dinastía Qing) Glosa: Wu Fu dijo :“Perdóname, mi señor. Digo palabras verdaderas, el calvo al que te refieres es Wu Yuanbao, el jefe de nuestro pueblo. Trad.: Wu Fu suplicó: “Perdóname, mi señor. Digo francamente que el calvo al que te refieres es Wu Yuanbao, el jefe de nuestro pueblo”. (68) 智爷微微一笑，说: “你说实话罢，到底是怎么件事情？” (CCL del chino clásico, Dinastía Qing) Glosa: Zhiye sonrió y dijo: "¿di la verdad, qué demonios está pasando? Trad.: Zhiye sonrió y dijo: “ Dime honestamente, ¿qué demonios está pasando? Junto con su sujeto agente, la expresión ¨ 说实话¨ ha ganado fuerza ilocutiva cuando se usa en la posición inicial de la oración. Al moverse a la posición inicial de la oración, seguido del objeto del verbo enunciativo, la expresión cambia de un componente interno de la cláusula al disyuntivo en una oración. Este cambio implica un nuevo análisis jerárquico, con el alcance de la expresión ampliada. El cambio de posición sintáctica es seguido por un cambio de comportamiento gramatical. El verbo ¨说¨ ahora se vuelve no finito y ya no se puede usar con la tercera persona (descategorización del verbo). El reanálisis jerárquico y la descategorización son evidencia de la gramaticalización de la expresión. Junto con el cambio de posición sintáctica y el comportamiento gramatical, también cambia la semántica de la expresión. La expresión pierde gradualmente su composición semántica, es decir, la expresión ya no es compositiva, o la suma del significado del verbo ¨ 说¨ y su objeto ¨ 实话¨. Semánticamente, la expresión está en su transición de ser compositivo a ser idiomático. Así que, podemos decir que la expresión está en proceso de lexicalización. También se ha vuelto pragmáticamente más significativo, con una fuerza ilocutiva que expresa la manera de hablar del emisor/hablante cuando se usa antes de una oración declarativa (67), o exige la forma de respuesta del receptor/oyente cuando se usa antes de una oración 246 Chen, C 陈晨. CLAC 79 2019: 217-256 interrogativa (68). La posición sintáctica prominente y la mejora de la función pragmática proporcionan las condiciones necesarias para que la expresión se convierta en un marcador pragmático. En la primera mitad del siglo XX (principios del mandarín moderno), la expresión se volvió más condensada en forma, con la pérdida del sujeto agente del verbo. Véase el ejemplo (69). (69) “说实话，紧不紧？” (CCL del chino moderno temprano, República de China) Glosa: Di palabras verdaderas, [¿está presionado (por dinero)]? Trad.: Honestamente, ¿estás presionado (por dinero)? El ejemplo (69) es uno de los casos que encontramos donde la expresión ¨说实话 ¨ se usa por separado en la posición inicial de una cláusula. En la oración (69), el significado conceptual y la composición semántica de ¨说实话 ¨ se vuelven aún más débiles, y su significado de procedimiento así como su función pragmática son más importantes, lo que indica que se encuentra en proceso de pragmaticalización. Con la pérdida de la composicionalidad semántica, la expresión, ahora se trata como un todo no analizable, lo que implica fuertemente que la expresión ha estado experimentando una lexicalización. Al igual que la expresión ¨说实话¨, otras EAHs en chino, como ¨说真的¨ (hablar verdad), ¨老实说¨ (honesto decir) también siguen un camino de evolución similar (Shen 2012; Kong 2017): al salir de la cláusula, se han sometido a la gramaticalización, luego las formas gramaticalizadas se vuelven más pragmáticas e idiomaticizadas. Sin embargo, estas expresiones aún no pueden considerarse como palabras en chino, o al menos no son palabras típicas en chino, aunque pueden comportarse sintácticamente como las palabras, por lo que se puede considerar que están lexicalizados. En definitiva, por lo visto hasta ahora en este apartado, los AAHs y las EAHs en las dos lenguas en contraste, en términos generales, siguen un camino similar en su evolución: primero experimentaron un proceso de gramaticalización: de los elementos internos de la cláusula a los adverbios oracionales en español/a los modificadores oracionales en chino; luego un proceso de lexicalización: de las expresiones compositivas a las formas léxicas o expresiones idiomáticas. Y todos ellos evolucionaron de formas no pragmáticas a formas pragmáticas (Fraser 1999, Anderson 2001, Feng 2004). Sin embargo, los AAHs en español y las EAHs en chino muestran diferentes patrones de lexicalización. En español, es la elipsis la que conduce a la reducción formal de una frase o una cláusula, que cuenta con varias palabras a un adverbio. En la reducción, los componentes semánticos intersubjetivos de eventos (el emisor, el receptor, el verbo enunciador y actos de habla) se representan por el adverbio a través de la metonimia. Al contrario, en la lengua china, el patrón típico de lexicalización es la pérdida de composicionalidad semántica (reanálisis de constituyentes internos de una frase o una cláusula), pero este proceso no da como resultado el producir una nueva palabra, como sucede en español. Teniendo en cuenta esto, decimos que los AAHs en español y las EAHs en chino se encuentran en diferentes etapas de lexicalización, y que las EAHs no están completamente lexicalizados, sino que solo están idiomaticizados. Pero cabe decir, según Chen, C. 陈晨 CLAC 79 2019: 217-256 247 Bussmann (1996) y Dong (2011), la idiomatización también es la etapa primaria de la lexicalización. Aparte de los AAHs, también nos llaman la atención que otros tipos adverbios en –mente también están más lexicalizados que sus paralelos en chino. Por ejemplo, en español, los adverbios evaluativos pueden recurrir a los recursos lingüísticos léxicos, y mientras que, en chino, generalmente se recurren a los recursos lingüísticos sintácticos (Kong 2018). Los equivalentes de los adverbios evaluativos (–mente), afortunadamente, sorprendentemente, posiblemente, asombrosamente, curiosamente, paradójicamente, comprensiblemente en chino suelen estar encajados en una estructura ¨X 的是¨ (Chen, Mario 2019). Por lo tanto, podemos especular que la razón de la diferencia del grado de lexicalización entre los AAHs y sus paralelos en chino podría ser que los dos idiomas son morfológicamente diferentes. El español, es rico en aspectos morfológicos, pues en el caso de los adverbios en –mente, es más capaz de combinar varias semánticas de eventos mediante recursos léxicos, y la entidad semántica de los verbos enunciados se puede codificar implícitamente. Mientras que, en chino, por otro lado, carece de aspectos morfológicos y, por lo tanto, no tiene capacidad de proyectar la semántica conceptual de los actos de habla mediante recursos léxicos en la estructura superficial, lo que significa que tiene que recurrir a los recursos sintácticos (construcciones verbales o cláusulas) para codificar el mismo concepto de evento. 6. Conclusiones En el presente estudio, basándose en la semántica de eventos, profundiza en la diferencia de los recursos lingüísticos utilizados en español y chino para expresar las semánticas conceptuales de evento de actos de habla de orientación al emisor o receptor, y observa la evolución diacrónica de los adverbios de actos de habla en español y sus paralelos en chino, con el fin de descubrir la motivación de la diferencia inter-lingüística de las dos lenguas en contraste. Los principales hallazgos del estudio son los siguientes: a) En cuanto al nivel intralingüístico, tanto en el español como en el chino se utilizan variados recursos lingüísticos para codificar determinadas semánticas conceptuales de eventos de actos de habla. b) En cuanto al nivel inter-lingüístico, para realizar la proyección de las mismas semánticas conceptuales de eventos de actos de habla, las dos lenguas en contraste, difieren en los recursos lingüísticos, que en español es la forma léxica (adverbio), mientras que en el chino no dispone de los adverbios de actos de habla, sino de la forma sintáctica (construcción verbal y cláusula). Gracias a las consultas avanzadas en el corpus de CCL, se ha podido comprobar, que las construcciones verbales, son recursos lingüísticos predominantes en chino, y entre ellas, la estructura Verbo enunciativo + NP¨ 说 / 讲实话 ¨ y Adjetivo adverbializado + Verbo enunciativo ¨老实说/讲¨ son las dos expresiones más representativas entre los 23 tipos de EAHs del chino. c) El desarrollo diacrónico de los AAHs en español y las EAHs en chino muestra un patrón de evolución similar, primero los AAHs y las EAHs experimentaron un proceso de gramaticalización, y luego un proceso de lexicalización. Todos ellos evolucionaron de formas no pragmáticas a formas 248 Chen, C 陈晨. CLAC 79 2019: 217-256 pragmáticas. Sin embargo, los AAHs en español y las EAHs en chino se encuentran en diferentes etapas de lexicalización y las EAHs no están completamente lexicalizadas, sino que solo están idiomatizadas. d) La razón de la diferencia del grado de lexicalización podría ser que los dos idiomas son morfológicamente diferentes. El español, rico en aspectos morfológicos, es más capaz de combinar varias semánticas de eventos mediante recursos léxicos, los AAHs se encargan de la proyección de eventos de actos de habla a la estructura superficial, pues, otros constituyentes conceptuales semánticos no necesitan obligatoriamente presentarse, y la entidad semántica de los verbos enunciados se puede codificar implícitamente. Mientras el chino, carece de aspectos morfológicos y, por tanto, no tiene capacidad de proyectar la semántica conceptual de los actos de habla mediante recursos léxicos en la estructura superficial, lo que significa que tiene que recurrir a los recursos sintácticos (construcciones verbales o cláusulas) para codificar el mismo concepto del evento. Cabe decir, que nuestro trabajo también pone de manifiesto la viabilidad y la efectividad de estructura semántica conceptual compartida por las dos lenguas en contraste, que sirve como tertium comparationis en el estudio inter-lingüístico y abre una nueva perspectiva del análisis contrastivo entre dos idiomas tipológicamente diferentes como el español y el chino. Agradecimientos Esta investigación está enmarcada en el proyecto 18CI08C financiado por la Oficina Central del Instituto Confucio, China. 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Expresiones adverbiales del evento de actos de habla de orientación hacia el emisor/hablante Semántica conceptual y su paráfrasis Indican una evaluación del hablante/emisor sobre su propia actitud en cuanto enunciador, la actitud del hablante/emisor ante lo que dice. P.E: (Yo) Soy franco / sincero / honesto / honrado al decirte que creo que está bien. Forma lógica de estructura conceptual y su interpretación (∃e) [E(e) & Agente (e, emisor-hablante) & Tema (e, P)]& ADJ (e, emisor-hablante) Existe un evento del acto de habla(E), cuyo agente es yo, el contenido es la proposición(P), y cuando el agente(yo) realiza la proposición, su característica es ADJ. Estructura superficial en español Estructura superficial en chino Recursos léxicos (AAHs) Adverbios Francamente, [P] Sinceramente, [P] Honestamente, [P] Honradamente, [P] Recursos sintácticos Otras expresiones adverbiales equivalentes Con (toda) franqueza, [P] Con sinceridad, [P] En confianza, [P] … Cláusula Digo francamente/sinceramente/ honestamente/honradamente, [P] … Recursos léxicos -- -- Recursos sintácticos (EAHs) Adjetivo adverbializad o + Verbo enunciativo: 老实说/讲, [P] 坦率地说/讲, [P] 坦白（DE）说/讲, [P] 诚实地说/讲, [P] Verbo enunciativo + NP 说/讲实话, [P] 说/讲老实话, [P] 说实在的, [P] 说/讲真的, [P] 说真话, [P] 说真心话, [P] Locución: 实话实说, [P] 坦诚相告, [P] Cláusula (我)坦率地告诉你, [P] Chen, C. 陈晨 CLAC 79 2019: 217-256 255 (我)坦白(DE)告诉你(吧), [P] (我)实话告诉你/您(吧), [P] (你/您)实话告诉我, [P] (我)跟你/您说实话(吧), [P] (你)给我说实话, [P] (你)跟我说实话, [P] (我)老实告诉你/您(吧), [P] (你)老实告诉我, [P] (我)老实跟你/您说(吧), [P] (你)老实跟我说, [P] Anexo 2. Los recursos lingüísticos de las expresiones adverbiales del evento de actos de habla de orientación hacia el receptor/oyente en español y chino. Expresiones adverbiales del evento de actos de habla de orientación hacia el receptor/oyente Semántica conceptual y su paráfrasis El hablante/emisor pide o espera una respuesta del oyente/receptor y le solicita que en ésta tenga la actitud denotada por el adverbio. P.E: (Tú) Sé franco / sincero / honesto / honrado al contestarme si crees que está bien. Forma lógica de estructura conceptual y su interpretación IMP(∃e) [E(e) [E´(e) & Agente (e, receptor-oyente) & Tema (e, P)] & ADJ (e*, receptor-oyente)] Existe un evento del acto de habla(E) que consiste un sub-evento de pedir la información (E´)-un emisor/hablante pide o espera una respuesta del receptor/oyente, el contenido de la información es la proposición(P), y cuando el agente (receptor) realiza la proposición, su característica se requiere ser ADJ. Estructura superficial en español Estructura superficial en chino Recursos léxicos (AAHs) Adverbios Francamente, [P] Sinceramente, [P] Honestamente, [P] Honradamente, [P] Recursos sintácticos Otras expresiones adverbiales equivalentes Con (toda) franqueza, [P] Con sinceridad, [P] En confianza, [P] … Cláusula Digo francamente/sinceramente/ honestamente/honradamente, [P] … Recursos léxicos -- -- Recursos Adjetivo 老实说/讲, [P] Chen, C 陈晨. CLAC 79 2019: 217-256 256 sintácticos (EAHs) adverbializa do + Verbo enunciativo: 坦率地说/讲, [P] 坦白（DE）说/讲, [P] 诚实地说/讲, [P] Verbo enunciativo + NP 说/讲实话, [P] 说/讲老实话, [P] 说实在的, [P] 说/讲真的, [P] 说真话, [P] 说真心话, [P] Locución: 实话实说, [P] 坦诚相告, [P] Cláusula (我)坦率地告诉你, [P] (我)坦白(DE)告诉你(吧), [P] (我)实话告诉你/您(吧), [P] (你/您)实话告诉我, [P] (我)跟你/您说实话(吧), [P] (你)给我说实话, [P] (你)跟我说实话, [P] (我)老实告诉你/您(吧), [P] (你)老实告诉我, [P] (我)老实跟你/您说(吧), [P] (你)老实跟我说, [P]
https://openalex.org/W2110677442	http://publikationen.ub.uni-frankfurt.de/files/31712/InTech-Refinement_of_protein_tertiary_structure_by_using_spin_spin_coupling_constants_from_nuclear_magnetic_resonance_measurements.pdf	English	null	Refinement of Protein Tertiary Structure by Using Spin-Spin Coupling Constants from Nuclear Magnetic Resonance Measurements	InTech eBooks	2,012	cc-by	12,217	Refinement of Protein Tertiary Structure by Using Spin-Spin Coupling Constants from Nuclear Magnetic Resonance Measurements Jürgen M. Schmidt1 and Frank Löhr2 1School of Biosciences, University of Kent, Canterbury, Kent 2Institute of Biophysical Chemistry, Center for Biomolecular Magnetic Resonance, Goethe-University, Frankfurt am Main 1United Kingdom 2Germany 4 Refinement of Protein Tertiary Structure by Using Spin-Spin Coupling Constants from Nuclear Magnetic Resonance Measurements Jürgen M. Schmidt1 and Frank Löhr2 1School of Biosciences, University of Kent, Canterbury, Kent 2Institute of Biophysical Chemistry, Center for Biomolecular Magnetic Resonance, Goethe-University, Frankfurt am Main 1United Kingdom 2Germany 4 1. Introduction Modelling protein structure seems a challenging enterprise because the number of structure parameters required ordinarily exceeds the amount of independent data points available from experimental observations. Expressing the predominant conformation of a protein in terms of a geometry model, a polypeptide chain consisting of N atoms would command 3N – 6 Cartesian coordinates be fixed. Even for small proteins, this becomes a daunting number. Fortunately, so-called holonomic constraints limit the number of variables, leaving substantially fewer, truly relevant parameters for folding the polypeptide chain into its native tertiary structure. For example, adjusting bond lengths and the many angles between the covalent bonds connecting the atoms is of little concern and appropriate standard values can be inserted from tableworks (Pople & Gordon, 1967; Engh & Huber, 1991, 2006). Table 1 exemplifies for the 147-residue protein Desulfovibrio vulgaris flavodoxin how the number of truly independent internal rotational degrees of freedom amounts to less than one-tenth of the Cartesian coordinate set size. IUPAC-IUB conventions (1970) define three mainchain torsions for the polypeptide backbone. Protein structure determination primarily seeks to adjust the values of those two mainchain torsion angles that chiefly determine the fold of the chain, that is,  and  in each amino-acid residue. Peptide-bond geometry, as described by mainchain torsion angle , is normally assumed to be trans- or cis-planar, fixed at 180° or 0° angles, respectively, and thus does not give rise to rotational variability. In a next instance, the sidechain torsion angle 1 is of interest. Nuclear magnetic resonance (NMR) spectroscopy is uniquely positioned to help determine internal orientational constraints in a molecule, be these atom-atom distances (Wüthrich, 1986; Neuhaus & Williamson, 1989), relative bond orientations (Reif et al., 1997, 2000; www.intechopen.com 96 Protein Structure Schwalbe et al., 2001), relative protein domain orientations (Tjandra & Bax, 1997; Fischer et al., 1999) or rotational states of torsion angles (Pachler, 1963, 1964; Hansen et al., 1975; Bystrov, 1976; Ejchart, 1999). Especially powerful at measuring short-range interaction, NMR thus complements X-ray crystallography that is stronger at determining correlations over longer distances. Approach Parameters Approach Parameters h Parameters Items Coord’s n Cartesian coordinates (3N – 6) 6411 less bond length constraints (N – 1) – 2138 4273 less bond angle constraints – at tetrahedral centers (5 bond angles to fix) – 417×5 2188 — N-terminus (1) — mainchain C (all residues) (147) — sidechain C (all residues excl Gly) (129) — sidechain C (select residues) (85) — sidechain C (select residues) (47) — sidechain C (Met, Lys) and N (Lys) (8) – at planar centers (3 bond angles to fix) – 450×3 838 — mainchain N’ and C’ (excl N- but incl C-terminus) (293) — sidechain nitrogen and carbon (157) – at angled centers (1 bond angle to fix) – 24×1 814 — sidechain hydroxyl / thiol groups / sulfide bonds (24) less fixed or irrelevant torsion angles — mainchain  of peptide bonds – 146 668 — sidechain methyl – 84 584 — sidechain amide / guanidinium groups – 26 558 — sidechain hydroxyl / thiol groups – 24 534 — N-/C-termini – 2 532 up Variable torsion angles of relevance — mainchain  and  (2R – 2) 292 292 — sidechain 1 (all residues excl Gly, Ala) + 112 404 — sidechain 2 (select residues) + 84 488 — sidechain 3 (select residues) + 30 518 — sidechain 4 (Arg, Lys, Pro) + 14 532 We here focus on high-field NMR in aqueous solution which yields best resolution of the protein signals. Concentrated at around 1 mM, the dissolved protein is exposed to strong Table 1. Coordinate statistics for D. vulgaris flavodoxin (R = 147 residues, N = 2139 atoms) 1. Introduction Approach Parameters Items Coord’s top-down Cartesian coordinates (3N – 6) 6411 less bond length constraints (N – 1) – 2138 4273 less bond angle constraints – at tetrahedral centers (5 bond angles to fix) – 417×5 2188 — N-terminus (1) — mainchain C (all residues) (147) — sidechain C (all residues excl Gly) (129) — sidechain C (select residues) (85) — sidechain C (select residues) (47) — sidechain C (Met, Lys) and N (Lys) (8) – at planar centers (3 bond angles to fix) – 450×3 838 — mainchain N’ and C’ (excl N- but incl C-terminus) (293) — sidechain nitrogen and carbon (157) – at angled centers (1 bond angle to fix) – 24×1 814 — sidechain hydroxyl / thiol groups / sulfide bonds (24) less fixed or irrelevant torsion angles — mainchain  of peptide bonds – 146 668 — sidechain methyl – 84 584 — sidechain amide / guanidinium groups – 26 558 — sidechain hydroxyl / thiol groups – 24 534 — N-/C-termini – 2 532 bottom-up Variable torsion angles of relevance — mainchain  and  (2R – 2) 292 292 — sidechain 1 (all residues excl Gly, Ala) + 112 404 — sidechain 2 (select residues) + 84 488 — sidechain 3 (select residues) + 30 518 — sidechain 4 (Arg, Lys, Pro) + 14 532 top-down Cartesian coordinates (3N – 6) bottom-up — mainchain  and  (2R – 2) Table 1. Coordinate statistics for D. vulgaris flavodoxin (R = 147 residues, N = 2139 atoms) We here focus on high-field NMR in aqueous solution which yields best resolution of the protein signals. Concentrated at around 1 mM, the dissolved protein is exposed to strong www.intechopen.com Protein Structure from NMR Coupling Constants 97 fields of 11.7-23.4 T magnetic flux density, giving rise to nominal resonance frequencies in the proton band (1H) of 500-1000 MHz. Other biologically relevant stable isotopes 13C and 15N, respectively, resonate at roughly one-quarter and one-tenth of 1H at a given field. fields of 11.7-23.4 T magnetic flux density, giving rise to nominal resonance frequencies in the proton band (1H) of 500-1000 MHz. Other biologically relevant stable isotopes 13C and 15N, respectively, resonate at roughly one-quarter and one-tenth of 1H at a given field. NMR experiments then consist of radio-frequency pulse trains that generate controlled transitions of spin states (Ernst et al., 1987). These transitions manifest in resonance lines whose spectrum location and detailed shape reveal information about the molecular environment in which the probed atoms are embedded. Rotatory states of torsion angles are accessible through measurement of so-called J-coupling constants which lend NMR signals a splitting fine structure due to bond-electron mediated pair interactions between NMR active nuclei. Put simply, the presence of each neighbouring (coupled) spin splits the resonance line of the observed spin into two. This splitting is field- independent (hence coupling ‘constant’) but does depend on local molecular geometry. Of particular interest to structure determination are three-bond 3JXY coupling constants as their magnitudes relate to dihedral angles subtended by the three covalent bonds connecting the coupled pair of nuclei X and Y, which can be any combination of 1H, 15N, or 13C, prevalent in the topology of the torsion considered (Fig. 1). Ci+1 α HΝ Hβ N Cα Cβ C’ Cγ Hβ ψ φ χ1 χ2 ω C’ Ni+1 i-1 Hα HΝ Hβ N Cα Cβ C’ Cγ Hβ ψ φ χ1 χ2 ω C’ i-1 Hα HΝ Hβ N Cα Cβ C’ Cγ Hβ ψ φ χ1 χ2 ω C’ Ni+1 i-1 Hα 3J(φ) 3J(ψ) 3J(χ1) Ni+1 HΝ HΝ HΝ Fig. 1. 3J spin-spin coupling topologies in 13C,15N enriched polypeptides. Arrows denote three-bond connectivities, grouped by conformational relevance to , , and  torsions. HΝ Fig. 1. top-down Cartesian coordinates (3N – 6) 3J spin-spin coupling topologies in 13C,15N enriched polypeptides. Arrows denote three-bond connectivities, grouped by conformational relevance to , , and  torsions. Fig. 1. 3J spin-spin coupling topologies in 13C,15N enriched polypeptides. Arrows denote three-bond connectivities, grouped by conformational relevance to , , and  torsions. Fig. 1. 3J spin-spin coupling topologies in 13C,15N enriched polypeptides. Arrows denote three-bond connectivities, grouped by conformational relevance to , , and  torsions. Multiple J data collected for a given torsion angle overdetermine that angle and thus allow its value to be retrieved. Six coupling constants, 3JHNH, 3JHNC’, 3JHNC, 3JC’H, 3JC’C’, and 3JC’C, www.intechopen.com www.intechopen.com 98 Protein Structure determine torsion angle (C’i–1–N’i–C i–C’i) of residue i (Wang & Bax, 1996; Blümel et al., 1998). Another three couplings, 3JHN’, 3JCN’, and 3JN’N’, would yield (N’i–C i–C’i–N’i+1). Of qualitative concern only, the planar trans- or cis-configured peptide bonds at torsion angle (C i–C’i–N’i+1–C i+1) can be verified by two couplings, 3JCHN and 3JCC, which appear to reflect  geometry also (Hennig et al., 2000). Of numerous sidechain torsions encountered in amino acids, 1(N’i–C i–C i–C i) impacts most on the spatial orientation of the sidechain. Up to nine distinct coupling constants are accessible in amino acids (Pérez et al., 2001). Karplus (1963) suggested the dependence of 3J on dihedral angle  subtended by the three bonds that connect the coupled nuclei follow the empirical relation 3J() = C0 + C1 cos  + C2 cos 2  3J() = C0 + C1 cos  + C2 cos 2 3J() = C0 + C1 cos  + C2 cos 2  where Cm are Karplus coefficients in Hz empirically calibrated for the 3J types encountered in amino acids (Bystrov, 1976; Wang & Bax, 1995, 1996; Hu & Bax, 1996, 1997; Schmidt et al., 1999, Pérez et al., 2001). Multiples of 60° increments to  establish the phase relation between the actual internuclear dihedral angle  and the IUPAC-defined torsion , , or 1 (Fig. 2). –150 –120 –90 –60 –30 0 30 60 90 120 150 1 2 3 4 5 6 7 8 9 3JHNHα 3JHNCβ 3JHNC' 3JC'Hα 3JC'Cβ 3JC'C' φ (deg) J (Hz) Fig. 2. Angular dependence of the protein -torsion related 3J coupling constants. 3J are at maximum when the bonds between the coupled nuclei are trans-oriented, and at minimum for perpendicular orientations. www.intechopen.com top-down Cartesian coordinates (3N – 6) For 3JHNH, the panels show from left to right the internuclear angle HNH =  – 60° in the situations ±180°, –90°, ±0°, and +90°. –150 –120 –90 –60 –30 0 30 60 90 120 150 1 2 3 4 5 6 7 8 9 3JHNHα 3JHNCβ 3JHNC' 3JC'Hα 3JC'Cβ 3JC'C' φ (deg) J (Hz) Fig. 2. Angular dependence of the protein -torsion related 3J coupling constants. 3J are at maximum when the bonds between the coupled nuclei are trans-oriented, and at minimum for perpendicular orientations. For 3JHNH, the panels show from left to right the internuclear angle HNH =  – 60° in the situations ±180°, –90°, ±0°, and +90°. www.intechopen.com 99 Protein Structure from NMR Coupling Constants The coefficients C0 signify mean J values obtained for a complete torsion-angle revolution, also referred to as conformation averaged J coupling constants. The differences (C2 – C1) are the largest deflections in J from the mean, where primary and secondary maxima of the curves at ±180° and ±0°, respectively, differ by 2C1. Given sets of up to six 3J parameters per torsion, the challenge is now to find that angle value in best agreement with the experimental data. Numerical methods exploiting the redundance present in large data pools permit the self-consistent calibration of Karplus coefficients during the course of the angle refinement (Schmidt et al., 1999). This obviates the need for traditional referencing of conformations derived from X-ray data. We previously collected for flavodoxin values for the six possible coupling constants 3J() and determined the protein’s mainchain torsion angles  (Schmidt et al., 1999). Discrepancy in  between our NMR solution structure and comparison coordinates from X-ray crystallography (Walsh et al., 1998; Artali et al. 2002) is only 5° on average, which is smaller than the molecular dynamical angular libration due to thermal effects, indicating that both NMR solution and X-ray crystal structures of flavodoxin are very similar indeed. Here, we record 3J data for the enzyme Ribonuclease T1, a 104-residue protein (11 kDa) from Aspergillus oryzae (RNase T1, EC 3.1.27.3) that cleaves single-stranded RNA 3’-side of guanine nucleotides, and determine the majority of the  torsion angles in the enzyme. 2. Materials and methods One stringent requirement for efficient protein NMR analysis is that the protein sample be artifically enriched in the stable non-radioactive isotopes 15N and 13C, a process nowadays commonly applied in protein expression by recombinant technologies (Kainosho, 1997). Whilst oxygen does not play any role in protein NMR practice, the 1H isotope offering greatest sensitivity is ubiquitous and abundant. Sometimes, it is being depleted by 2H replacement in order to alleviate adverse signal relaxation effects occuring in large protein samples (above approximately 250 amino acids) exhibiting slow rotational tumbling rates (longer than approximately 10 ns rad–1). 2.1 Protein sample Uniformly 13C,15N-labeled RNase T1 (Lys25 isoenzyme) was obtained following established protocols (Quaas et al. 1988a,b; Spitzner et al., 2001) and used at 2-mM concentration in aqueous solution of pH 5.5 (containing 10% D2O). All NMR spectra were recorded at 308 K. Prerequisite to any protein NMR analysis is the assignment of resonance signals to individual nuclei, not unlike a fingerprint of the molecule. Values quoted are chemical shifts in parts per million (ppm) from the respective 1H, 13C, or 15N band base frequency. Resonance assignments for RNase T1 (Fig. 3) are available from the BioMagRes database for 1H (BMRB-133; Hoffmann & Rüterjans, 1988) and for 15N (BMRB-1658; Schmidt et al., 1991), and 13C chemical shifts were given by Pfeiffer et al. (1996b). Comparison  torsion angles were calculated from crystal coordinates, resolved at 0.15 nm, of RNase T1 complexed with Ca2+ (PDB-9RNT; Martinez-Oyanedel et al., 1991). www.intechopen.com 100 Protein Structure 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 100 105 110 115 120 125 130 F2 / 1HN (ppm) F1 / 15N (ppm) C2 D3 Y4 T5 C6 G7 S8 N9 C10 Y11 S12 S13 S14 D15 V16 S17 T18 A19 Q20 A21 A22 G23 Y24 K25 L26 H27 E28 D29 G30 E31 T32 V33 G34 S35 N36 S37 Y38 H40 K41 Y42 N43 N44 Y45 E46 G47 F48 D49 F50 S51 V52 S53 S54 Y56 Y57 E58 W59 I61 L62 S63 S64 G65 D66 V67 Y68 S69 G71 S72 G74 A75 D76 R77 V78 V79F80 N81 E82 N83 N84 Q85 L86 A87 G88 V89 I90 T91 H92 T93 G94 A95 S96 G97 N98 N99 F100 V101 E102 C103 T104 Fig. 3. 1H,15N-chemical shift correlation spectrum of RNase T1 at 900 MHz and 308 K. Fig. 3. 1H,15N-chemical shift correlation spectrum of RNase T1 at 900 MHz and 308 K. 2.3 Data evaluation Whichever the chosen approach, the particular method applied to extract J values as structure parameters from NMR spectra must be considered an integral part of the analysis also. The authors gathered experience with both methods, albeit with an undeniable preference for E.COSY-type spectra which perform particularly robust in connection with computer-assisted lineshape analysis (Schmidt, 1997a; Löhr et al., 2000). Contour plots of NMR signals for J analysis recorded in E.COSY mode show characteristic tilts (Griesinger et al., 1987), where a prominent large one-bond coupling spreads out the multiplet along the vertical spectrum dimension, so as to permit reading the sought smaller 3J coupling off the frequency difference between the two multiplet halves in the horizontal dimension. In the example of Fig. 4, the 1JCH of typically 143 Hz is exploited to split the signal into two halves along the F1 dimension, given here by the 13C resonance frequency, and the small 3JC’H coupling results from the frequency difference between both halves along the perpendicular F2 dimension, given here by the 13C’ carbonyl resonance. The parameter record lists optimized values for coupling constants 1JCH and 3JC’H as the primary E.COSY components responsible for the tilted appearance of the signal shape in vertical and horizontal directions, respectively. Other parameters include, apart from amplitude scaling, line widths and line asymmetries in both dimensions, a second unresolved splitting pair. F2 / CO [Hz] F1 / Cα [Hz] RNase T1 - Asn44 @ F1=53.78, F2=175.38 ppm, F3=5.73 ppm LogScale= 6.0195 LW(Ca)= 11.00 Hz LW(CO)= 7.21 Hz dLW(Ca)= 3.79 Hz dLW(CO)= 1.69 Hz seco. ECOSY J(Ca,Ha) = 4.73 Hz J(CO,Ha) = 7.08 Hz prim. ECOSY J(Ca,Ha) = 131.12 Hz J(CO,Ha) = 6.67 Hz –40 –20 0 20 40 –160 –120 –80 –40 0 40 80 120 160 Experiment –40 –20 0 20 40 Simulation –40 –20 0 20 40 Difference 3JC'Hα 1JCαHα Fig. 4. Contour plot of a 500-MHz H(N)CA,CO[HA]-E.COSY multiplet recorded for RNase T1. Example analysis for extracting the 3JC’H coupling constant from the 2D 13C,13C’ correlation signal (F1,F2) taken at the resonance of the amide 1HN proton in the third dimension (F3). The three nuclei eliciting the three spectrum dimensions all couple with the 1H spin (Fig. 1.), whose presence, following the E.COSY principle, is seen only as splittings, not as another frequency dimension. 2.2 Data collection The focus of this chapter lies on protein structure and interpretation of NMR data. The reader interested in the details of NMR pulse-sequence design and the setup of multi- dimensional NMR experiments is referred to specialist textbooks (Roberts, 1993; Evans, 1995; Keeler, 2005; Hoch & Stern, 1996; Cavanagh et al., 2007) and to the literature including contained bibliography on the measurement of each of the six protein  related 3J coupling constants, as given in the separate NMR Experiments section at the end of the chapter. Measurements of J coupling constants are subject to two principal concepts. One family of NMR experiments exploits frequency modulation, such as COSY, DQF-COSY, and E.COSY (Griesinger et al., 1987), giving rise to characteristic multiplet line splittings (Fig. 4). The other technique uses amplitude modulation, subdivided into J-modulation (Billeter et al., 1992) and J-correlation (Bax et al., 1994), encoding J in time-dependent signal intensity. Both approaches naturally have their pros and cons. While frequency-modulation schemes produce NMR signals of weaker intensity and larger space requirement, at times leading to signal overlap, amplitude-modulation schemes chiefly fail to measure small coupling constants. To minimize signal overlap, protein NMR spectra are often recorded in 3D mode by exploiting the resonance of an auxiliary spin and selecting 2D projections at that frequency. Example graphs in Fig. 4 show such an experimental multiplet pattern, together with its best-fit least-squares lineshape reconstruction and the residual error signal. www.intechopen.com www.intechopen.com 101 Protein Structure from NMR Coupling Constants 3.1 RNase T1 Using a uniformly 15N and 13C stable-isotope labeled sample of the enzyme RNase T1, a total 512 3J values were collected, related to the polypeptide mainchain torsion angles  in 82 out of the total 104 amino-acid residues. Even without fitting quantitative torsion angles to the 3J data, qualitative inspection of the J values permits insights into some details of the protein’s secondary structure already. For example, residue Asn44 in RNase T1 exhibits a very large 3JC’H coupling of 6.67 Hz (Fig. 4), second only to that seen in Asn84. This is irreconcilable with a  torsion in the negative value range (Fig. 2). In addition, the 1JCH coupling of only 131 Hz (Fig. 4) falls well short of the expected average and supports a positive  torsion, too. Consequently, Asn44 must exhibit a positive value for its  torsion. Signals for consecutive residues Asn43 and Asn44 in RNase T1 could hardly be more different. A value of 2.94 Hz for 3JHNC in Asn43 (Fig. 5A) contrasts the lowly 0.47 Hz in Asn44 (Fig. 5B). Very different 1JCC couplings also suggest differing backbone geometries. While the near-average 1JCC coupling of 35.8 Hz in Asn43 is common with negative  torsions, the unusually large 43.7-Hz coupling in Asn44 agrees better with a positive  value (Schmidt et al., 2009). Both being asparagines, this cannot be a residue-type specific effect on the J couplings (Schmidt, 2007a). The above-average value of 2.66 Hz seen for 3JC’C in Tyr45 (Fig. 5C) is consistent with a type-I  turn spanning both Tyr45 and Glu46. This tyrosine’s aromatic ring system is a critical component in nucleotide recognition and binding. The consecutive residues Ala87 and Gly88 in RNase T1 form a  bulge (Chan et al., 1993). An unusually small 3JHNC' coupling near zero in Ala87 (Fig. 5D) and an unusually large 2.9- Hz 3JC’C’ coupling in Gly88 (Fig. 5E) hint at  torsion angles near +90°/–90° and 180° (Fig. 2), respectively, corroborating the distorted geometry in the central portion of a  strand. Coupling type  C0 C1 C2 trans J(180°) gauche J(±60°) (deg) (Hz) (Hz) (Hz) (Hz) (Hz) 3JHNH –60° 5.67 –0.71 3.37 9.74 3.63 3JHNC’ 180° 1.79 –0.75 1.56 4.09 0.64 3JHNC 60° 2.32 –1.64 1.91 5.86 0.54 3JC’H 120° 3.21 –2.17 2.05 7.43 1.10 3JC’C’ 0° 1.43 –0.96 0.76 3.15 0.58 3JC’C –120° 1.33 –0.51 0.91 2.75 0.62 Table 2. 2.3 Data evaluation F2 / CO [Hz] F1 / Cα [Hz] RNase T1 - Asn44 @ F1=53.78, F2=175.38 ppm, F3=5.73 ppm LogScale= 6.0195 LW(Ca)= 11.00 Hz LW(CO)= 7.21 Hz dLW(Ca)= 3.79 Hz dLW(CO)= 1.69 Hz seco. ECOSY J(Ca,Ha) = 4.73 Hz J(CO,Ha) = 7.08 Hz prim. ECOSY J(Ca,Ha) = 131.12 Hz J(CO,Ha) = 6.67 Hz –40 –20 0 20 40 –160 –120 –80 –40 0 40 80 120 160 Experiment –40 –20 0 20 40 Simulation –40 –20 0 20 40 Difference 3JC'Hα 1JCαHα Fig. 4. Contour plot of a 500-MHz H(N)CA,CO[HA]-E.COSY multiplet recorded for RNase T1. Example analysis for extracting the 3JC’H coupling constant from the 2D 13C,13C’ correlation signal (F1,F2) taken at the resonance of the amide 1HN proton in the third dimension (F3). The three nuclei eliciting the three spectrum dimensions all couple with the 1H spin (Fig. 1.), whose presence, following the E.COSY principle, is seen only as splittings, not as another frequency dimension. www.intechopen.com 102 Protein Structure e 2. Karplus coefficients for Eq. 1, optimized against experimental 3J data for RNase T1 3.1 RNase T1 Karplus coefficients for Eq. 1, optimized against experimental 3J data for RNase T1 www.intechopen.com 103 Protein Structure from NMR Coupling Constants F2 / HN [Hz] F1 / Cα [Hz] RNase T1 - Asn43 @ F1=62.71, F2=8.53 ppm, F3=125.32 ppm LogScale= 6.6491 LW(Ca)= 14.47 Hz LW(HN)= 13.11 Hz dLW(Ca)= 0.91 Hz dLW(HN)= 0.75 Hz seco. ECOSY J(Ca,Cx) = 0.98 Hz J(HN,Cx) = 6.29 Hz prim. ECOSY J(Ca,Cb) = 35.80 Hz J(HN,Cb) = 2.94 Hz –40 –20 0 20 40 –60 –40 –20 0 20 40 60 Experiment –40 –20 0 20 40 Simulation –40 –20 0 20 40 Difference A 3JHNCβ 1JCαCβ F2 / HN [Hz] F1 / Cα [Hz] RNase T1 - Asn44 @ F1=61.82, F2=5.69 ppm, F3=113.92 ppm LogScale= 6.8681 LW(Ca)= 10.62 Hz LW(HN)= 19.91 Hz dLW(Ca)= 0.08 Hz dLW(HN)= 0.41 Hz seco. ECOSY J(Ca,Cx) = 0.69 Hz J(HN,Cx) = 3.62 Hz prim. ECOSY J(Ca,Cb) = 43.67 Hz J(HN,Cb) = 0.47 Hz –40 –20 0 20 40 –60 –40 –20 0 20 40 60 Experiment –40 –20 0 20 40 Simulation –40 –20 0 20 40 Difference B 3JHNCβ 1JCαCβ Cα [Hz] RNase T1 - Tyr45 @ F1=59.79, F2=177.47 ppm, F3=8.58 ppm LogScale= 6.4622 LW(Ca)= 9.03 Hz LW(CO)= 4.93 Hz dLW(Ca)= 0.23 Hz dLW(CO)= 0.10 Hz seco. ECOSY J(Ca,Cx) = 1.05 Hz J(CO,Cx) = 3.46 Hz i 0 20 40 60 Experiment Simulation Difference C 1JCαCβ F2 / HN [Hz] F1 / Cα [Hz] RNase T1 - Asn43 @ F1=62.71, F2=8.53 ppm, F3=125.32 ppm LogSca LW(Ca) LW(HN) dLW(Ca dLW(HN seco. J(Ca,C J(HN,C prim. J(Ca,C J(HN,C –40 –20 0 20 40 –60 –40 –20 0 20 40 60 Experiment –40 –20 0 20 40 Simulation –40 –20 0 20 40 Difference A 3JHNCβ 1JCαCβ A F2 / HN [Hz] F1 / Cα [Hz] g LW(Ca)= 14.47 Hz LW(HN)= 13.11 Hz dLW(Ca)= 0.91 Hz dLW(HN)= 0.75 Hz seco. ECOSY J(Ca,Cx) = 0.98 Hz J(HN,Cx) = 6.29 Hz prim. ECOSY J(Ca,Cb) = 35.80 Hz J(HN,Cb) = 2.94 Hz –40 –20 0 20 40 –60 –40 –20 0 20 40 60 Experiment –40 –20 0 20 40 Simulation –40 –20 0 20 40 Difference 3JHNCβ 1JCαCβ F2 / HN [Hz] F1 / Cα [Hz] RNase T1 - Asn44 @ F1=61.82, F2=5.69 ppm, F3=113.92 ppm LogScale= 6.8681 LW(Ca)= 10.62 Hz LW(HN)= 19.91 Hz dLW(Ca)= 0.08 Hz dLW(HN)= 0.41 Hz seco. ECOSY J(Ca,Cx) = 0.69 Hz J(HN,Cx) = 3.62 Hz prim. 3.1 RNase T1 ECOSY J(Ca,Cb) = 43.67 Hz J(HN,Cb) = 0.47 Hz –40 –20 0 20 40 –60 –40 –20 0 20 40 60 Experiment –40 –20 0 20 40 Simulation –40 –20 0 20 40 Difference B 3JHNCβ 1JCαCβ F2 / CO [Hz] F1 / Cα [Hz] RNase T1 - Tyr45 @ F1=59.79, F2=177.47 ppm, F3=8.58 ppm LogScale= 6.4622 LW(Ca)= 9.03 Hz LW(CO)= 4.93 Hz dLW(Ca)= 0.23 Hz dLW(CO)= 0.10 Hz seco. ECOSY J(Ca,Cx) = 1.05 Hz J(CO,Cx) = 3.46 Hz prim. ECOSY J(Ca,Cb) = 34.00 Hz J(CO,Cb) = 2.66 Hz –40 –20 0 20 40 –60 –40 –20 0 20 40 60 Experiment –40 –20 0 20 40 Simulation –40 –20 0 20 40 Difference C 3JC'Cβ 1JCαCβ F2 / HN [Hz] F1 / Cα [Hz] RNase T1 - Asn44 @ F1=61.82, F2=5.69 ppm, F3=113.92 ppm LogScale= 6.8681 LW(Ca)= 10.62 Hz LW(HN)= 19.91 Hz dLW(Ca)= 0.08 Hz dLW(HN)= 0.41 Hz seco. ECOSY J(Ca,Cx) = 0.69 Hz J(HN,Cx) = 3.62 Hz prim. ECOSY J(Ca,Cb) = 43.67 Hz J(HN,Cb) = 0.47 Hz –40 –20 0 20 40 –60 –40 –20 0 20 40 60 Experiment –40 –20 0 20 40 Simulation –40 –20 0 20 40 Difference B 3JHNCβ 1JCαCβ B F2 / CO [Hz] F1 / Cα [Hz] RNase T1 - Tyr45 @ F1=59.79, F2=177.47 ppm, F3=8.58 ppm LogScale= 6 LW(Ca)= 9. LW(CO)= 4. dLW(Ca)= 0. dLW(CO)= 0. seco. ECOSY J(Ca,Cx) = J(CO,Cx) = prim. ECOSY J(Ca,Cb) = J(CO,Cb) = –40 –20 0 20 40 –60 –40 –20 0 20 40 60 Experiment –40 –20 0 20 40 Simulation –40 –20 0 20 40 Difference C 3JC'Cβ 1JCαCβ C www.intechopen.com 104 Protein Structure F2 / HN [Hz] F1 / Cα [Hz] RNase T1 - Ala87 @ F1=52.38, F2=9.08 ppm, F3=110.78 ppm LogScale= 6.8253 LW(Ca)= 0.00 Hz LW(HN)= 10.69 Hz dLW(Ca)= 0.72 Hz dLW(HN)= 0.10 Hz seco. ECOSY J(Ca,CO) = 0.36 Hz J(HN,CO) = 9.04 Hz prim. ECOSY J(Ca,CO) = 52.98 H J(HN,CO) = 0.16 H –40 –20 0 20 40 –80 –60 –40 –20 0 20 40 60 80 Experiment –40 –20 0 20 40 Simulation –40 –20 0 20 40 Difference D 1JCαC' 3JHNC' D E F2 / CO [Hz] F1 / Cα [Hz] RNase T1 - Gly88 @ F1=63.54, F2=177.03 ppm, F3=7.06 ppm LogScale= 6.1999 LW(Ca)= 0.00 Hz LW(CO)= 3.78 Hz dLW(Ca)= 0.12 Hz dLW(CO)= 0.07 Hz seco. ECOSY J(Ca,CO) = 1.28 Hz J(CO,CO) = 2.29 Hz prim. 3.1 RNase T1 ECOSY J(Ca,CO) = 50.61 Hz J(CO,CO) = 2.93 Hz –40 –20 0 20 40 –80 –60 –40 –20 0 20 40 60 80 Experiment –40 –20 0 20 40 Simulation –40 –20 0 20 40 Difference E 3JC'C' 1JCαC' Fig. 5. Example 2D E.COSY multiplet sections from 3D NMR spectra recorded for RNase T1. Panels A-C (previous page): 3JHNC and 3JC’C evaluations exploiting the 1JCC coupling of typically 35 Hz; Panels D-E: 3JHNC' and 3JC’C' evaluations exploiting the 1JCC' coupling of typically 53 Hz. Solid and dotted lines are positive and negative contours, respectively. E F2 / CO [Hz] F1 / Cα [Hz] RNase T1 - Gly88 @ F1=63.54, F2=177.03 ppm, F3=7.06 ppm LogScale= 6.1999 LW(Ca)= 0.00 Hz LW(CO)= 3.78 Hz dLW(Ca)= 0.12 Hz dLW(CO)= 0.07 Hz seco. ECOSY J(Ca,CO) = 1.28 Hz J(CO,CO) = 2.29 Hz prim. ECOSY J(Ca,CO) = 50.61 Hz J(CO,CO) = 2.93 Hz –40 –20 0 20 40 –80 –60 –40 –20 0 20 40 60 80 Experiment –40 –20 0 20 40 Simulation –40 –20 0 20 40 Difference E 3JC'C' 1JCαC' RNase T1 - Gly88 @ F1=63.54, F2=177.03 ppm, F3=7.06 ppm Fig. 5. Example 2D E.COSY multiplet sections from 3D NMR spectra recorded for RNase T1. Panels A-C (previous page): 3JHNC and 3JC’C evaluations exploiting the 1JCC coupling of typically 35 Hz; Panels D-E: 3JHNC' and 3JC’C' evaluations exploiting the 1JCC' coupling of typically 53 Hz. Solid and dotted lines are positive and negative contours, respectively. Eventually, the 82  torsion angles were fitted simultaneously to the pool of all 512 3J data. Self-consistently optimized in conjunction with the torsion-angle values, the Karplus curves shown in Fig. 6 and respective coefficients summarized in Table 2 represent the best fit to the 3J data available for RNase T1 exclusively, yet, would be similar with other proteins also. www.intechopen.com www.intechopen.com Protein Structure from NMR Coupling Constants 105 φ (deg) J (Hz) 0 3 6 9 XR (initial) 0 2 4 6 0 2 4 6 0 2 4 6 0 1 2 3 –120 –60 0 60 120 0 1 2 3 3JHNHα SC (final) 3JHNC' 3JHNCβ 3JC'Hα 3JC'C' –120 –60 0 60 120 3JC'Cβ → → → Fig. 6. Optimization of protein  torsions on the basis of 3J coupling constants. XR panels Experimental data for RNase T1 (dots) plotted against initial torsion values calculated fr crystal structure coordinates (PDB-9RNT). 3.1 RNase T1 SC panels: The same data plotted against torsi values iteratively optimized by referring exclusively and simultaneously to all 3J data, adjusting both torsion angles and Karplus coefficients in a self-consistent manner (Schm et al., 1999). Arrows point at Asn44 data that appear to be outliers initially. SC (final) Fig. 6. Optimization of protein  torsions on the basis of 3J coupling constants. XR panels: Experimental data for RNase T1 (dots) plotted against initial torsion values calculated from crystal structure coordinates (PDB-9RNT). SC panels: The same data plotted against torsion values iteratively optimized by referring exclusively and simultaneously to all 3J data, adjusting both torsion angles and Karplus coefficients in a self-consistent manner (Schmidt et al., 1999). Arrows point at Asn44 data that appear to be outliers initially. www.intechopen.com 106 Protein Structure –120 –60 0 60 120 –120 –60 0 60 120 D3 T4 T5C6 S8 N9 C10 Y11 S12 S14 D15 V16 S17 T18 A19 Q20 A21 A22 Y24 K25 L26 H27 E28 D29 E31 T32 V33 S37 Y38 H40 K41 Y42 N43 N44 Y45 E46 F48 D49 F50 S51 V52 S53 S54 Y56 Y57 E58 W59 I61 L62 S63 S64 D66 V67 Y68 S69 A75 D76 R77 V78 V79 F80 N81 E82 N83 N84 Q85 L86 A87 V89 I90 T91 H92 T93 A95 S96 N98N99 F100 V101 E102 C103 T104 φNMR (deg) φX-ray (deg) Fig. 7. Comparison of mainchain torsions  in RNase T1 as inferred from self-consistent J coupling analysis and from crystallographic data (PDB-9RNT) by Martinez-Oyanedel et al. (1991). The majority of items agree within a tolerance of ±30° (dashed bounds). In the final optimized set, the Asn44  torsion angle has flipped from –86° to +37°. –120 –60 0 60 120 –120 –60 0 60 120 D3 T4 T5C6 S8 N9 C10 Y11 S12 S14 D15 V16 S17 T18 A19 Q20 A21 A22 Y24 K25 L26 H27 E28 D29 E31 T32 V33 S37 Y38 H40 K41 Y42 N43 N44 Y45 E46 F48 D49 F50 S51 V52 S53 S54 Y56 Y57 E58 W59 I61 L62 S63 S64 D66 V67 Y68 S69 A75 D76 R77 V78 V79 F80 N81 E82 N83 N84 Q85 L86 A87 V89 I90 T91 H92 T93 A95 S96 N98N99 F100 V101 E102 C103 T104 φNMR (deg) φX-ray (deg) Fig. 7. Comparison of mainchain torsions  in RNase T1 as inferred from self-consistent J coupling analysis and from crystallographic data (PDB-9RNT) by Martinez-Oyanedel et al. 3.1 RNase T1 (1991). The majority of items agree within a tolerance of ±30° (dashed bounds). In the final optimized set, the Asn44  torsion angle has flipped from –86° to +37°. Two facts rationalize the markedly reduced scatter seen in the right-hand-side 3JHH panel in Fig. 6 compared with the other five coupling types: Firstly, being larger than 3JHC or 3JCC on an absolute scale, 3JHH values dominate the fit and, secondly,  torsion values in regular protein structure typically fall on the steep slope of the 3JHH curve, making regression sensitive to small changes in 3JHH. The residual scatter is due to unaccounted substituent effects in the different amino-acid types (Schmidt, 2007a), unaccounted torsion-angle dynamics (Brüschweiler & Case, 1994; Pérez et al., 2001), and random experimental error. Two facts rationalize the markedly reduced scatter seen in the right-hand-side 3JHH panel in Fig. 6 compared with the other five coupling types: Firstly, being larger than 3JHC or 3JCC on an absolute scale, 3JHH values dominate the fit and, secondly,  torsion values in regular protein structure typically fall on the steep slope of the 3JHH curve, making regression sensitive to small changes in 3JHH. The residual scatter is due to unaccounted substituent effects in the different amino-acid types (Schmidt, 2007a), unaccounted torsion-angle dynamics (Brüschweiler & Case, 1994; Pérez et al., 2001), and random experimental error. Assigned to Asn44 in RNase T1, the solitary data point in the left-hand-side 3JC'H() panel in Fig. 6, near the upper border, is elevated by around 3 Hz above the crowd. Apparently, other large 3JC'H coupling constants around 6-7 Hz connect with positive torsion angles. Contrasting the negative value found for Asn44 in crystal structure PDB-9RNT (Martinez- Oyanedel et al., 1991), the conformation in solution that emerged from our J coupling analysis differs from that in the solid state (Fig. 7). Similarity between relative J coupling values for Asn44 and those residues that exhibit positive  angles clearly suggest that 44 be rotated to a positive value also. Indeed, self-consistent optimization of  torsions referencing the set of six -related coupling types converges at +37° for Asn44, while improving dramatically the error between observed coupling constants and those predicted from the crystal-structure angles. The region around residues 42-48 of the enzyme engages in the recognition and binding of the substrate nucleotide (Fig. 8). 3.1 RNase T1 Inspection of eight crystal structures of RNase T1 in a variety of complexes reveals that Asn44 adopts a negative  value in only those structures www.intechopen.com www.intechopen.com 107 Protein Structure from NMR Coupling Constants presenting a free binding site or, rather, a calcium, zinc, or vanadate metal ion bound to the apo-enzyme (9RNT, 8RNT, 3RNT). Similarly, the inadequate adenosine nucleotide complex (6RNT) also resembles the apo-enzyme. Even though the distant ribose phosphate interacts with catalytic residues His40 and Glu58 in this latter structure, the nucleobase is directed away from the enzyme, so as to leave the recognition loop unoccupied, for RNase T1 binding is G not A specific. presenting a free binding site or, rather, a calcium, zinc, or vanadate metal ion bound to the apo-enzyme (9RNT, 8RNT, 3RNT). Similarly, the inadequate adenosine nucleotide complex (6RNT) also resembles the apo-enzyme. Even though the distant ribose phosphate interacts with catalytic residues His40 and Glu58 in this latter structure, the nucleobase is directed away from the enzyme, so as to leave the recognition loop unoccupied, for RNase T1 binding is G not A specific. However, four other crystal structures of RNase T1 in various complexes, notably those with a bound guanine nucleotide (1RLS, 1RNT, 2RNT, 5RNT), do exhibit positive  angles for Asn44 (Fig. 7). Also  angles in the adjacent positions Asn43 and Tyr45 differ between the two groups, by approximately 40° and 10°, respectively. The observations suggest that binding of the correct substrate elicits a concerted conformation change involving torsion 44, and likely 44, too, as well as torsions 43 and 45 in the preceding and subsequent residue, respectively. Yet, the NMR evidence tells a different story: Torsion angle 44 is positive already in our sample of the free enzyme in aqueous solution that was subjected to NMR measurement! Conformational variability of the Asn44 backbone, such as continual flips between positive and negative values, can be ruled out on the basis of the large 3JC'H coupling value observed, as such conformational averaging would reduce the J value towards its mean of about half the size (Table 2). A likely explanation for the negative angle value in some of the crystal structures would be packing effects through molecules in adjacent grid cells distorting the conformation of the rather exposed 42-48 loop region at the protein surface. 3.1 RNase T1 Another possible explanation could be that the metal agents added to aid the crystallization process obstruct the binding site differently than the natural substrate would do. Fig. 8. Stereo view of residues 42-48 in the crystal structure of the RNase T1:2’GMP inhibitor complex (PDB-1RNT, Arni et al., 1988). Involved in nucleotide recognition and binding, Asn43 and Asn44 backbone amide groups interact with the guanine five- and six-ring, respectively, while the Glu46 sidechain carboxylate probes the presence of the correct hydrogen bonding capacity at the Watson-Crick edge of the guanine. Tyr42 and Tyr45 sandwich the guanine ring plane. Fig. 8. Stereo view of residues 42-48 in the crystal structure of the RNase T1:2’GMP inhibitor complex (PDB-1RNT, Arni et al., 1988). Involved in nucleotide recognition and binding, Asn43 and Asn44 backbone amide groups interact with the guanine five- and six-ring, respectively, while the Glu46 sidechain carboxylate probes the presence of the correct hydrogen bonding capacity at the Watson-Crick edge of the guanine. Tyr42 and Tyr45 sandwich the guanine ring plane. 3.2 Flavodoxin 3J coupling data from a similar study targeting backbone torsion angles  in flavodoxin suggested subtle differences between the structures of this protein in aqueous solution www.intechopen.com www.intechopen.com 108 Protein Structure (NMR) and in the crystalline solid state (X-ray). For example, on the basis of J coupling constants the orientation of peptide plane Ala39/Ser40 shown in the X-ray structure needed to be adjusted by about +10° in order to fulfil the experimental data (Löhr et al., 2001). (NMR) and in the crystalline solid state (X-ray). For example, on the basis of J coupling constants the orientation of peptide plane Ala39/Ser40 shown in the X-ray structure needed to be adjusted by about +10° in order to fulfil the experimental data (Löhr et al., 2001). A conformational hot spot is found in flavodoxin’s so-called “60-loop”, the region protruding around the isoalloxazine ring system, made up of Gly61-Asp62-Asp63-Ser64. Results from our analysis on the exclusive basis of J-coupling constants agree with what has been known for some time already from X-ray studies on flavodoxin (Watenpaugh et al., 1976). These crystal structures of flavodoxin can be grouped according to 2FX2, 3FX2, 1J8Q, and ‘WFX2’1, on one side, and 4FX2, 5FX2, 1BU5A, and 1BU5B, on the other. The former group represents oxidized species of the FMN cofactor, the latter includes reduced (5FX2) and semi-reduced (4FX2) quinone forms. 1BU5A and 1BU5B (Walsh et al., 1998) are two chains in an asymmetric unit of apo-flavodoxin-riboflavin complex lacking the ribose phosphate. Conformations of torsions 61 and 62 in flavodoxin differ by almost 180° between both structure sets, implying a flip of the intervening peptide plane with connected changes in the hydrogen bonding network. Our NMR data on the oxidized species support the torsion angles derived from the first group. Most notably, Asp62 adopts a rare positive torsion angle responsible for an elevated 3JC'H coupling constant of 5.1 Hz. As it is also connected with an unusual negative  torsion, this data point stands out in a graph of 3JC'H() (not shown). Albeit a non-glycine residue, Asp62 exhibits a secondary-structure feature normally characteristic of glycine as frequently found in a type-II’  turn motif (Creighton, 1993). But then, the surrounding torsion angles are too distorted to form a proper reverse turn. Variability in this loop region is also corroborated by an analysis of coupling constants related to the sidechain 1 torsion angle (Schmidt, 2007b). 4. Discussion, conclusion and scope Complete determination of a protein fold from scratch is inherently impossible on the sole basis of J coupling restraints alone, owing to their short-range nature of interaction. Even though  torsion angles can be determined fairly accurately, and values of  can at least be narrowed to plausible ranges, while fixing  torsions at the planar 180° value, the chaining of the distinct amino-acid fragments is likely to cause errors to accumulate in the process. A few long-range restraints, such as those provided by the measurement of NOE effects, will normally be required to ensure the correct fold over the whole polypeptide chain. In fact, the present results derived from our J-coupling data are consistent with an independent previous investigation into the RNase T1 solution structure by means of the traditional measurement of NOE effects that were subsequently converted into proton-proton distance constraints (Pfeiffer et al., 1996a). 1 ‘WFX2’ signifies a preliminary X-ray coordinate set of D. vulgaris flavodoxin not available from the PDB and kindly provided by Martin Walsh, University College Galway, Ireland. 2 Diagonal: mean and standard deviation (square-root of variance, in Hz). Upper triangle: standard deviation attributed to joint variation in both J types (square-root of covariance, in Hz), negative signs indicating antivariance. Lower triangle: pair-correlation coefficients. 4.1 Data correlation and redundance The  torsion angle in each amino-acid residue is supported by up to six 3J coupling constants, which, in aggregate, determine just one internal rotational degree of freedom of the molecular model, the value of . Even though the J data are all independent www.intechopen.com Protein Structure from NMR Coupling Constants 109 observations, they do not represent six independent structure parameters. Owing to their simultaneous dependence on the same torsion, their values are highly correlated, or anti- correlated, for that matter (Table 3). Somewhat limited by pairs of couplings exhibiting correlated angle dependence (Fig 2), the amount of independent structure information available reduces to effectively 3 or 4, rather than 6, yet still exceeds the single rotational degree sought to be fixed. Precisely this redundance, however, is key to determining torsion angles accurately. 3JHNH 3JHNC’ 3JHNC 3JC’H 3JC’C’ 3JC’C 3JHNH 6.77 ± 2.12 –0.82 –1.20 1.18 0.81 –1.13 3JHNC’ –44% 0.89 ± 0.74 –0.11 0.27 0.27 –0.19 3JHNC –79% –2% 1.62 ± 0.85 –0.82 –0.62 0.77 3JC’H 54% 8% –61% 2.32 ± 1.29 0.47 –0.84 3JC’C’ 58% 16% –76% 30% 0.97 ± 0.58 –0.59 3JC’C –79% –6% 89% –69% –77% 1.83 ± 0.78 Table 3.Variance-covariance matrix for 512 -related 3J coupling constants in RNase T12 4.2 Molecular dynamics effects At times, the excess information contained in a set of observables can give insights into dynamic effects that may prevail in the molecular structure (Schmidt, 1997b). Conformational dynamics frequently complicate the analysis of amino-acid sidechain torsion angles 1 and need be taken into account for satisfactory interpretation of experimental J data related to that angle type. A variety of angular-mobility models can be applied to cases in which a single fixed torsion does not explain the observed data satisfactorily. Analyses commonly assume the 1 torsion either to dwell preferentially in energetically favourable staggered states (Pachler, 1963, 1964; Hansen et al., 1975) or to librate about a mean value according to a Gaussian probability profile (Jardetzky, 1980; Karimi-Nejad et al., 1994; Brüschweiler & Case, 1994). In the first approach, amino-acid sidechain torsions are analyzed assuming the presence of interconverting staggered-rotamer conformations of 1 = –60°, ±180° and +60° to solve for the respective populations, p1, p2, and p3, by linear combinations of so-called trans and gauche coupling values (Table 2). The Gaussian model, however, typically limits the torsion to one predominant conformation, yet, allowing for larger angular variability, at times. To this end, angular standard deviations of approximately 60° can be considered to represent a fully revolving torsion. Both models are somewhat complementary, the staggered-rotamer one being easier to apply, whereas the Gaussian one usually fits the data better (Schmidt, 1997b). 2 Diagonal: mean and standard deviation (square-root of variance, in Hz). Upper triangle: standard deviation attributed to joint variation in both J types (square-root of covariance, in Hz), negative signs indicating antivariance. Lower triangle: pair-correlation coefficients. www.intechopen.com 110 Protein Structure 4.3 1J and 2J couplings in protein structure analysis Interest is arising in short-range 1J and 2J coupling constants (Wienk et al., 2003; Löhr et al., 2011) as these are comparatively easier to measure than 3J, albeit less well understood regarding their conformational dependence (Schmidt et al., 2009, 2010). However, by simply recognizing qualitative classes of small, medium, and large magnitudes for the respective parameters, it was feasible to pinpoint the boundaries of secondary-structure elements in a protein (Schmidt et al., 2011). The suggested procedure of J-indexing also exploits data redundance as each torsion angle is surrounded by large numbers of 1J and 2J coupling constants (Fig. 9). Fig. 9. The ten types of 2J coupling constants encountered in the protein backbone. Fig. 9. The ten types of 2J coupling constants encountered in the protein backbone. Fig. 9. The ten types of 2J coupling constants encountered in the protein backbone. Protein 1J-, 2J-, and 3J-coupling constant data related to one-, two-, and three-bond interaction, respectively, continue being deposited by NMR spectroscopists with the Biological Magnetic Resonance Data Bank (BMRB, Ulrich et al., 2008) (Fig. 10). 4.4 Accuracy issues Quantitative 3J-coupling analysis in proteins appears to have reached a level of detail and accuracy at which a change of a few degrees in a torsion angle, comparable to thermal librational amplitudes, makes a noticeable difference, allowing, for example, genuine differences between NMR-based solution and X-ray based crystal structures to be detected. The process of defining useful conformational constraints on the basis of J coupling constants usually requires, first, extracting accurate values for the spin-system related property J from NMR spectra and, second, translating these values into molecular-geometry related dihedral-angle values within the framework of a specified model of molecular structure and possibly dynamics. Naturally, both these stages come with their inherent inaccuracies which will impact on the final result (Fig. 11). www.intechopen.com www.intechopen.com Protein Structure from NMR Coupling Constants 111 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 0 5000 10000 15000 20000 25000 30000 Year Data Fig. 10. Cumulative data volume submitted to the BMRB in category protein J coupling constants. The current number of deposited sets is around 350, totaling more than 25,000 values, the majority of which are 3JHNH coupling constants. Recent leaps reflect large-scale depositions of hitherto less popular 1J and 2J parameters which the authors of this chapter have set out to explore regarding utility in protein structure determination (Schmidt et al., 2009, 2010). 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 0 5000 10000 15000 20000 25000 30000 Year Data Data Fig. 10. Cumulative data volume submitted to the BMRB in category protein J coupling constants. The current number of deposited sets is around 350, totaling more than 25,000 values, the majority of which are 3JHNH coupling constants. Recent leaps reflect large-scale depositions of hitherto less popular 1J and 2J parameters which the authors of this chapter have set out to explore regarding utility in protein structure determination (Schmidt et al., 2009, 2010). g. 10. Cumulative data volume submitted to the BMRB in category protein J coupling nstants. The current number of deposited sets is around 350, totaling more than 25,000 p , g , he majority of which are 3JHNH coupling constants. Recent leaps reflect large-scale ons of hitherto less popular 1J and 2J parameters which the authors of this chapter out to explore regarding utility in protein structure determination (Schmidt et al., 10). Fig. 11. 5.1 3J(H,H) coupling constants 3JHNH coupling constants originated from 3D-HA[HB,HN](CACO)NH quantitative J correlation spectra recorded at 500 MHz (Löhr et al., 1999). The coupling constant for residue i was evaluated in spectrum planes at the resonance Ni+1 by summing the data points comprising the HNi,HNi+1 cross peak and referring its intensity to the H i,HNi+1 auto peak according to Icross/Iauto = –tan2(J), with the magnetization-transfer delay, , set at 17 ms. Raw coupling constants were corrected for H/D exchange by an average factor of 1.049, due to scaling all Icross/Iauto ratios for the 10-% D2O solvent component needed for field-frequency locking. Both datasets yielded 77 values of corrected 3JHNH averaging 6.77 ± 2.17 ± 0.24 Hz. 4.4 Accuracy issues Progression of procedural errors and random uncertainties into molecular structure. ogression of procedural errors and random uncertainties into molecular structure. 112 Protein Structure A word of caution is advised though. Even if both above mentioned evaluation stages as well as the recording of NMR spectra were void of systematic error, i.e., not limited by their technical approach, molecules don’t always do us the favour of revealing an unambiguous structure. If conformational dynamics are at play, such ambiguity is not to be confused with lack of accuracy or precision, and the geometry of the molecule must not be squeezed into a ‘strait-jacket’, as it were. Rather we need to develop our understanding of molecular structure, if not re-define the term, in a way that integrates both dynamic as well as static aspects of conformation. Research into partially or intrinsically disordered protein structures is witness to such development (Dyson & Wright, 1998). 5. NMR Experiments J coupling measurements were carried out on NMR spectrometers manufactured by Bruker, Rheinstetten, Germany, with nominal fields ranging from 500 to 900 MHz proton frequency. All instruments were fitted with cryogenically cooled triple-resonance z-axis pulsed-field- gradient probes, except the 500-MHz instruments were equipped with room-temperature triple-resonance three-axis pulsed-field-gradient probes. Experiment names of the exclusive correlation spectroscopy (E.COSY) follow a convention that identifies by uppercase lettering those protein nuclei that are active in generating the spectrum dimensions, in round parentheses those used for magnetization relay only, and in square brackets the presence of those passive spins that give rise to the sought J coupling interaction (Wang & Bax, 1995). Each coupling constant was measured at least twice, and mean values quoted carry two standard deviations, the former denoting the intra-spectrum, i.e., variation across all amino- acid residues, and the latter denoting the standard error or inter-spectrum rms variation between the repeat experiments, i.e., a measure of reproducibility. 5.2 3J(H,C’) coupling constants 3JHNC' coupling constants were determined by least-squares fitting 2D projections of E.COSY-type C,H multiplets (Schmidt et al., 1997a) recorded at 900 MHz using a [15N,1H]- TROSY variant (Pervushin et al., 2000) of 3D-(H)CANNH[CO] spectra (Löhr & Rüterjans, 1995). To minimize transverse relaxation effects during the constant-time period, C www.intechopen.com Protein Structure from NMR Coupling Constants 113 chemical shifts and 1JCC' couplings were simultaneously evolved in a 1H,13C multiple- quantum constant-time t1 period while spin-locking 1H magnetization using a 3-kHz spin- lock field. Insufficient to cover the complete H shift range uniformly, poorly decoupled C resonances near the excitation band edges were omitted from evaluation. Glycine patterns were disregarded entirely as the NMR experiment is not optimized for their twin-H spin topology. The highly reproducible results comprised 70 values of 3JHNC' averaging 0.75 ± 0.71 ± 0.06 Hz. For many of those residues that needed to be ignored in (H)CANNH evaluations for reasons of lineshape distortion or overlap, 3JHNC' coupling constants were obtained using a simpler 3D-heteronuclear relayed-E.COSY approach at 500 MHz in combination with more sophisticated antiphase 2D-multiplet line-shape analysis (Schmidt et al., 1996). The 76 values averaged 0.86 ± 0.72 ± 0.17 Hz, slightly larger than those from (H)CANNH. Additional values were extracted from a [15N,1H]-TROSY version of ct-HNCA[CO]-E.COSY spectra (Wang & Bax, 1995) recorded at 600 MHz, averaging 0.81 ± 0.79 ± 0.21 Hz and bringing the total number of items to 92, including data for glycine. Rmsd between all six spectra was 0.22 Hz. chemical shifts and 1JCC' couplings were simultaneously evolved in a 1H,13C multiple- quantum constant-time t1 period while spin-locking 1H magnetization using a 3-kHz spin- lock field. Insufficient to cover the complete H shift range uniformly, poorly decoupled C resonances near the excitation band edges were omitted from evaluation. Glycine patterns were disregarded entirely as the NMR experiment is not optimized for their twin-H spin topology. The highly reproducible results comprised 70 values of 3JHNC' averaging 0.75 ± 0.71 ± 0.06 Hz. For many of those residues that needed to be ignored in (H)CANNH evaluations for reasons of lineshape distortion or overlap, 3JHNC' coupling constants were obtained using a simpler 3D-heteronuclear relayed-E.COSY approach at 500 MHz in combination with more sophisticated antiphase 2D-multiplet line-shape analysis (Schmidt et al., 1996). The 76 values averaged 0.86 ± 0.72 ± 0.17 Hz, slightly larger than those from (H)CANNH. 5.3 3J(H,C) coupling constants 3JHNC coupling constants were determined by simulating 2D projections of C,H multiplets resulting from a [15N,1H]-TROSY version of the 3D-HNCA[CB]-E.COSY experiment (Wang & Bax, 1996) carried out at 800 MHz. Glycines generally lacking the C nucleus were ignored. Overlapping C i and C i–1 resonances prevented a few coupling constants from being obtained. Coincident C and C chemical-shift ranges in serine and threonine residues precluded selective excitation of either nucleus and respective data were disregarded. Thus, the two recordings yielded only 58 values, averaging 1.50 ± 0.94 ± 0.09 Hz. Additional 3JHNC coupling constants resulted however from 3D [15N,1H]-TROSY-HNCB quantitative J correlation experiments (F. Löhr, unpublished) performed at 600 and 800 MHz. Dephasing of 1HN coherence due to passive 3JHNH couplings during J evolution delay , which was set at either 40 or 45 ms, was avoided by employing BIRD refocusing elements (Garbow et al., 1982) in the centre of the  period. Three spectra yielded 68 values of 3JHNC averaging 1.71 ± 0.79 ± 0.18 Hz between quantitative J correlation data. Between all five sets, 81 values were determined at 1.62 ± 0.85 ± 0.14 Hz. 5.4 3J(C'i–1,H i) coupling constants 3JC’H coupling constants resulted from fitting 2D projections of C i,C'i–1 multiplets in 3D- H(N)CA,CO[HA]-E.COSY spectra (Löhr & Rüterjans, 1997; Löhr et al., 1997) recorded at 500 MHz. The total 81 values collected averaged 2.33 ± 1.29 ± 0.16 Hz. 5.2 3J(H,C’) coupling constants Additional values were extracted from a [15N,1H]-TROSY version of ct-HNCA[CO]-E.COSY spectra (Wang & Bax, 1995) recorded at 600 MHz, averaging 0.81 ± 0.79 ± 0.21 Hz and bringing the total number of items to 92, including data for glycine. Rmsd between all six spectra was 0.22 Hz. 5.6 3J(C'i–1,C i) coupling constants 3JC’C coupling constants were measured at 500 MHz by H(N)CA,CO[CB]-E.COSY (Löhr et al., 1997), yielding only 59 values averaging 1.96 ± 0.86 ± 0.16 Hz. As with 3JHNC, determining 3JC’C coupling constants with these E.COSY-type experiments fails for serine, threonine, and some leucine residues, all exhibiting downfield-shifted 13C resonances, so as to overlap with C chemical-shift ranges, preventing selective excitation. In contrast, HN(CO)CB quantitative J correlation (Hu & Bax, 1997) provides values for all residue types as long as the active coupling magnitude exceeds a certain threshold determined primarily by the signal-to-noise ratio. Spectra were acquired at 500 MHz with delays  set at either 37.5 or 38.5 ms to match 2/1J(C',C). The 13C carrier frequency was positioned at either 26 or 60 ppm. Quantitative J correlation yielded 74 data, averaging 1.83 ± 0.75 ± 0.10 Hz. Additional 3JC’C values including those in Ser and Thr residues were obtained from C i,C'i–1 multiplet projections in H(N)CO,CA[CA]-E.COSY recorded at 800 MHz (F. Löhr, unpublished). The 76 values averaged 1.66 ± 0.80 ± 0.20 Hz, and the grand average over all ten sets was 1.82 ± 0.78 ± 0.20 Hz, totaling 82 values. 5.5 3J(C'i–1,C’i) coupling constants 3JC’C’ coupling constants resulted from fitting 2D projections of C i,C'i–1 multiplets in 3D- H(N)CA,CO[CO]-E.COSY spectra (Löhr et al., 1997) recorded at 500 MHz. Glycine couplings included, the two datasets comprised 90 3JC’C’ values averaging 0.84 ± 0.65 ± 0.16 Hz. Additional 3JC’C’ coupling constants resulted from quantitative J-correlation experiments www.intechopen.com 114 Protein Structure using a [15N,1H]-TROSY version of the 3D HN(CO)CO pulse sequence (Hu & Bax, 1996). Two spectra were acquired at 500 MHz with the J-evolution period  set at either 50 or 60 ms. The spectra allowed both sequential coupling constants 3J(C’i–2,C’i–1) and 3J(C’i–1,C’i) to be evaluated, yielding 78 values averaging 1.03 ± 0.61 ± 0.23 Hz. The final set comprised 98 coupling constants of 0.95 ± 0.63 ± 0.26 Hz. 7. Acknowledgements Norman Spitzner (Frankfurt university) is thanked for preparing a doubly-labelled RNase T1 sample. Robert Pritchard (graduate from Kent university) is thanked for helping evaluate RNase T1 spectra in the course of his undergraduate final-year project. Financial support by the Access to Research Infrastructures activity in the 7th Framework Programme of the EC (Project number: 261863, Bio-NMR) is gratefully acknowledged. 6. Supplementary material 3J coupling constants related to the -torsion angles in RNase T1 as determined in the present work are deposited with the BioMagRes Database (accession number BMRB-16469), available at http://www.bmrb.wisc.edu/cgi-bin/explore.cgi?format=raw&bmrbId=16469 8. References Arni, R., Heinemann, U., Maslowska, M., Tokuoda, R. & Saenger, W. (1988) Restrained least- squares refinement of the crystal structure of the ribonuclease T1*2'-guanylic acid complex at 1.9 Å resolution. Acta Crystallographica Section B, Vol. 43, pp. 534-554, ISSN 0108-7681 (p) 1600-5740 (e) www.intechopen.com Protein Structure from NMR Coupling Constants 115 Artali, R., Bombieri, G., Meneghetti, F., Gilardi, G., Sadeghi, S.J., Cavazzini, D. & Rossi, G.L. 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(2003) Simultaneous measurement of protein one-bond and two-bond nitrogen-carbon coupling constants using an internally referenced quantitative J- correlated [15N,1H]-TROSY-HNC experiment. Journal of Biomolecular NMR, Vol. 25, pp. www.intechopen.com 8. References 133-145, ISSN 0925-2738 (p) 1573-5001 (e) Wüthrich, K. (1986) NMR of proteins and nucleic acids, John Wiley and Sons, New York, ISBN 0-471-82893-9 www.intechopen.com www.intechopen.com Protein Structure Edited by Dr. Eshel Faraggi Protein Structure Edited by Dr. Eshel Faraggi ISBN 978-953-51-0555-8 Hard cover, 396 pages Publisher InTech Published online 20, April, 2012 Published in print edition April, 2012 Published in print edition April, 2012 Since the dawn of recorded history, and probably even before, men and women have been grasping at the mechanisms by which they themselves exist. Only relatively recently, did this grasp yield anything of substance, and only within the last several decades did the proteins play a pivotal role in this existence. In this expose on the topic of protein structure some of the current issues in this scientific field are discussed. The aim is that a non-expert can gain some appreciation for the intricacies involved, and in the current state of affairs. The expert meanwhile, we hope, can gain a deeper understanding of the topic. How to reference In order to correctly reference this scholarly work, feel free to copy and paste the following: Jürgen M. Schmidt and Frank Löhr (2012). Refinement of Protein Tertiary Structure by Using Spin-Spin Coupling Constants from Nuclear Magnetic Resonance Measurements, Protein Structure, Dr. Eshel Faraggi (Ed.), ISBN: 978-953-51-0555-8, InTech, Available from: http://www.intechopen.com/books/protein- structure/refinement-of-protein-tertiary-structure-by-using-spin-spin-coupling-constants-from-nuclear-magnetic InTech China Unit 405, Office Block, Hotel Equatorial Shanghai No.65, Yan An Road (West), Shanghai, 200040, China Phone: +86-21-62489820 Fax: +86-21-62489821 InTech Europe University Campus STeP Ri Slavka Krautzeka 83/A 51000 Rijeka, Croatia Phone: +385 (51) 770 447 Fax: +385 (51) 686 166 www.intechopen.com
https://openalex.org/W2114960294	https://www.revistas.usp.br/anaismp/article/download/80842/84478	Portuguese	null	Arte Nova e Eclectismo no palacete projectado por Ernesto Korrodi para a família Bouhon	Anais do Museu Paulista	2,013	cc-by	18,506	Arte Nova e Eclectismo no palacete projectado por Ernesto Korrodi para a família Bouhon Ana Mónica Pereira Reis de Matos Romãozinho1 Ana Mónica Pereira Reis de Matos Romãozinho1 1. Licenciada em Arquitectura, Mestre e Doutorada em Design pela Faculdade de Arquitectura da Universidade Técnica de Lisboa; docente da Escola Superior de Artes Aplicadas do Instituto Politécnico de Castelo Branco. Integra desde 2009 a rede i n t e r n a c i o n a l d e investigadores Réseau Art Nouveau Network. E-mail: <monica.romaoz@gmail. com>. RESUMO: No início do século XX, o design de interiores resultava do trabalho desenvolvido por arquitectos, decoradores, pintores ou entalhadores. Embora então integrado no universo das artes decorativas, acreditamos que já existia como prática profissional regulada por uma rede coerente de princípios de concepção e pelo entendimento do espaço interior como teia global de relações que se estabelece entre planos, ornamento, iluminação e mobiliário. No final do século anterior, perante a necessidade de proceder a uma reforma do ensino industrial, o governo português abrira um concurso para contratação de professores estrangeiros. Evidenciamos o papel de Ernesto Kor rodi (1870-1944). As suas obras decorrem de uma atitude paradoxalmente ecléctica e moderna, apoiando-se por um lado, numa reinterpretação de soluções referenciadas no período medieval ou na Renascença; por outro, em fórmulas sediadas nos movimentos Arts and Crafts, Arte Nova (Art Nouveau) ou Secessão. Nos seus projectos persistem determinadas dependências e hierarquias, mas estas se cruzam com as necessidades despertadas pelas inovações técnicas, o que faz com que se preocupe também com a resposta à eclosão de novas funções e mobiliário na casa, a par das exigências higienistas do momento. O presente artigo consiste na análise da casa da família Bouhon, localizada na cidade da Covilhã, Portugal. Se a Arte Nova marca as suas fachadas, no interior deparamo-nos com espaços mais depurados, marcados por apontamentos decorativos, incorporados na azulejaria e nos tectos estucados ou em caixotões. O ornamento contribui, deste modo, para a dignificação dos planos das paredes, lambris e tectos, assumindo um papel funda mental na composição espacial. PALAVRAS-CHAVE: Arte Nova (Art Nouveau). Design de Interiores. Eclectismo. Funcionalidade. Or namento. Portugal. ABSTRACT: In the early twentieth century, Interior Design resulted from the work of architects, decora tors, painters and carvers. Anais do Museu Paulista. São Paulo. N. Sér. v.21. n.2. p. 79-124. jul.- dez. 2013. 3. Cf. Maria João Rocha Simões Fernandes (1985, p.43). 2. Cf. Rui Afonso Santos (1995, p.144). Arte Nova e Eclectismo no palacete projectado por Ernesto Korrodi para a família Bouhon Although then integrated into the world of decorative arts, we believe that already existed as a profession regulated by a coherent network design principles and the under standing of interior space as global web of relationships established between plans, ornament, light ing and furniture solutions. At the end of the previous century, before the need for a reform of industrial training, the Portuguese Government opened a tender for hiring of foreign teachers. We emphasize 79 the role of Ernesto Korrodi (1870-1944). His works arise from an attitude paradoxically modern and eclectic, leaning one hand on a reinterpretation of solutions referenced in the medieval period or the Renaissance, on the other, based on formulas from Arts and Crafts movement, Art Nouveau or Secession. In his projects certain rooms and hierarchies remain, but these are intersect with the needs aroused by technical innovations, which causes an attempt to answer to the emergence of new func tions and features in the house, along with the hygienists demands of the moment. This article consists in the analysis of Bouhon home, located in the city of Covilhã, Portugal. If Art Nouveau is visible in the elevations, inside we find a cleaner space, marked by decorative notes which we can find in tiles and embedded in the stucco or coffered ceilings. The ornament thus contributes to the dignity of the walls, wainscots and ceiling plans, assuming a key role in the spatial composition. KEYWORDS: Art Nouveau Interior Design Eclecticism Functionality Ornament Portugal YWORDS: Art Nouveau. Interior Design. Eclecticism. Functionality. Ornament. Portugal. KEYWORDS: Art Nouveau. Interior Design. Eclecticism. Functionality. Ornament. Portugal. Introdução No final do século XIX, a procura de originalidade dependia da composição mais ou menos inventiva criada a partir de vocabulários de estilos anteriores e, consoante o cliente, o lugar e restantes condicionantes programáticas, o projecto poderia seguir uma linguagem Renascença (revivalismo sobretudo abraçado pela Inglaterra e Alemanha), próxima aos modelos de Luís XV ou Luís XVI, neo-românica e cada vez mais raramente neo-manuelina ou neo-gótica à medida que avançamos no séc. XX. As obras associadas à Arte Nova (Art Nouveau), assinalam o início de um processo de viragem, simbolizando uma ruptura com um século XIX, marcado por imitações e formas estrangeiradas, em que as “as artes menores caminhavam ao acaso, hesitantes entre o tradicionalismo estafado [os chamados “estilos históricos”] e um mal disciplinado naturalismo”2. Observa-se então, em Portugal, uma atitude projectual diferenciada consoante a formação académica de cada arquitecto, decorador, pintor ou entalhador. Se por exemplo, Ventura Terra (1866–1919), Norte Júnior (1878-1962) ou Adães Bermudes (1864-1947) estudaram em França, já Raul Lino (1879-1974) ou Ernesto Korrodi (1870-1944) fizeram um percurso afastado dos modelos franceses. De acordo com Maria João Fernandes, num núcleo de carácter mais erudito encontraremos José Coelho, Miguel Nogueira, Adães Bermudes, Norte Júnior, Ernesto Korrodi e Ventura Terra, “que a par de exemplos de arquitectura historicista produziram obras marcadas pela nova estética Arte Nova segundo a particular expressão de cada”; e acrescenta ainda a este grupo autores consagrados na região de Aveiro, como José de Pinho, Jaime Inácio dos Santos e Francisco Augusto da Silva Rocha (1864-1957)3. Parece-nos, contudo, pertinente acrescentar que alguns destes autores “de carácter mais erudito”, nomeadamente Ernesto Korrodi e Raul Lino, combinaram nas suas arquitecturas uma linguagem chã mais portuguesa marcada por soluções tradicionais da arquitectura portuguesa e por apontamentos Arte Nova mais sintetizados que os aproximam de uma Arte Nova inglesa e/ou Secessão vienense e do espírito da corrente Arts and Crafts sobretudo no caso do design de interiores. Por outro lado, a difusão dos princípios e conceitos proclamados por movimentos internacionais seria reforçada pelo contributo dos arquitectos e 80 Anais do Museu Paulista. v. 21. n.2. Jul.- Dez. 2013. decoradores estrangeiros que se instalaram em Portugal no final do século XIX, na sequência do concurso para contratação de professores estrangeiros, promovido pelo Governo Português. Introdução O processo de análise de estudos nacionais sobre a temática Arte Nova e a consulta de imprensa e de documentação dos Arquivos Municipal e Distrital de Leiria, levou-nos a constatar que os interiores domésticos projectados por Ernesto Korrodi, arquitecto e designer de origem suíça, contratado para leccionar no ensino industrial, constituíam fontes de análise não exploradas. O seu percurso e obra arquitectónica foram primeiramente analisados nos estudos pioneiros intitulados Ernesto Korrodi: 1889-1944: arquitectura, ensino e restauro do património4 e Rota de arquitectura Korrodi: contributo para o conhecimento da vida e obra do arquitecto (1870-1944)5. Em 1889, Korrodi seria colocado na Escola Industrial de Braga e mais tarde, em 1894, na Escola Domingos Sequeira, em Leiria, onde foi professor da disciplina de Desenho Ornamental e Modelação. Ernesto Korrodi nascera em Zurique em 30 de Janeiro de 1870 e, aos quinze anos, ingressara na Escola de Arte Industrial, onde concluiu os cursos de escultor-decorador e de professor de desenho6. Em 1898, publicou um primeiro estudo intitulado Estudos de Reconstrucção sobre o Castello de Leiria: Reconstituição graphica de um notável exemplo de construcção civil e militar portuguesa7, graças aos quais lhe seria atribuído, sob a proposta do Ministro e Secretário de Estado das Obras Públicas, Comércio e Indústria, o título de Comendador da Real Ordem do Mérito Civil. Em 1905, Korrodi foi nomeado director interino da Escola Domingos Sequeira e, em 1906, assumiu a direcção efectiva da escola, cargo que exerceu até 1917. No mesmo ano, montou uma oficina de cantaria junto à sua casa de Leiria, a Villa Hortênsia, que oferecia cantarias de calcário e lioz. Projectou obras para todo o país: Paços de Concelho, Sedes de Bancos, Garagens, Hotéis, Prédios de Rendimento e Habitações Unifamiliares. Duas obras da sua autoria receberiam o Prémio Valmor: a Casa do Dr. António Macieira (Avenida Fontes Pereira de Melo, Lisboa) em 1910 e o Prédio de Rendimento da viúva Estefânia Macieira (Rua Viriato, Lisboa) em 1917. Em 1926, foi-lhe atribuído o Diploma de Arquitecto pela Escola de Belas Artes de Lisboa. 4. Ver Lucília Verdelho da Costa (1997). 4. Ver Lucília Verdelho da Costa (1997). 5. Ver Maria Genoveva Moreira Oliveira (2005). 6. Ver Sousa Viterbo (1899, p.46). 7. “Contém 26 estampas, de r e p r o d u c ç õ e s photolithographicas dos desenhos originaes (plantas, perspectivas, apontamentos do estado actual, etc)”, cf. Annals of Museu Paulista. v. 21. n.2. Jul.-Dec. 2013. 9. Em França, entre 1890 e 1900, toda uma geração de designers arquitectos ou escultores lançava-se no aprofundamento de novas soluções no âmbito do Design: Tony Selmersheim (1871-1971), Charles Plumet (1861-1928), Hector Guimard (1867-1942), 4. Ver Lucília Verdelho da Costa (1997). Introdução carta autobiográfica de Ernesto Korrodi, apud Sousa Viterbo (1899, p.47). 7. “Contém 26 estampas, de r e p r o d u c ç õ e s photolithographicas dos desenhos originaes (plantas, perspectivas, apontamentos do estado actual, etc)”, cf. carta autobiográfica de Ernesto Korrodi, apud Sousa Viterbo (1899, p.47). 8. Em meados de 1850, alguns artistas ingleses pretenderiam a renascença de uma arte mais próxima do gótico e dos primitivos. Para John Ruskin (1819- 1900), o teórico do movimento Arts and Crafts, as descobertas científicas e industriais seriam a causa da decadência artística. Avanços tecnológicos como os caminhos-de-ferro e os telégrafos eram inimigos do progresso no âmbito da produção criativa. Mas um dos contributos inegáveis seria a criação de corporações do tipo medieval, à cabeça das quais surgiriam operários excepcionais tais como William Morris e Walter Crane (1845-1915). Madsen (1923-2007) defende precisamente que a Arte Nova apareceu em Inglaterra e atribuí-lhe a designação de Proto-Art Nouveau, no fundo uma derivação do movimento Arts and Crafts com ênfase na decoração linear e floral. Ver S. Tschudis Madsen (1967, p.14). As suas obras revelam-nos, por um lado, registos apoiados numa reinterpretação de soluções referenciadas no período medieval ou na Renascença, por outro fórmulas sediadas nos movimentos Arts and Crafts ou Proto-Arte Nova8, Arte Nova e Secessão vienense9. Korrodi assinava revistas da especialidade, nomeadamente a Academy Architecture and Annual Architectural Review (1895- 1922) ou a Schweizerishe Bauzeitung (1900-1935), veículos difusores daquilo que se passava lá fora10. Os seus estudos arqueológicos permitiram-lhe criar obras particularmente fundamentadas sob o ponto de vista do léxico decorativo nacional e do uso cuidado de materiais e técnicas tradicionais11. Algumas das suas obras, representativas da influência da corrente Arte Nova em Portugal, não tinham sido visitadas ou estudadas e, neste sentido, pretendemos demonstrar, por meio da leitura de um estudo de caso inédito, que a sua atitude projectual, essencialmente ecléctica, denuncia um sentido de modernidade, presente em apontamentos decorativos sua 9. Em França, entre 1890 e 1900, toda uma geração de designers arquitectos ou escultores lançava-se no aprofundamento de novas soluções no âmbito do Design: Tony Selmersheim (1871-1971), Charles Plumet (1861-1928), Hector Guimard (1867-1942), 81 Annals of Museu Paulista. v. 21. n.2. Jul.-Dec. 2013. autoria, soluções industriais, novas funcionalidades e espacialidades mais fluídas, resultante da influência exercida pelos padrões estéticos e funcionais propagados quer pela imprensa internacional quer por obras literárias de referência12. 13. Informação cedida pelo Eng.º Miguel Korrodi, bisneto de Ernesto Korrodi. Introdução Eugène Gaillard (1862- 1933), George de Feure (1868-1943), Eugène Colonna, Alexandre Charpentier (1859-1909), entre outros. Émile Gallé (1846-1904) impulsionava este novo movimento a partir da procura de elementos de inspiração renovada na natureza, a Art Nouveau. Na Bélgica, d e s t a c a v a - s e a experimentação formal desenvolvida por Victor Horta (1861-1947), Paul Hankar (1859-1901), Gustave Serrurier-Bovy (1858-1910) e Henry Van de Velde (1863-1957). Por fim, na Áustria, no âmbito da Secessão vienense, explorava-se um estilo rectilíneo e apesar de incorporar elementos essenciais da Art Nouveau, a sua repulsa face à ornamentação opõe-se inteiramente ao espírito desta corrente, conduzindo a uma abstracção crescente dos próprios motivos que integravam o design de interiores e respectivo mobiliário e abrindo o caminho ao movimento modernista; destacam-se Otto Wagner (1841-1918), Joseph Hoffmann (1870- 1956), Adolf Loos (1870- 1933) e Joseph Olbrich (1867-1908). No âmbito do presente estudo, evidenciamos não só a literatura relevante, como o trabalho de campo, possível graças à disponibilidade da família Carneiro (actuais proprietários) e a elaboração de uma ficha analítica de projecto no âmbito da qual procedemos a uma sistematização do levantamento fotográfico e análise escrita/desenhada das soluções ornamentais, construtivas e de mobiliário. Estamos perante uma tipologia de habitação unifamiliar situada na Covilhã, centro industrial então em franca expansão e resultado de uma encomenda por parte do belga Joseph Bouhon, proprietário da Fábrica do Sineiro. Korrodi conheceu Bouhon em 1895, pois Frida Korrodi, a irmã mais velha de Ernesto, casara-se com Camille Bouhon (1872-1952), irmão de Joseph e químico que trabalhava numa fábrica de tecidos na Covilhã13. Por sua vez, a filha de Joseph Bouhon, Susana (1908-1991), casar-se-ia em 1929 com Ernesto Camilo Korrodi (1905-1985), filho de Ernesto Korrodi, também arquitecto (Figura 1). A casa da família Bouhon terá sido projectada entre 1919 e 1922. A fachada revela a influência exercida pelos modelos renascentistas, embora nem Anais do Museu Paulista. v. 21. n.2. Jul.- Dez. 2013. Figura 1 – Vista da fachada principal do palacete. Fotografia de Mónica Romãozinho. 10. Ver Lucília Verdelho da Costa (1997, p.302). 10. Ver Lucília Verdelho da Costa (1997, p.302). 11. Ver, José Manuel Pedreirinho (1981, p. 60). 12. Ver Ana Mónica Romãozinho (2013). Anais do Museu Paulista. v. 21. n.2. Jul.- Dez. 2013. Figura 1 – Vista da fachada principal do palacete. Fotografia de Mónica Romãozinho. 82 Anais do Museu Paulista. v. 21. n.2. Jul.- Dez. 2013. Introdução todos os vãos sigam alinhamentos rígidos: o aparelho rusticado do soco, a demarcação dos pisos através de frisos de pedra, as cantarias dos vãos simples ou tripartidos com os seus medalhões. No entanto, são notáveis os apontamentos Arte Nova que vêm ritmar a composição destes planos exteriores: o recurso a arcos abatidos com remates em voluta presentes na varanda de ângulo, no medalhão da janela do salão ou no vão tripartido da sala de almoços orientada a tardoz; o arqueamento de alguns lintéis; os planos de azulejaria com rosas damascenas e caules em movimento ou rostos femininos emoldurados por cercaduras como os mesmos motivos, sempre em tonalidades pastel. A serralharia artística surge também inspirada na natureza, mas de modo mais sintetizado, obedecendo, contudo, a uma composição simétrica. As fachadas encontram-se revestidas a ladrilho branco e o coroamento a ladrilhos cor marfim e preto, dispostos de modo alternado. Ao longo desta faixa final, os ladrilhos pretos formam cruzes que recebem azulejos Arte Nova com acantos de contorno linear. Toda a azulejaria que reveste o exterior terá sido importada da Bélgica14. As soluções afastam-se aqui de uma arquitectura de raízes portuguesas e, deste modo, Korrodi enfatiza a vertente mais internacional desta obra, em resposta ao gosto do cliente belga que se enquadrava numa burguesia emergente. Contudo, é-nos impossível tipificar o seu perfil de cliente, pois o mesmo autor projectou para industriais e estrangeiros como é o caso, além de comerciantes, políticos (António Macieira, por exemplo), médicos (Egas Moniz, a quem seria atribuído um Prémio Nobel pelas suas descobertas no campo da neocirurgia), entre outros clientes portugueses, sendo a influência da Arte Nova transversal a muitas destas encomendas independentemente da nacionalidade do cliente. 14. “Korrodi pôde dar provas do seu entendimento graças à encomenda de um estrangeiro – como ele também o era, afinal -, que para a sua moradia mandou vir da Bélgica os azulejos dos painéis decorativos das paredes, de estilizações Arte Nova”; cf. Lucília Verdelho da Costa (1997, p.282). Na perspectiva da sua volumetria arquitectónica, constatamos que a casa Bouhon projecta-se numa planta em forma de “L”, destacando-se dois volumes salientes: a varanda com o pequeno alpendre e a bow-window. 14. “Korrodi pôde dar provas do seu entendimento graças à encomenda de um estrangeiro – como ele também o era, afinal -, que para a sua moradia mandou vir da Bélgica os azulejos dos painéis decorativos das paredes, de estilizações Arte Nova”; cf. Lucília Verdelho da Costa (1997, p.282). Annals of Museu Paulista. v. 21. n.2. Jul.-Dec. 2013. Introdução Existe uma separação clara entre esfera social e privada da casa que apresenta quatro pisos: sub-cave (garagem, lavagem de carros e garrafeira); cave (bilhar, armazéns, cozinha e dependências complementares); rés-do-chão (funções de recepção e convívio familiar); primeiro andar (quartos da família e de hóspedes) e último piso em águas furtadas (alojamento do pessoal doméstico e da preceptora). Os próprios acessos verticais permitem a separação no âmbito da circulação de pessoal doméstico e da própria família. As plantas desenhadas denunciam a influência que os modelos clássicos exercem não só ao nível das volumetrias sólidas e bem definidas, mas também na definição de uma geometria reguladora de todo o projecto. Korrodi explora mais uma vez estratégias históricas de composição espacial como a axialidade e a simetria, respondendo às exigências funcionais e programáticas (Figura 2). O hall é definido como espaço gerador da casa, apresentando uma forma octogonal, seccionada no piso de recepção e completa no piso privado, que se aproxima dos modelos do Renascimento italiano. A entrada faz-se através de um pequeno vestíbulo (Figura 3) que integra uma escadaria de acesso ao hall de distribuição. Este projecto difere de todos os outros não só esteticamente mas também 83 Annals of Museu Paulista. v. 21. n.2. Jul.-Dec. 2013. Figura 2 – Planta do piso térreo. Acervo do Arquivo Distrital de Leiria. Figura 2 – Planta do piso térreo. Acervo do Arquivo Distrital de Leiria. ao nível funcional e o vestíbulo é exemplo disso, pois apresenta uma pequena porta lateral que conduz a uma escada exterior de serviço que dá acesso à rouparia. Debaixo do tapete que antecede a escadaria, encontra-se um alçapão onde era largada a lenha, para depois ser recolhida ao nível da cave e transportada para o respectivo compartimento. O vestíbulo encontra-se ainda hoje pintado de azul petróleo, que contrasta com o lambril e o tecto claro em cor marfim. O tratamento do tecto reporta-se essencialmente ao estilo Luís XVI, pela sua cor mas também pelos apontamentos dourados e léxico decorativo como os acantos visíveis nas mísulas e as molduras com óvulos e dardos, embora o centro se apresente pintado de azul, enfatizando a leitura do lampião que segue o mesmo estilo. Uma faixa de papel com rosas vermelhas e folhas de louro estilizadas, atadas por fitas, antecede a cornija. Introdução Os caixotões apresentam faixas de papel com rosas, caules e folhas estilizadas em cores esbatidas (rosa e verde) sobre fundo dourado, que correspondem a apontamentos Arte Nova, também visíveis ao nível do lambril, que apresenta superiormente vasos com rosas e acantos, executados em estuque e pintados de creme. A verticalidade surge acentuada pelas pilastras pintadas de verde, que acabam por ritmar o lambril e que estabelecem uma relação de continuidade com as ombreiras e lintéis das portas, da mesma cor. O desenho da porta encontra-se sediado numa linguagem Renascença, pelo seu traçado rectilíneo, pela canelura das suas ombreiras, pelo seu lintel que se eleva sob a forma de frontão 84 Anais do Museu Paulista. v. 21. n.2. Jul.- Dez. 2013. rectangular, pelo jogo geométrico formado pelos pinázios que se traduz numa composição de quadrados e quadrados chanfrados. Na casa Bouhon, o hall (Figura 4) não apresenta uma vocação como espaço de permanência e a sua linguagem compensa a maior exuberância habitualmente presente nas salas ou salões, à semelhança do que era aconselhado em certos manuais e tratados de savoir vivre, publicados na França no mesmo período: “(…) é aqui que ocorrem os empregados a fim de receberem as ordens dos donos ou para anunciarem as visitas; a sua decoração é geralmente sóbria e severa”15. Nesta altura, estes espaços adoptavam frequentemente cânones estéticos da Renascença ou do Gótico e a primeira tendência é observável neste caso, ao nível do tecto com os seus caixotões, do lambril com as suas pilastras, das portas e respectivos puxadores, assim como dos próprios pavimentos. Motivos florais marcam cada um dos caixotões de fundo azul do tecto, sob a forma de festões que estabelecem uma ligação visual entre os vários apainelados e que surgem animados por rosas e fitas entrelaçadas em movimento. Estes apainelados surgem emoldurados por frisos com óvulos e dardos, dando continuidade à solução adoptada na pequena antecâmara. O caixotão central, demarcado nos seus cantos por pinhas salientes, reveste-se de particular valor simbólico, apresentando três cartelas emolduradas por Annals of Museu Paulista. v. 21. n.2. Jul.-Dec. 2013. Figura 3 – Vestíbulo de chegada: vista da escada e porta de acesso ao hall; pormenor do óculo com serralharia Arte Nova e tecto. Fotografias de Mónica Romãozinho. Figura 3 – Vestíbulo de chegada: vista da escada e porta de acesso ao hall; pormenor do óculo com serralharia Arte Nova e tecto. Fotografias de Mónica Romãozinho. 15. “(…) là que se tiennent les domestiques pour répondre aux ordres de leurs maîtres ou pour annoncer les visiteurs; sa décoration est généralement sobre et sévère. (…)”; cf. Ris-Paquot (1894, p.194). 15. “(…) là que se tiennent les domestiques pour répondre aux ordres de leurs maîtres ou pour annoncer les visiteurs; sa décoration est généralement sobre et sévère. (…)”; cf. Ris-Paquot (1894, p.194). Introdução 85 Annals of Museu Paulista. v. 21. n.2. Jul.-Dec. 2013. Annals of Museu Paulista. v. 21. n.2. Jul.-Dec. 2013. coroas entrelaçadas, lendo-se na cartela central AD. Gloria Belgivm. Anno Domini. MCMXXIII e nas laterais Labor e Honor, mensagens respectivamente complementadas pelas imagens de um fuso e de uma palma, também conhecida como acácia maçónica16, símbolo da imortalidade. O fuso constitui uma referência simbólica à profissão de Joseph Bouhon, ligada aos lanifícios17. A cornija revela um trabalho de entalhe rigoroso e apresenta um friso de contas douradas. Acantos, volutas e rosas seguem movimentos assimétricos. Um friso de papel, com rosas abertas, destaca-se sobre o azul das paredes. No desenho técnico respeitante à Decoração do hall, a decoração geométrica seria executada em estuque a imitar madeira e constituída por pequenos motivos quadrados idênticos aos do papel da sala de jantares. 16. Símbolo de vitória, de ascensão, da imortalidade da alma: “É o que acontece com o ramo de ouro de Eneias e com os mistérios de Elêusis; com o salgueiro chinês, com o sakaki japonês; com a acácia maçónica; com o visco druídico; com, mais uma vez, os ramos de Salgueiro, de que fala o Pastor de Hermas; com o buxo que em França se planta nos túmulos no domingo de Ramos. As palmas de Ramos prefiguram a Ressurreição de Cristo, após o drama do calvário (…) A coroa de espinhos de Cristo teria sido entrançada com espinhos de acácia. Por fim, no ritual maçónico, um ramo de acácia é colocado sobre o manto do recipiendário, para recordar aquele que foi plantado no túmulo do Hirão.”; cf. Jean Chevalier e Alain Cheerbrandt (1994, p.37, 502). O lambril que percorre todo o hall surge revestido a papel oleado com relevo, eventualmente proveniente da fábrica britânica Lincrusta, ainda hoje existente. A primeira opção teria correspondido a um trabalho de estuque a imitar madeira, de acordo com o desenho respeitante à decoração do hall. É importante relembrar que o período vitoriano alimentara toda uma indústria de produção de revestimentos decorativos. A planta de pavimentos, em mosaico cerâmico e de origem certamente belga, desdobra-se em dois tapetes, o central que se inscreve na forma poligonal, e outro, de configuração rectangular, que antecede o lance de escadas de acesso ao piso inferior. Introdução O pavimento central, idêntico ao do vestíbulo de entrada, é constituído por mosaicos quadrados com padrão formado por folhas e volutas que obedecem a uma simetria absoluta, ao mesmo tempo em que simulam mosaicos romanos de pequena escala e formato irregular. Uma rica cercadura, constituída por três frisos, remata esta composição, apresentando na faixa central mosaicos similares com volutas e, nos frisos de menor espessura, um jogo de linhas curvas e contracurvas. No tapete que demarca o arranque da escadaria, observamos uma composição central idêntica à anterior, emoldurada por um friso igual aos menos espessos da composição anterior. À volta deste tapete, sucedem-se mosaicos com o padrão de uma estrela e formas quadrifoliadas. As cores dominantes são a terracota, sangue- de-boi, verde e marfim. Este mesmo pavimento cerâmico pode ter sido importado da Bélgica, pois a fábrica Gilliot Hemiksen produzia padrões similares18. 17. Joseph estudou até aos 16 anos num colégio na Bélgica, de onde regressou para a Covilhã com o curso de tintureiro. Tanto ele como o irmão Camille estudaram em Liège e em vários documentos são descritos como “Quimicos”. Em Dezembro de 1921 houve um incêndio na fábrica e Joseph morreu de ataque cardíaco, tendo sido a casa posteriormente vendida ao Dr.Fernando Carneiro. Informações cedidas pelo Eng.º Miguel Korrodi, bisneto de Korrodi e de Joseph Bouhon. As portas do hall apresentam novamente frontões estilo Renascença, mas neste caso com remates laterais curvos, revelando festões que pendem verticalmente ao longo das suas folhas ou em arco no caso dos frontões, assim como conchas explodidas, elementos que surgem evidenciados pelo Rococó e que persistiriam no período de Luís XVI. Os puxadores em bronze remetem-nos para o período Barroco, com os seus movimentos ondulantes, conchas explodidas, acantos, uma pequena flor no remate inferior, mas ainda dominados pela simetria. O manípulo da porta da antecâmara diferencia-se dos outros pelo jogo de cubos, paralelepípedos, discos e elementos esféricos. 18. Encontrámos um exemplo similar da marca G i l l i o t H e m i k s e n , pertencente a uma casa próxima de Namur na Bélgica. C.1910. Disponível e m : h t t p : / / w w w . theantiquefloorcompany. com. Acesso em: 11 ago. 2011. Todas as portas que confinam com o hall seriam pormenorizadas no respectivo mapa de vãos (Figura 5), elaborado à escala 1:10 (versão inicial datada de 1920). Introdução Apresentam-se neste mapa quatro modelos de porta: tipo A (porta para 86 Anais do Museu Paulista. v. 21. n.2. Jul.- Dez. 2013. Figura 4 – Vista do hall e pormenor da serralharia Arte Nova da escadaria principal. Fotografias de Mónica Romãozinho. Figura 4 – Vista do hall e pormenor da serralharia A h Figura 4 – Vista do hall e pormenor da serralharia Arte Nova da escadaria principal. Fotografias de Mónica Romãozinho. a divisão do monta-pratos), tipo B (porta de dois batentes para o corredor e armário) e tipo D (quatro portas de dois batentes correspondentes aos vãos mais largos). Com excepção do modelo C (porta de um só batente para o salão e escada de serviço) que aparece representado no desenho da decoração do hall, seriam sempre desenhados cortes horizontais e verticais à escala natural de cada um dos vãos, com as respectivas orientações assinaladas (lado do hall ou lado das salas) onde surgem legendadas componentes fundamentais como o pinázio, com a indicação pregado depois da aplicação do vidro, ou o vidro biselado. A escada destaca-se pelo trabalho elaborado da serralharia Arte Nova, visível quer no corrimão quer no suporte para vasos que demarca o seu arranque. Os caules orgânicos inscrevem-se numa malha ortogonal demarcada por suportes verticais, afastando-se do movimento mais livre associado a este trabalho no caso dos interiores franceses ou belgas. O janelão, com três batentes e encimado por bandeira em arco de volta perfeita, é uma solução recorrente nas habitações projectadas por Korrodi. O hall corresponde à única dependência detalhada (Figura 6), o que enfatiza a sua importância como espaço cénico que deveria impressionar pelo seu 87 Annals of Museu Paulista. v. 21. n.2. Jul.-Dec. 2013. carácter mais pesado, massivo e simples, sendo encarado como um espelho da dignidade familiar apregoada pelo dono da casa19. A preocupação relativa à unidade espacial é inclusivamente evidente quando constatamos que a própria geometria octogonal do espaço se estende ao desenho dos caixilhos dos vidros das portas que, por sua vez, seguem o alinhamento do lambril. Introdução Na planta desta divisão, Korrodi não desenhou nenhuma peça de mobiliário específica, mas os tratados da época aconselhavam “uma arca em madeira esculpida ou coberta de tapeçaria, algumas cadeiras, cabides, um bengaleiro para pendurar os casacos, um porta- guarda-chuvas, uma consola com tudo o que é necessário para escrever, um espelho para que as senhoras possam reparar a desordem da sua toilette, eis o mobiliário que comporta uma antecâmara”20. 19. Ver Juliet Kinchin (2008, p.169). 19. Ver Juliet Kinchin (2008, p.169). 20. “(…) un coffre à bois sculpté ou recouvert de tapisserie, quelques chaises, des patères, un porte-manteau pour accrocher les vêtements, un porte-parapluie, une table avec tout ce qu’il faut pour écrire, une glace pour que les dames puissent réparer le désordre de leur toilette, voilà tout l’ameublement q u e c o m p o r t e l’antichambre.” ; cf. Ris- Paquot (1894, p.194). Nos dois cantos chanfrados do hall, emergem os pontos de acesso quer ao escritório quer à sala de almoços, complementada por um zonamento de serviços, constituído pela copa, um pequeno WC e um lava-mãos (canto superior esquerdo da sala). Neste projecto, a copa integra um monta-pratos e um móvel trinchante que se destina a apoiar a recepção das travessas. 21. À semelhança da bay- window que consistia numa reentrância de planta poligonal, também muito frequente nas salas ou quartos de tipologias habitacionais inglesas. Ao contrário de outros projectos, vemos aqui emergir duas salas de refeições, inter-comunicantes e que seguem, na sua disposição, uma lógica de axialidade. Embora a planta aponte para uma relação de total fluidez entre elas, na fase construtiva foi inserida uma porta envidraçada, que Korrodi designa de “guarda-vento”. A sala de jantar é iluminada quer por uma janela de sacada que conduz a uma pequena varanda de ângulo quer por uma bow-window de planta semi- circular, solução espacial importada da Inglaterra21, que em planta correspondia à varanda principal e que comunicaria com o interior através de duas portas simétricas de dupla folha, entre as quais estava prevista uma mesa de apoio (Figura 7). É interessante confirmar através da planta do “pavimento nobre” que as colunas dessa varanda foram já executadas em betão. 22. Azulejaria Arte Nova na varanda de Casa em Blankenberge, Bélgica. Disponível em: http:// matinlumineux.blogspot. com/2011/08/art-nouveau. html. Acesso em: 11 ago. 2011. Anais do Museu Paulista. v. 21. n.2. Jul.- Dez. 2013. Introdução A bow-window acabaria por funcionar como extensão da sala de jantares mas os zonamentos continuariam a ser diferenciados através do próprio tratamento do pavimento: soalho de madeira no caso da zona de refeições, mosaico com padrão geometrizado (castanho, marfim, sangue-de-boi) no caso da bow-window. Relativamente à varanda, é de evidenciar o valor plástico dos seus painéis de azulejaria Arte Nova, cuja temática naturalista e paleta cromática os aproxima de uma casa situada em Blankenberge na Bélgica22. O lambril que percorre toda a sala, surge demarcado por elementos verticais e revestido a papel castanho oleado que imita o couro, fabricado em Inglaterra pela Lincrusta, também ele marcado por divisões verticais, ao longo das quais de dispõe sempre o mesmo motivo floral geometrizado colocado a alturas diferentes, atribuindo ritmo a este mesmo plano. Regras de higiene mais pesadas aplicavam-se então mais à sala de jantar do que a qualquer outro espaço de recepção e enfatizava-se a importância de revestimentos laváveis, a exemplo do que ocorria na França: “a sala de jantar deve ter de preferência paredes pintadas a óleo, podendo ter também as suas 88 Anais do Museu Paulista. v. 21. n.2. Jul.- Dez. 2013. paredes revestidas com tapeçaria de cor neutra, panneaux decorativos representando assuntos simples: paisagens ou naturezas mortas”23. O embasamento apresenta maior profundidade com as suas almofadas e a cimalha, mais alta do que era comum, integra quer um friso marcado por linhas horizontais quer uma faixa composta de elementos florais, trabalhados em stencil e com a paleta pastel característica do movimento Arte Nova (azul, salmão, vermelho em pequenas quantidades). 23. “(…) la salle a manger doit avoir, de préférence, des murs peints à l’huile, elle peut aussi avoir ses murs tendus d’une tapisserie de teinte neutre, ou mieux de panneaux décoratifs représentant de très simples sujets : paysages ou natures mortes”; cf. Monique Eleb e Anne Debarre (1995, p.110). 23. “(…) la salle a manger doit avoir, de préférence, des murs peints à l’huile, elle peut aussi avoir ses murs tendus d’une tapisserie de teinte neutre, ou mieux de panneaux décoratifs représentant de très simples sujets : paysages ou natures mortes”; cf. Monique Eleb e Anne Debarre (1995, p.110). A parede, pintada de castanho, integra frisos com trevos pintados a castanho e creme (em stencil novamente), de grande abstracção. Introdução É o caso de Julian Guadet, que criticava a iluminação zenital; ver Monique Eleb e Anne Debarre (1995, p.108). 24. É o caso de Julian Guadet, que criticava a iluminação zenital; ver Monique Eleb e Anne Debarre (1995, p.108). 25. Argentários que se destinam à exposição das pratas. 26. “La mode française de l’emploi généralisé des grandes surfaces de verre est venu avec l’Art nouveau, ou ‘Modern Style’, comme ils l’appellent” ; cf. Peter Thornon (1986, p.315). O mobiliário consistia nas seguintes peças: um móvel trinchante, um bufete integrado na parede contígua à varanda da frontaria, outro bufete de desenho similar, dois argentiers25 de dimensão idêntica que ocupam a parede lateral esquerda, uma mesa centralizada com seis lugares previstos e mais uma mesa ou aparador de apoio. A Arte Nova revela-se ainda nos cachorros que sustentam a grande viga. Estes suportes revelam uma composição escultórica de troncos e caules secos entrelaçados, parecendo os mesmos caules assumir a forma de ossos na parte frontal numa linguagem que os aproxima de certos pormenores manuelinos (Figura 8). Uma porta envidraçada, atravessada em toda a sua extensão por um arco abatido invertido e que apresenta quatro batentes com pequenos vidros quadrados verdes e superfícies espelhadas, separa a sala de jantares da sala de almoços, seguindo tendência internacional: “A moda francesa do emprego generalizado de grandes superfícies envidraçadas chegou com a Art Nouveau ou Modern Style”26. Cada batente da porta é pontuado superiormente por um arco de volta perfeita e os puxadores em bronze apresentam um contorno orgânico e fechado sediado na Arte Nova (Figura 8). A relação de maior fluidez entre espaços foi bastante propiciada pela Arte Nova assim como o recurso ao vidro colorido ou ao vitral. Neste caso, o vidro colorido verde apresentava um acabamento martelado que se relacionava de modo harmonioso com a paleta cromática escolhida em ambas as salas de refeições. À À semelhança do que Korrodi fizera em outras casas, são trabalhadas alternâncias cromáticas de divisão para divisão e essa diferenciação revela-se desde logo nos vãos de acesso. Neste caso, a mesma porta apresenta os batentes em madeira com cores diferentes de um lado e do outro: envernizadas no lado que confina com a sala de jantares, e pintadas de marfim e rosa no lado que confina com a sala de almoços. 26. “La mode française de l’emploi généralisé des grandes surfaces de verre est venu avec l’Art nouveau, ou ‘Modern Style’, comme ils l’appellent” ; cf. Peter Thornon (1986, p.315). Annals of Museu Paulista. v. 21. n.2. Jul.-Dec. 2013. 25. Argentários que se destinam à exposição das pratas. Introdução Mísulas de recorte quadrado, pontuadas no meio por uma roseta e inferiormente por uma sequência de esferas, prolongam-se sob a forma de frisos nervurados em direcção ao tecto, atribuem-lhe ritmo e quando se intersectam dão origem a pequenos quadrados que a 5 – Detalhes de portas interiores da casa da família Bouhon, Covilhã (mapa de vãos). Esc.1:10 e escala natural. Abril de 1922 o do Arquivo Distrital de Leiria. Figura 5 – Detalhes de portas interiores da casa da família Bouhon, Covilhã (mapa de vãos). Esc.1:10 e escala natural. Abril de 1922. Acervo do Arquivo Distrital de Leiria. gura 5 – Detalhes de portas interiores da casa da família Bouhon, Covilhã (mapa de vãos). Esc.1:10 e escala natural. Abril de 1922. d A D l d L ores da casa da família Bouhon, Covilhã (mapa de vãos). Esc.1:10 e escala natural. Abril de 192 89 Annals of Museu Paulista. v. 21. n.2. Jul.-Dec. 2013. gura 6 – Decoração do hall (vistas de porta de duplo batente e lambril, porta simples e corte construtivo). Esc.1:10. Acervo do Arquivo istrital de Leiria. s de porta de duplo batente e lambril, porta simples e corte construtivo). Esc.1:10. Acervo do Arqu Figura 6 – Decoração do hall (vistas de porta de duplo batente e lambril, porta simples e corte construtivo). Esc.1:10. Acervo do Arquivo Distrital de Leiria. Figura 6 – Decoração do hall (vistas de porta de duplo batente e lambril, porta simples e corte construtivo). Esc.1:10. Acervo do Arquivo Distrital de Leiria Anais do Museu Paulista. v. 21. n.2. Jul.- Dez. 2013. 90 Figura 7 – Bow-window da sala de jantar. Fotografia de Mónica Romãozinho. 90 Anais do Museu Paulista. v. 21. n.2. Jul.- Dez. 2013. acolhem os quatro pontos de iluminação e que se inscrevem em formas circulares demarcadas a estuque. Estes mesmos frisos, marcados por sucessivas linhas horizontais, definem a cimalha. O dourado faz a sua aparição quer nas mísulas quer nos quadrados do tecto, pormenores que se evidenciam deste modo face à cor dominante que é o marfim. As proporções da sala também favorecem a integração da mesa de refeições e respectivas cadeiras e, deste modo, os espaços residuais seriam minimizados. Por outro lado, críticos da época aconselhavam também a que a sala de jantar se desenvolvesse em profundidade a fim de evitar criar sombra sobre si mesma24. 24. Introdução A madeira envernizada dá lugar à madeira pintada de marfim com pormenores pintados a rosa velho, nomeadamente frisos ao nível dos aros das portas e de contorno às próprias almofadas dos batentes e dos envidraçados (em vidro biseauté) e ainda nos limites superiores do rodapé e do lambril. Os envidraçados assumem aqui uma maior verticalidade e os cantos dos seus losangos alinham com o friso superior do lambril, também este em rosa velho e marfim. Na sala de almoços, o papel que reveste o lambril é o mesmo da sala de jantares, mas agora pintado de casca de ovo, o que parece evidenciar mais os seus motivos que consistem em folhas com caules rectilíneos e interligados assim 91 como pequenos quadrados subdivididos, todos inscritos nas listas verticais da composição (Figura 9). Também o castanho das paredes dá lugar a um azul pontuado logo acima do limite do lambril, por frisos em papel com rosas (cor de mel) e folhas de pétalas azuis e verdes sobre vermelho, e acima da verga da porta por apontamentos mais pequenos (lírios azuis e folhas em dois tons de verde). Esta prática de substituir motivos escultóricos em estuque por aplicações de papel era comum na Inglaterra e Escócia no séc. XIX como podemos ver na figura anexada. O pavimento da sala de jantares é em soalho corrido de pinho e o da sala de almoços é mais delicado, em solho à inglesa, com uma cercadura em parquet e mosaico de madeira, num jogo enriquecedor de molduras e de tonalidades. Na sala de almoços, implantou um bufete e outro móvel trinchante. gura 8 – Pormenores: cachorros que sustentam o vigamento da sala de jantar e manípulo da porta que separa as salas de refeições. esenho e fotografia de Mónica Romãozinho. Figura 8 – Pormenores: cachorros que sustentam o vigamento da sala de jantar e manípulo da po Desenho e fotografia de Mónica Romãozinho. e manípulo da porta que separa as salas de refeições. Figura 8 – Pormenores: cachorros que sustentam o vigamento da sala de jantar e manípulo da porta que separa as salas de refeições. Desenho e fotografia de Mónica Romãozinho. Figura 8 – Pormenores: cachorros que sustentam o vigamento da sala de jantar e manípulo da porta que separa as salas de refeições. Desenho e fotografia de Mónica Romãozinho. 92 Anais do Museu Paulista. v. 21. n.2. Jul.- Dez. 2013. Anais do Museu Paulista. Introdução v. 21. n.2. Jul.- Dez. 2013. Anais do Museu Paulista. v. 21. n.2. Jul.- Dez. 2013. Há que apontar um pormenor funcional, um pedal localizado no pavimento que acciona a campainha, permitindo chamar as criadas à hora das refeições segundo padrões em voga na França: “Nos apartamentos burgueses, uma campainha localizada na sala de jantar permite chamar a criada encarregue do serviço de refeições. O contacto pode ser feito por um interruptor, ou de modo mais Annals of Museu Paulista. v. 21. n.2. Jul.-Dec. 2013. Figura 9 – Sala de almoços: porta de ligação à sala de jantar (em cima) e vão tripartido com varanda (em baixo). Fotografias de Mónica Romãozinho. Figura 9 – Sala de almoços: porta de ligação à sala de jantar (em cima) e vão tripartido com varanda (em baixo). Fotografias de Mónica Romãozinho. Figura 9 – Sala de almoços: porta de ligação à sala de jantar (em cima) e vão tripartido com varanda (em baixo). Fotografias de Mónica Romãozinho. 93 Annals of Museu Paulista. v. 21. n.2. Jul.-Dec. 2013. discreto, por um pedal de parquet. Estas invenções permitidas pelos progressos técnicos, respondem a uma exigência dos habitantes, a da eficácia do serviço do pessoal doméstico ‘invisível’ ”27. 27. “Dans les appartements bourgeois, une sonnette placée dans la salle à manger permet d’appeler le domestique chargé du service du repas. Le contact peut être donné par une poire ou, de façon plus discrète, par une pédale de parquet. Ces inventions, permises par le progrès techniques, répondent à une demande des habitants, celle de l’efficacité du service de domestiques ‘invisibles’ ”; cf. Monique Eleb e Anne Debarre (1995, p.402). 27. “Dans les appartements bourgeois, une sonnette placée dans la salle à manger permet d’appeler le domestique chargé du service du repas. Le contact peut être donné par une poire ou, de façon plus discrète, par une pédale de parquet. Ces inventions, permises par le progrès techniques, répondent à une demande des habitants, celle de l’efficacité du service de domestiques ‘invisibles’ ”; cf. Monique Eleb e Anne Debarre (1995, p.402). Embora o tecto da sala de almoços apresente um desenho similar ao anterior, surge aqui complementado por faixas pintadas, com motivos e cores que estabelecem uma relação de continuidade temática em relação aos frisos florais das paredes (roseiras com rosas na sua cor natural e também amarelas, complementadas pelos seus caules espinhosos e folhas). Introdução Mais uma vez, verificamos que os próprios frisos lineares partem do alinhamento das ombreiras das portas ou da respectiva mediatriz. Em ambas as salas, surgem desenhados em planta dois rectângulos a tracejado que corresponderiam a duas mesas, o que significa que Korrodi terá pensado num conjunto de salas suficientemente versátil ao ponto destas funcionarem como uma só sempre que a família Bouhon reunisse um maior número de convidados. O lava-mãos de apoio não chegaria a ser inserido, dando lugar a uns arrumos. Existe, indiscutivelmente, um contraste entre estas duas dependências, diferença que se faz sentir no acabamento dos materiais e uso da cor e padrões e que podemos associar à alternância entre domínio masculino e feminino que se fazia sentir de modo particular no design de interiores. A influência exercida pelos modelos ingleses evidencia-se novamente no modo como tradição e novas soluções surgem articuladas, salvaguardando sempre os valores específicos que se pretendia comunicar no âmbito da concepção de um espaço de recepção: 28. “By long tradition the English dining-room is serious and dignified in character, its colour-scheme rather dark than light, its furniture heavy and made of polish mahogany; it has a Turkish carpet on the floor and oil-paintings, preferably family portraits, in heavy gilt frames, on the walls. But in recent times a more joyful atmosphere has entered this room also. Here too the decoration is becoming ever lighter; furniture modeled on that of Chippendale and Sheraton period has become light and pleasing; the heavy curtains in dark material have given way to curtains of a lighter and brighter material.”; cf. Hermann Muthesius (2007 [1904], p.206). Na sequência de uma longa tradição, a sala de jantar inglesa é séria e digna no seu carácter, a sua composição de cores é mais escura do que leve, a sua mobília é pesada e feita de nogueira polida, apresenta um tapete turco no pavimento e pinturas a óleo, preferencialmente retratos familiares, em pesadas molduras douradas nas paredes. Nos tempos mais recentes, uma atmosfera mais alegre invadiu também esta dependência. Também aqui a decoração se está a tornar mais leve; o mobiliário baseado no período de Chippendale e Sheraton tornou-se mais mais leve e aprazível; as pesadas cortinas em material escuro deram lugar a cortinas de material mais leve e luminoso28 (Figura 10). 29. Desenho original da fábrica alemã Grohn (c.1905), ainda hoje sediada em Bremen. Annals of Museu Paulista. v. 21. n.2. Jul.-Dec. 2013. Introdução Existia então uma forte cooperação entre as suas fábricas e muitos padrões da Helman Céramique correspondem a desenhos da fábrica Grohn. Disponível em: http://cgi. ebay.com.sg/ ORIGINAL- ANTIQUE-Art-Nouveau- Majolica-tile-150-PCS- /220684921022? pt= A r c h i t e c t u r a l _ G a r d e n & h a s h = item3361d8a8be. Acesso em: 11 ago. 2011. Três portas laterais dão acesso a um pequeno corredor, copa com monta-pratos e arrumos de apoio. O WC social revela uma escolha cuidadosa de acabamentos. O branco é a cor dominante, pontuado apenas por detalhes a dourado associados à faiança monocromática com decoração moldada e vidrada, importada da Bélgica e fabricada pela Helman Céramique Sint - Agatha - Berchem Belgium29. A parede divide-se em três níveis: um rodapé de azulejo com festões, uma faixa de azulejo com folhas enroladas, o corpo central revestido a ladrilho vidrado (solução utilizada na Bélgica de que são exemplo os projectos de Victor Horta), um friso de azulejos de padrão idêntico ao seguido no WC que se lhe sobrepõe no piso dos quartos (conjunto simétrico de folhas e rosas estilizadas, circunscritas em círculo, que lembram os padrões de Mackintosh), encimado pela última faixa composta por contas, rosetas 94 Anais do Museu Paulista. v. 21. n.2. Jul.- Dez. 2013. Figura 10 – Soluções decorativas empregues no tecto e paredes da sala de almoços. Fotografia de Mónica Romãozinho. Exemplo similar de aplicação de frisos em papel na cimalha e tecto. Drawing- room de casa situada em Hatfield Drive, Kelvinside, Glasgow. Fonte: Academy Architecture and Annual A hit t l R i L d 29 93 j j l 1909 Figura 10 – Soluções decorativas empregues no tecto e paredes da sala de almoços. Fotografia de Mónica Romãozinho. Exemplo similar de aplicação de frisos em papel na cimalha e tecto. Drawing- room de casa situada em Hatfield Drive, Kelvinside, Glasgow. Fonte: Academy Architecture and Annual Architectural Review, London, v.29, p.93, jan.-jul. 1909. 95 também integradas em círculos e por um novo alinhamento de folhas enroladas (Figura 11). 30. Numa planta datada de 1919, não constava nenhuma divisória envidraçada. O vão aparece desenhado em plantas posteriores, mas era menor e consistia numa porta ladeada de duas janelas simétricas, surgindo posteriormente alterado no mapa de vãos final. 31. Informação que figura n o d e s e n h o d e pormenorização do guarda- vento da sala e do salão que integra o mapa de vãos do projecto. 32. Informação cedida por Andy Sarson (Lincrusta Technical Manager). Introdução O pavimento apresenta um plano central de mosaicos coloridos similares aos das varandas (em castanho, marfim, azul, sangue de boi e ocre) que formam desenhos geométricos como uma roza-cruz ou um trevo de quatro folhas. A moldura deste plano apresenta geometrismos a duas cores e o plano rectangular junto à janela dá continuidade à mesma paleta (castanho escuro e marfim). 31. Informação que figura n o d e s e n h o d e pormenorização do guarda- vento da sala e do salão que integra o mapa de vãos do projecto. 31. Informação que figura n o d e s e n h o d e pormenorização do guarda- vento da sala e do salão que integra o mapa de vãos do projecto. A saleta e a sala ou salão, como aparece designado no desenho de pormenorização, comunicam entre si através de um guarda-vento, de duplo batente, idêntica à que separa as salas de refeições. O arco de circunferência que determina um das travessas curvas, apresenta o seu centro no alinhamento dos dois arcos superiores e termina no ponto de partida da linha do lambril. Esta relação métrica dos caixilhos e lambris manifesta-se em todas as dependências e também ao nível das janelas de sacada cujas curvaturas não são aleatórias30. As suas paredes apresentam um lambril em tela relevada e pintada de casca de ovo da marca Lincrusta31, igualmente ritmado por linhas verticais em relevo, mas agora fragmentadas e alternadas com sequências de pequenos pontos que também encimam o motivo integrado em cada uma dessas faixas fragmentadas, constituído por uma folha. No canto superior direito do desenho Anais do Museu Paulista. v. 21. n.2. Jul.- Dez. 2013. Figura 11: Pormenores: monta-pratos localizado na copa e azulejaria / ladrilho vidrado do WC social. Fotografias de Mónica Romãozinho. Figura 11: Pormenores: monta-pratos localizado na copa e azulejaria / ladrilho vidrado do WC social. Fotografias de Mónica Romãozinho. 96 Anais do Museu Paulista. v. 21. n.2. Jul.- Dez. 2013. Anais do Museu Paulista. v. 21. n.2. Jul.- Dez. 2013. em vista da porta envidraçada e de parte do lambril, observa-se a pormenorização do arranque deste último. Aparece representado em vista ampliada e também seccionado horizontalmente, vendo-se a cantoneira assim como uma baguette que seria aplicada após a aplicação do revestimento (Figura 12). Introdução ‘Art for art’s sake’ was its battle cry, a slogan that originated with the French poet Théophile Gautier.”; cf. Fiona Maccarthy (2011). 33. O Aesthetic movement, dedicado à Beleza no seu estado mais puro, eclodiria n o s a n o s 1 8 6 0 , correspondendo a uma corrente de transição para o movimento Arts and Crafts, amplamente influenciada pelo Oriente. Entre os seus seguidores encontravam-se Dante Gabriel Rossetti (1828- 1882) e William Morris: “The aesthetic movement stood in stark and sometimes shocking contrast to the crass materialism of Britain in the 19th century. ‘Art for art’s sake’ was its battle cry, a slogan that originated with the French poet Théophile Gautier.”; cf. Fiona Maccarthy (2011). Mais uma vez, Korrodi opta por duas fontes de luz simetricamente posicionadas ao invés de uma em posição centralizada (o que já acontecera na sala de almoços). Esses pontos de iluminação demarcam o centro de semi- circunferências divididas em oito partes, parecendo evocar a visão de pérgolas vistas de baixo, ao longo das quais se entrelaçam rosas com corolas pontuadas a dourado e os seus caules e folhas dispostos num movimento orgânico. Duas molduras enquadram toda a composição: a mais interior formada por nervuras e quadrados com motivos nos pontos de intersecção (como na sala de almoços), a exterior por um friso com rosas silvestres brancas, caules e folhas. O tecto da sala ou salão revela o mesmo tipo de enquadramento e linguagem: quartos de circunferências com rosas, caules e espinhos que rematam os cantos dos tectos, frisos com rosas silvestres vermelhas, caules e folhas. Embora este tipo de frisos apareça com o movimento Arts and Crafts, estes são valorizados e integrados em interiores Estéticos33 ou Arte Nova. A sala (Figuras 15 a 17) encontra-se estruturada em duas zonas: a de maior área, que integra uma lareira e que se destinava à família, assim como um recanto ao fundo, separado da área anterior por uma arcada, constituída por um arco central abatido e por dois laterais com balaustrada, onde as crianças da família Carneiro tocavam pianola. Esta última zona assumia uma vocação de espaço de encenação e apresentava um banco fixo de apoio, em continuidade com a linha do lambrim da sala. De acordo com a planta, esta pequena área receberia inicialmente um sofá fixo e perfeitamente ancorado na métrica da arcada, mas esta solução seria abandonada. Introdução Segundo informações cedidas pela fábrica em causa o papel com em vista da porta envidraçada e de parte do lambril, observa-se a pormenorização do arranque deste último. Aparece representado em vista ampliada e também seccionado horizontalmente, vendo-se a cantoneira assim como uma baguette que seria aplicada após a aplicação do revestimento (Figura 12). Segundo informações cedidas pela fábrica em causa, o papel com Segundo informações cedidas pela fábrica em causa, o papel com relevo, da marca Lincrusta, teria sido primeiramente aplicado através de uma cola (goma arábica n.º1 ou dextrina) e posteriormente oleado e envernizado segundo o seguinte processo: lubrificação da superfície, aplicação de primário à base de óleo, aplicação de uma ou duas camadas de pigmento misturado com óleo (neste caso uma base de casca de ovo misturada com linhaça ou terebentina), uma demão muito fina de verniz opaco (scumble glaze) enquanto a tinta ainda está molhada e, no final, uma camada de verniz tradicional a fim de proteger a base oleada32. O papel de parede que cobre a restante parede até à sanca apresenta of Museu Paulista. v. 21. n.2. Jul.-Dec. 2013. 97 2 – Pormenorização do guarda-vento da sala de jantar e salão. Esc. 1:10. Acervo do Arquivo Distrital de Leiria. Figura 12 – Pormenorização do guarda-vento da sala de jantar e salão. Esc. 1:10. Acervo do Arquivo Distrital de Leiria. Annals of Museu Paulista. v. 21. n.2. Jul.-Dec. 2013. 97 Annals of Museu Paulista. v. 21. n.2. Jul.-Dec. 2013. Annals of Museu Paulista. v. 21. n.2. Jul.-Dec. 2013. motivos florais dourados simetricamente dispostos sobre fundo azul, cuja linguagem faz lembrar alguns papéis de William Morris (1834-1896), em particular o padrão St. James. A saleta (Figuras 13 e 14) evidencia-se ainda pela sua planta de tectos e pela originalidade dos seus estuques, referenciados numa tradição clássica mas contrariados por aplicações de papel de parede e interpretações orgânicas de flora. 33. O Aesthetic movement, dedicado à Beleza no seu estado mais puro, eclodiria n o s a n o s 1 8 6 0 , correspondendo a uma corrente de transição para o movimento Arts and Crafts, amplamente influenciada pelo Oriente. Entre os seus seguidores encontravam-se Dante Gabriel Rossetti (1828- 1882) e William Morris: “The aesthetic movement stood in stark and sometimes shocking contrast to the crass materialism of Britain in the 19th century. Introdução A droite et à gauche de la cheminée, surmontée d’une glace, un canapé et des fauteuils” ; cf. Ris-Paquot (1894, p.195). O branco começa-se a generalizar sobretudo uma tendência para o marfim mas ele não foi somente utilizado por Voysey, Mackintosh e progressistas desta geração, pois, por exemplo, certos móveis da rainha Alexandra em Sandrigham, eram pintados em branco – quase sempre ligeiramente pontuados a dourado - e os estilos “Luís XVI” e “Adam” pintados de branco tinham-se tornado uma realidade inegável no final do século36. 35. “The oak is man, in firmness drest, / With strength of fondness in his breast, / Delighting in the lie:-/the ivy is the gentle wife.”; cf. Scots Magazine (Marc.1789), apud, Juliet Kinchin (2008, p.170). O conjunto formado pelas salas obedece também a uma orientação solar cuidadosa. Já Raul Lino37 expressara essas preocupações na sua obra escrita: “Casas de estar ficam bem quando recebam bastante luz do sol durante a tarde, por isso se orientam de preferência com suas janelas ao Sudoeste ou Oeste”38. 36. “Le blanc commence à se généraliser, souvent avec une tendance vers l’ivoire, mais il ne fut pas seulement utilisé par Voysey, Mackintosh et les progressistes de cette génération, puisque, par exemple, certains meubles du Sandringham de la reine Alexandra étaient peints en blanc en blanc – légèrement rechampis d’or toutefois – et que les styles ‘Louis Seize’ et ‘Adam’ peints en blanc étaient devenus une réalité indéniable à la fin du siècle” ; cf. Peter Thornon (1986, p.312). 36. “Le blanc commence à se généraliser, souvent avec une tendance vers l’ivoire, mais il ne fut pas seulement utilisé par Voysey, Mackintosh et les progressistes de cette génération, puisque, par exemple, certains meubles du Sandringham de la reine Alexandra étaient peints en blanc en blanc – légèrement rechampis d’or toutefois – et que les styles ‘Louis Seize’ et ‘Adam’ peints en blanc étaient devenus une réalité indéniable à la fin du siècle” ; cf. Peter Thornon (1986, p.312). O escritório segue o então chamado estilo Renascença, tendência que é visível ao nível do remate superior do lambril, ritmado por pequenos quadrados com volutas ou apainelados quadrados ou rectangulares, pontualmente em ponta de diamante, que se integram na malha de almofadas pré-estabelecida. Quer os cachorros, pontuados por acantos, quer a cimalha que apresenta um trabalho de relevo com sucessivos arabescos executados a estuque, reflectem o mesmo tipo de linguagem. Introdução O tecto segue a linha formal dos dois tectos anteriores, mas aqui as semi-circunferências dão lugar a uma composição mais rectilínea, mas igualmente decorada por roseiras, em que os seus caules e folhas transgridem pontualmente as molduras geometrizadas que regulam a composição. Se observarmos atentamente a planta de tectos do conjunto de recepção formado pelas várias salas, é-nos possível arriscar em associá-los à Arte Nova, não só pela criatividade das suas composições inspiradas na natureza que oscilam entre um registo mais orgânico ou mais linear, mas também pelo afastamento em relação a outros estilos que eram então reinterpretados ou simplesmente copiados. Em ambas as salas, observamos pavimentos de mosaicos de madeira, que formam uma composição alternada de formas quadrangulares e quadradas. O tapete central formado por este jogo é envolvido por uma cercadura em parquet espinhado. A restante integração do mobiliário no salão segue também algumas “modas” ditadas na altura e divulgada pela literatura internacional de especialidade: “Em relação à lareira, coloca-se à sua frente uma mesa de centro, ornada com um 98 Anais do Museu Paulista. v. 21. n.2. Jul.- Dez. 2013. Annals of Museu Paulista. v. 21. n.2. Jul.-Dec. 2013. Figuras 13 e 14 – Perspectiva e tecto da saleta. Fotografias de Mónica Romãozinho. Annals of Museu Paulista. v. 21. n.2. Jul.-Dec. 2013. Figuras 13 e 14 – Perspectiva e tecto da saleta. Fotografias de Mónica Romãozinho. Annals of Museu Paulista. v. 21. n.2. Jul.-Dec. 2013. Figuras 13 e 14 – Perspectiva e tecto da saleta. Fotografias de Mónica Romãozinho. Annals of Museu Paulista. v. 21. n.2. Jul.-Dec. 2013. Figuras 13 e 14 – Perspectiva e tecto da saleta. Fotografias de Mónica Romãozinho. Figuras 13 e 14 – Perspectiva e tecto da saleta. Fotografias de Mónica Romãozinho. 99 Annals of Museu Paulista. v. 21. n.2. Jul.-Dec. 2013. relógio e dois candelabros; de cada lado múltiplas cadeiras. À direita e à esquerda da lareira, complementada por um espelho, um canapé e poltronas”34. Os tons dominantes são claros e o branco marfim ou casca de ovo, paleta mais suave e feminina35, voltam a invadir os lambris e tectos desta sucessão de espaços sociais destinados à família, aqui como no estrangeiro: 34. “Vis-à-vis la cheminée, lui faisant face, se place la table de milieu, ornée d’une pendule et de deux candélabres ; de chaque côté plusieurs sièges. Introdução O tratamento atribuído às paredes, neste caso pintadas de verde, é similar ao da sala de jantar, tendo sido pintado um friso através de stencil, cuja temática são os trevos. O próprio interruptor foi integrado no lambril. Dois tons de madeira são alternados quer no lambril quer nas almofadas das folhas da porta. 37. Raul Lino e Ernesto Korrodi cruzam-se em 1926 no dia em que ser-lhes-ia atribuído o título de arquitecto, na sequência da saída de um decreto de lei (1925) que determinava que ninguém podia usar a designação de arquitecto sem ter o diploma oficial de uma das Escolas de Belas- Artes do país. E será Raul Lino, como presidente da Academia das Belas-Artes que elaborará o discurso póstumo quando Korrodi morre. Raul Lino estudou em Inglaterra e na Alemanha, onde frequentou a Technische Hochschule e trabalhou no atelier do Prof. Albrecht Haupt. Neste sentido compreende-se a proximidade da sua obra aos modelos anglo- Dois dos cantos do escritório são chanfrados, recebendo um deles a porta e correspondendo o outro a um nicho em arco de volta perfeita, pintado de azul que acolheria um móvel classeur (arquivador). Entre estes cantos, desenha- se na parede uma grande reentrância em arco destinada a um sofá ladeado de duas pequenas mesas. Aqui, o fogão não apresenta uma coifa e apresenta-se absolutamente integrado no desenho do lambril. O tecto, em caixotões de madeira, apresenta as suas superfícies reentrantes pintadas na cor das paredes e contornadas por frisos Arte Nova. O caixotão central recebe a base circular de fixação da fonte de luz, elemento que segue a mesma linguagem Renascença, com o seus acantos simetricamente posicionados. Dada à localização, dimensão e nobreza do tratamento decorativo de superfícies, bem como maior contenção decorativa, subentende-se que este escritório e biblioteca assumiriam também a vocação de espaço de convívio do domínio fundamentalmente masculino, tendência que também era observada na Inglaterra: “Nas grandes casas que possuíam uma biblioteca, tem-se a impressão que esta dependência servia mais como sala de estar do que como local para 100 Anais do Museu Paulista. v. 21. n.2. Jul.- Dez. 2013. Figura 15 – Pequeno recanto que se forma para lá da arcada tripartida e que podia funcionar como espaço de representação para a família ou convidados. Fotografia de Mónica Romãozinho. saxónicos apreciados na Alemanha e também determinantes para Korrodi. 38. Cf. Raul Lino (1992 [1941], p.35). Introdução Figura 15 – Pequeno recanto que se forma para lá da arcada tripartida e que podia funcionar c espaço de representação para a família ou convidados. Fotografia de Mónica Romãozinho. Figura 15 – Pequeno recanto que se forma para lá da arcada tripartida e que podia funcionar como espaço de representação para a família ou convidados. Fotografia de Mónica Romãozinho. Annals of Museu Paulista. v. 21. n.2. Jul.-Dec. 2013. Figura 16 – Plantas de tecto do salão e recanto. Desenhos de Mónica Romãozinho. Annals of Museu Paulista. v. 21. n.2. Jul.-Dec. 2013. Figura 16 – Plantas de tecto do salão e recanto. Desenhos de Mónica Romãozinho. 101 101 “Dans les grandes ons qui possédaient bibliothèque, on a ression que cette pièce t tellement de la salle éjour que la personne souhaitait lire ou er devait probablement tirer ailleurs, l’homme son « antre » peut-être, a femme dans son oir” ; cf. Peter Thornon 6, p.316). O Dr. Fernando eiro utilizaria esta ão como escritório que unicava com o azém dos tecidos vés de um pequeno bulo que, por sua vez, sentava uma segunda da acessível através do m. Deste modo, a a da e saída de clientes -se sem que a esfera de lho jamais se cruzasse a da família. Figura 17 – Recanto do salão. Fotografia de Mónica Romãozinho. Figura 17 – Recanto do salão. Fotografia de Mónica Romãozinho. Figura 17 – Recanto do salão. Fotografia de Mónica Romãozinho. 39. “Dans les grandes maisons qui possédaient une bibliothèque, on a l’impression que cette pièce tenait tellement de la salle de séjour que la personne qui souhaitait lire ou étudier devait probablement se retirer ailleurs, l’homme dans son « antre » peut-être, et la femme dans son boudoir” ; cf. Peter Thornon (1986, p.316). 40. O Dr. Fernando Carneiro utilizaria esta divisão como escritório que comunicava com o armazém dos tecidos através de um pequeno vestíbulo que, por sua vez, apresentava uma segunda escada acessível através do jardim. Deste modo, a a entrada e saída de clientes fazia-se sem que a esfera de trabalho jamais se cruzasse com a da família. Figura 17 – Recanto do salão. Fotografia de Mónica Romãozinho. onde se retirava quem quisesse ler ou estudar, o homem no seu “antro” talvez, e a mulher no seu boudoir”39 (Figuras 18 a 20). Introdução No piso inferior (planta do subsolo), encontra-se localizada a sala de bilhar, precisamente sob o escritório, mas que na realidade acabaria por assumir mais tarde a função de escritório, eventualmente ainda no tempo de Joseph Bouhon40. Uma porta com um pequeno postigo (que não figura na planta) estabelecia a passagem desta divisão para um pequeno corredor ligado a um vestíbulo que, por sua vez, conduzia ao armazém dos tecidos e apresentava uma segunda escada em madeira de acesso ao jardim (Figura 21). O pavimento deste pequeno vestíbulo é em mosaicos quadrados pretos, sangue de boi e brancos. A entrada e saída de clientes ocorriam sem que a esfera do trabalho se cruzasse com a esfera da família. Em obra, o lavatório rodaria 90º no seu posicionamento e o espaço inferior das escadas acabaria por dar lugar a uns arrumos. O nicho destinava-se a um sofá, enquanto a área central seria ocupada pela mesa de jogo. O pavimento, em mosaico cerâmico, apresenta flores e acantos enquadrados numa composição geometrizada e assente num paleta de cinzas e azuis animados por contornos a preto. No mesmo piso e quase simetricamente colocada relativamente à sala de bilhar, situa-se a cozinha, assim como a casa de banho de serviço, que se desdobra em dois compartimentos: o primeiro destinado ao lava-mãos e acessível 102 102 Anais do Museu Paulista. v. 21. n.2. Jul.- Dez. 2013. através da cozinha, outro que integra ainda hoje a banheira em pedra lioz e que é acessível através do vestíbulo. Este último destinava-se às criadas, preocupação que começava a assolar alguns projectistas portugueses de então, como Raul Lino: “Quando o tamanho e categoria da casa o permitam, não devemos esquecer o quarto de banho para as criadas. (…) É fácil neste capítulo estabelecer um mínimo de comodidades – pelo menos a água fria e o despejo da tina encanados; para a água quente, onde não a possa haver encanada directamente da caldeira, depressa se transportam uma ou duas cafeteiras desde o fogão da cozinha”41. A cozinha (Figuras 22 e 23) apresenta-se revestida em toda a altura por ladrilho vidrado, apresentando três secções fundamentais: a mais baixa, delimitada por faixas de ladrilho cinzento e azul (rodapé) e definida no meio por alternâncias de ladrilhos brancos dispostos no sentido horizontal ou vertical; a intermédia formada pelo mesmo tipo de mosaico aplicado horizontalmente; a cimalha, constituída por uma faixa de azulejos de “Delft”. 41. Cf. Raul Lino (1992, p.45). Annals of Museu Paulista. v. 21. n.2. Jul.-Dec. 2013. Anais do Museu Paulista. v. 21. n.2. Jul.- Dez. 2013. Introdução Estes últimos surgem dispostos de modo pontuado e enquadrados por ladrilhos colocados nos sentidos vertical e horizontal, delimitada inferiormente por um friso liso em azul e superiormente por uma cercadura azul composta por motivos fitomórficos mais abstractos. A azulejaria azul e branca, em faiança de “Delft” ou inspirada na mesma, retrata temáticas de um quotidiano doméstico em família ligado ao campo e à pesca. Avós, pais com crianças ou crianças de colo, camponeses ou pescadores, personificam aquelas vivências, correspondendo cada azulejo a uma nova “história” que encantava as crianças da casa já no tempo da família Carneiro. No Figura 18 – Lareira do escritório, integrada em lambril estilo Renascença. Fotografia de Mónica Romãozinho. Figura 18 – Lareira do escritório, integrada em lambril estilo Renascença. Fotografia de Mónica R ã i h Figura 18 – Lareira do escritório, integrada em lambril estilo Renascença. Fotografia de Mónica Romãozinho. 103 Annals of Museu Paulista. v. 21. n.2. Jul.-Dec. 2013. Figura 19 – Vista do escritório e respectivo tecto em caixotões. Fotografia de Mónica Romãozinho. gura 19 – Vista do escritório e respectivo tecto em caixotões. Fotografia de Mónica Romãozinho Figura 20 – Decoração de tecto (em relevo). Professor J.Regl., Zurique. Fonte: Academy Architecture and Annual Architectural Review, London, v.23 , p.144, Jan./June 1913. Figura 20 – Decoração de tecto (em relevo). Professor J.Regl., Zurique. Fonte: Academy Architecture and Annual Architectural Review, London, v.23 , p.144, Jan./June 1913. projecto original, a cozinha apresentava uma copa de dimensões e localização idênticas às da copa do piso superior, mas na fase construtiva, seria apenas implementado o monta-pratos, com quadrados envidraçados e guarnição em madeira pintada de cinzento, com o telefone e campainha. Foi construído um lava-mãos na mesma prumada do da sala de jantar, com um pequeno armário pintado de cinzento. A cozinha é iluminada por uma composição simétrica de janelas com pequenos 104 Anais do Museu Paulista. v. 21. n.2. Jul.- Dez. 2013. quadrados envidraçados, em que a janela central assume uma altura mais elevada através da introdução de uma bandeira. O pavimento dos nichos das janelas laterais corresponde a um novo tapete de mosaicos policromos, dotado de cercadura. Persiste ainda hoje o forno de lenha e respectiva prateleira que ocupava um dos cantos da cozinha. Introdução Existe uma saída para uma conduta vertical junto ao forno o que significa que o calor que este libertava seria rentabilizado no âmbito do aquecimento da água da banheira do WC do piso superior. Lateralmente ao forno, situavam-se barras que sustentavam os talheres de cozinhar, assim como o saleiro. O nicho existente na cozinha, que correspondia em planta a uma janela, recebia uma estrutura em traves de madeira onde eram pendurados os tachos, panelas e frigideiras. Uma mesa com tampo de mármore completava este conjunto. 42. Pintor e retratista nascido na Covilhã. Conquistou em 1936 o Prémio Columbano, e no ano seguinte, a medalha de ouro da Exposição Internacional de Paris. Frequentou a Escola de Belas Artes do Porto. Ainda aparentado com o escultor Manuel Morais, e com o escritor Raul Brandão. Fez a instrução primária na sua terra, e, embora a família se tivesse fixado no Porto, foi em 1921 na Covilhã que desenhou os cenários para a revista “o País da Guedelha”. Retratou personalidades como Teixeira de Pascoais, Aquilino Ribeiro e Augusto de Castro. Os retratos de Salazar e do Cardeal Cerejeira, da sua autoria, pertencem ao Instituto Nacional de Estatística. O Presidente do Brasil Getúlio Vargas e o político espanhol José António Primo de Rivera, posaram para ele. Escreveu as obras Do Meu Ofício de Pintar e Retratos e Retratados. Disponível em: http://www.cm- covilha.pt/simples/?f=2395. Acesso em: 15 maio 2011. A cozinha encontra-se directamente ligada à dispensa e à rouparia, ambas acessíveis pelo pequeno vestíbulo ligado ao corredor e, no segundo caso, foi também prevista uma acessibilidade directa pelo exterior. Na rouparia, de planta semi-circular e integrada na bow-window, a parede recta incorpora uma fila de armários encimados por vãos em vidrinhos que iluminam e ventilam a despensa. A parede surge revestida pelo mesmo ladrilho branco, mas agora inserido na posição vertical até 1,50 m de altura aproximadamente e rematado superiormente por um friso de azulejaria igual ao do WC social e inferiormente por um rodapé em mosaico branco. O pavimento é composto por geometrismos nas cores laranja, castanho e branco. Apesar de, em planta, não existir nenhuma comunicação entre a despensa e a cave, acabaria por ser aberta uma porta que permite aceder a uma estreita escada que conduz à garagem localizada numa segunda cave e não em anexo (situado a Poente) como nos sugerem os desenhos. Introdução O espaço privado começa, de certo modo, a adivinhar-se a partir do patamar da escadaria. É precisamente ao nível deste patamar que nos é possível contemplar uma pintura de Eduardo Malta (1900-1967)42, que retrata um cenário pastoril, temática recomendada pelos tratados e manuais da época (Figura 24). A pintura não corresponde a um elemento pontual que fixamos na parede, mesmo que segundo uma lógica de centralidade ou alinhamento, passando a ocupar toda a parede e tornando-se deste modo mais uma componente integrante do design de interiores como também ocorria na Inglaterra: 43. “The picture painter will be the first to admit that the beauty of the picture depends on the relation of its parts; but if the relation of one colour to another is so important, the logical inference is that the picture itself must depend for its beauty in the scheme of things. And so one finds in the earlier ages of painting the painter at his best was a wall decorator.(...) Pictures, then, in the house, it may be said, should possess a decorative quality which should be brought into harmony with the whole scheme of which they form a part”; cf. M.H. Baillie Scott (1906, p.52- 53). O pintor de quadros será o primeiro a admitir que a beleza da pintura depende da relação entre as suas partes; mas se a relação entre uma cor e outra cor é tão importante, a lógica dedução é a de que a pintura em si própria deve depender da beleza da composição de tudo. E encontramos, de facto, nos primórdios da pintura, o pintor no seu melhor como decorador mural. (...) Nesta perspectiva, podemos constatar que as pinturas, numa casa, deveriam possuir qualidades decorativas que façam com que estas se relacionem em harmonia com a composição global da qual fazem parte43. Por outro lado, a própria temática da pintura relacionava-se com as necessidades psicológicas do dono da casa, que retornava a casa após um dia de trabalho nos moldes das sociedades burguesas que se generalizavam na Europa 105 Annals of Museu Paulista. v. 21. n.2. Jul.-Dec. 2013. their surroundings will at once enfold him as in an atmosphere which soothes and charms like harmonious music.”; cf. M.H. Baillie Scott (1906, p.53). Introdução Ocidental: A meta desejada, simplesmente estabelecida, era a criação de um reino pastoral para o relaxamento físico e mental em que o decor contribuiria para, mais do que cansar, o refrescar do cérebro cansado do trabalhador44.O universo do trabalho era então uma questão do domínio masculino e cabia à dona de casa, responsável pela decoração, garantir a harmonia e tranquilidade no lar: 44. “The desired goal, simply stated, was the creation of a pastoral realm for physical and mental reflection in which the décor contributed to, rather than detracted from, the refreshing of the weary brain-worker.”; cf. Joyce Henry Robinson (2008, p.297). Se nós imaginarmos, por um instante, o homem de negócios cansado retornando à sua casa de subúrbios no final do dia, dificilmente se esperará que esteja preparado para desenvolver um esforço mental especial na análise dos seus quadros; mas qualquer que seja a qualidade decorativa que expressam em conjunto com a sua envolvente, irão pelo menos envolvê-lo numa atmosfera que transmite doçura e charme como uma música harmoniosa45. 45. “If we imagine, for instance, the tired man of business returning to his suburban home in the evening, it can hardly be supposed that he will be prepared to make the special mental effort involved in an inspection of his pictures; but whatever decorative quality they express in conjunction with O tecto correspondente à escadaria, em estuque pintado de branco, apenas visível ao olhar de quem sobe em direcção ao piso dos quartos, consiste numa moldura percorrida por uma sucessão de linhas e rematada em ambos os topos por semi-círculos que demarcam o eixo central do tecto e que aparecem ladeados por pequenas contra-curvas. Figura 21 – Planta do primeiro subsolo: versão quase final apesar de alguns desajustes relativos ao construído. Acervo do Arquivo Distrital de Leiria. Figura 21 – Planta do primeiro subsolo: versão quase final apesar de alguns desajustes relativos ao construído. Acervo do Arquivo Distrital de Leiria. Anais do Museu Paulista. v. 21. n.2. Jul.- Dez. 2013. Anais do Museu Paulista. v. 21. n.2. Jul.- Dez. 2013. 106 Anais do Museu Paulista. v. 21. n.2. Jul.- Dez. 2013. Figura 22 – Vista da cozinha. Vê-se o monta-pratos em primeiro plano (lado esquerdo). Fotografia de Mónica Romãozinho Annals of Museu Paulista. v. 21. n.2. Jul.-Dec. 2013. Figura 23: – Interior da cozinha, na qual se encontra Maria Rita, criada que trabalhara 20 anos para a família Bouhon. Anais do Museu Paulista. v. 21. n.2. Jul.- Dez. 2013. Introdução Coleção privada de João Korrodi. Figura 22 – Vista da cozinha. Vê-se o monta-pratos em primeiro plano (lado esquerdo). Fotografia de Mónica Romãozinho. Annals of Museu Paulista. v. 21. n.2. Jul.-Dec. 2013. Figura 23: – Interior da cozinha, na qual se encontra Maria Rita, criada que trabalhara 20 anos para a família Bouhon. Coleção privada de João Korrodi. Figura 22 – Vista da cozinha. Vê-se o monta-pratos em primeiro plano (lado esquerdo). Fotografia de Mónica Romãozinho. Figura 22 – Vista da cozinha. Vê-se o monta-pratos em primeiro plano (lado esquerdo). Fotografia de Mónica Romãozinho. Figura 22 – Vista da cozinha. Vê-se o monta-pratos em primeiro plano (lado esquerdo). Fotografia de Mónica Romãozinho. Figura 23: – Interior da cozinha, na qual se encontra Maria Rita, criada que trabalhara 20 anos para a família Bouhon. Coleção privada de João Korrodi. 107 Annals of Museu Paulista. v. 21. n.2. Jul.-Dec. 2013. Annals of Museu Paulista. v. 21. n.2. Jul.-Dec. 2013. A supressão quase geral de todo o ornamento aproxima esta solução das propagadas quer pelos progressistas austríacos e alemães quer pelos franceses inspirados pelo estilo Império. A luminária de parede, em ferro, não integra mais as tulipas de vidro ou globos, mas subsiste a sua serralharia Arte Nova, composta por linhas ondulantes e folhagens assimétricas (Figura 25). Contudo, é invulgar a introdução de uma janela geminada em madeira escurecida, associada a um ponto poético de contemplação da escadaria principal. O próprio arco abatido que une os dois arcos de volta perfeita das janelas, apresenta novamente uma sucessão de frisos que atravessam as paredes, formando uma sanca de altura considerável e suscitando, ao mesmo tempo, uma continuidade de linguagem em relação às linhas da moldura do tecto. Porém, a planta sugeria um vão tripartido de largura equivalente à do janelão (Figuras 26 e 27). Figura 24 – Patamar de chegada. À esquerda: Quadro de Eduardo Malta.Fotografia de Mónica Romãozinho. Figura 24 – Patamar de chegada. À esquerda: Quadro de Eduardo Malta.Fotografia de Mónica Romãozinho. 108 108 Anais do Museu Paulista. v. 21. n.2. Jul.- Dez. 2013. Figura 25 – Luminária do hall, similar a outros modelos internacionais, de que é exemplo a luminária na Casa de Victor Horta, Bruxelas, 1898 (à direita). Fotografias de Mónica Romãozinho. Figura 25 – Luminária do hall, similar a outros modelos internacionais, de que é exemplo a luminária na Casa de Victor Horta, Bruxelas, 1898 (à direita). Annals of Museu Paulista. v. 21. n.2. Jul.-Dec. 2013. Introdução Fotografias de Mónica Romãozinho. Como fora referido anteriormente, o hall assume aqui a sua forma completa de um octógono e o seu tecto segue a mesma técnica e linguagem do tecto da escadaria, embora os frisos sejam complementados por conchas explodidas e festões e apresente, neste caso, um elemento central constituído por uma roseta e por alinhamentos radiais que recebe a luminária. As rosas continuam a constituir um elemento decorativo dominante no tema do papel de parede (rosas vermelhas e azuis) que, à semelhança dos frisos dos tectos da área social, se enquadram no movimento Arts and Crafts, como pudemos constatar assim que comparámos o padrão em causa com outro da autoria de William Morris, o padrão Trellis. Quer o lambril quer as portas com os seus lintéis de remates quadrangulares, seguem novamente um estilo Renascença. O lambril é dominado pela sucessão de pilastras nervuradas e revestido por papel em cor neutra. O pavimento em soalho não obedece a nenhuma composição em particular, revelando apenas uma cercadura que acompanha a forma do hall. Os pavimentos das restantes dependências seguem a mesma solução construtiva, mas sem molduras envolventes. No lado interno das dependências, as molduras das portas simplificam-se e a cor natural da madeira é substituída por 109 Annals of Museu Paulista. v. 21. n.2. Jul.-Dec. 2013. Figura 26 – Vista do hall do piso dos quartos e respectiva loggia. Fotografia de Mónica Romãozinho. l lh d h ll l d l d d d d Anais do Museu Paulista. v. 21. n.2. Jul.- Dez. 2013. Figura 26 – Vista do hall do piso dos quartos e respectiva loggia. Fotografia de Mónica Romãozinho. Exemplo semelhante de hall com janela geminada, contextualizado em interior da autoria de Ed.Cuypers. Fonte: Academy Architecture and Annual Architectural Review, London, v.44 p.136, july/dec. 1913. Anais do Museu Paulista. v. 21. n.2. Jul.- Dez. 2013. Figura 26 – Vista do hall do piso dos quartos e respectiva loggia. Fotografia de Mónica Romãozinho. Exemplo semelhante de hall com janela geminada, contextualizado em interior da autoria de Ed.Cuypers. Fonte: Academy Architecture and Annual Architectural Review, London, v.44 p.136, july/dec. 1913. Figura 26 – Vista do hall do piso dos quartos e respectiva loggia. Fotografia de Mónica Romãozinho. Exemplo semelhante de hall com janela geminada, contextualizado em interior da autoria de Ed.Cuypers. Fonte: Academy Architecture and Annual Architectural Review, London, v.44 p.136, july/dec. 1913. 110 Anais do Museu Paulista. v. Annals of Museu Paulista. v. 21. n.2. Jul.-Dec. 2013. Introdução 21. n.2. Jul.- Dez. 2013. 7 – Planta do segundo pavimento à escala 1:50. Acervo do Arquivo Distrital de Leiria. Figura 27 – Planta do segundo pavimento à escala 1:50. Acervo do Arquivo Distrital de Leiria. acabamentos em branco ou por um jogo cromático de marfim e ocre. Há sempre um sentido de diferenciação ou hierarquização entre espaços de passagem e espaços mais íntimos. À semelhança do que acontecera com a planta do piso social, mais uma vez Korrodi propõe a localização precisa de todas as peças de mobiliário essenciais para cada actividade ou tarefa. O quarto dos donos da casa ocupa uma localização privilegiada, pois não só usufrui de uma orientação solar a sudeste, usufruindo-se deste modo do nascer do sol, mas também integra a varanda semi-circular que se sobrepõe à bow-window da sala de jantar. Reunia fundamentalmente todos os requisitos necessários em termos de área, iluminação natural e ventilação, segundo ditames em voga na época: “A condição essencial para que um quarto de dormir seja 111 Annals of Museu Paulista. v. 21. n.2. Jul.-Dec. 2013. saudável, sobretudo quando é ocupado por duas pessoas, é que este seja vasto e espaçoso, bem iluminado e bem arejado: que o ar se possa renovar facilmente. Devem-se evitar as alcovas, que têm o inconveniente de diminuir a massa de ar respirável ao longo da noite”46. Nesta mesma perspectiva, uma reentrância, situada entre as duas portas de acesso, destina-se à cama e a duas mesas-de-cabeceira colocadas simetricamente. 46. “La condition essentielle, pour qu’une chambre à coucher soit saine, surtout quand on doit l’habiter à deux, c’est qu’elle soit vaste et spacieuse, bien éclairée et bien aérée ; que l’air puisse s’y renouveler facilement. On doit éviter les alcôves, qui ont l’inconvénient de diminuer la masse d’air respirable pendant la nuit.” ; cf. Ris-Paquot (1894, p.206). 46. Já Mackay Hugh Baillie Scott (1865-1945)47 sugeria que se abandonasse a solução da cama em ferro trabalhado ou em latão moldado, constatando que esta deveria ser, sempre que possível colocada num recesso, ou pelo menos numa posição em que parecesse uma parte integrante do quarto48. O vão tripartido é similar ao que ilumina e ventila quer a cozinha quer a sala de almoços (Figura 28). O plano inferior das janelas apresenta-se revestido a apainelado almofadado que dá continuidade ao das portadas quando fechadas. 48. Cf. M.H. Baillie Scott (1897). Figura 28 – Vão tripartido que ilumina o quarto dos pais. Fotografia de Mónica Romãozinho. Introdução Por outro lado, a colocação da cama em frente à janela evitava receber o ar que entra pelas frestas e permitia 47. Acreditamos que Baillie Scott estaria na génese daquilo que Madsen classifica de Proto-Arte Nova e que os seus trabalhos correspondem a verdadeiros projectos Arte Nova que se anteciparam aos de Victor Horta. O modo como interpreta o ornamento orgânico como elemento plástico capaz de interligar superfícies e mobiliário, fazem de Baillie Scott um verdadeiro precursor da Arte Nova e também uma referência para os progressistas alemães. Anais do Museu Paulista. v. 21. n.2. Jul.- Dez. 2013. ram . O ta o omo z de s e aillie eiro va e ncia stas cott Figura 28 – Vão tripartido que ilumina o quarto dos pais. Fotografia de Mónica Romãozinho. Anais do Museu Paulista. v. 21. n.2. Jul.- Dez. 2013. 48. Cf. M.H. Baillie Scott (1897). Anais do Museu Paulista. v. 21. n.2. Jul.- Dez. 2013. Figura 28 – Vão tripartido que ilumina o quarto dos pais. Fotografia de Mónica Romãozinho. Figura 28 – Vão tripartido que ilumina o quarto dos pais. Fotografia de Mónica Romãozinho. 112 Anais do Museu Paulista. v. 21. n.2. Jul.- Dez. 2013. receber luz quando se pretendesse ler49. A planta sugeria ainda a localização de um sofá e de várias cadeiras, situação que se repetirá nos outros quartos e que demonstra que estes continuavam a assumir, ainda nesta altura, uma função de estar. Um grande guarda-fato, uma cómoda e um armário integrado no vão central, eventualmente um toucador (que daria lugar a uma janela de sacada) a fim de que houvesse luz suficiente para a senhoras se pentearem, completam este conjunto. As portas e guarnições de madeira estão pintadas de branco, contrastando mais uma vez com as paredes, neste caso cobertas de branco. O tecto, referenciado fundamentalmente no Estilo Império, apresenta grinaldas nos quatro cantos, formadas por folhas de louro e acantos que circunscrevem uma composição formada urnas com flores e novamente acantos. Frisos constituídos por uma sequência de linhas ou por óvulos e dardos, emolduram o tecto que apresenta um motivo central com flores e uma bordadura também composta por alinhamentos paralelos. Introdução O quarto era, aliás, uma das dependências onde o leque de estilos variava mais, de acordo com a moda de então: “Todos os estilos se acomodam na perfeição para a decoração de um quarto de dormir, desde do gótico, a Renascença, o Luís XIV, o Luís XV, o Luís XVI até ao estilo Império”50. 49. Ver Monique Eleb e Anne Debarre (1995, p.156). 49. Ver Monique Eleb e Anne Debarre (1995, p.156). 50. “Tous les styles s ’ a c c o m m o d e n t parfaitement pour la décoration d’une chambre à coucher, depuis le gothique, la Renaissance, le Louis XIV, le Louis XV, le Louis XVI jusqu’au style Empire.”; cf. Ris-Paquot (1894, p.197). 51. “Il est un des signes du statut de l’habitant et aussi de son rapport à la propreté, élément de la respectabilité. Il n’est pas une pièce de service et encore moins un espace fonctionnel” ; cf. Monique Eleb e Anne Debarre (1995, p.224). 51. “Il est un des signes du statut de l’habitant et aussi de son rapport à la propreté, élément de la respectabilité. Il n’est pas une pièce de service et encore moins un espace fonctionnel” ; cf. Monique Eleb e Anne Debarre (1995, p.224). 52. “Il semble, selon les rares traités de savoir-vivre qui abordent cette question, que la plupart d’entre eux utilisent le cabinet de toilette de leur épouse, le matin, avant elle. Cependant, chacun des époux a son service spécial sur la même table de toilette.” ; cf. Monique Eleb e Anne Debarre (1995, p.225). No gabinete de toilette, o gomil e a bacia dão lugar a um armário lavatório. Encontra-se revestido a ladrilho vidrado marfim, com um friso de azulejaria Arte Nova, idêntico ao do WC social. Dada a profundidade das peças de mobiliário, tudo indica que Korrodi optou por dois guarda-fatos, que permitem a separação do vestuário do casal, restando uma peça que certamente corresponderia a um toucador. Um sofá, não uma chaise longue como era habitual em França, completa este conjunto. O Gabinete de toilette assume, como na França, um duplo papel: “Ele é um dos signos do estatuto do habitante e também da sua ligação à limpeza, elemento de respeitabilidade. Não é uma dependência de serviço, muito menos um espaço funcional”51. “facilita o serviço e representa inapreciáveis comodidades, pois assim que se sai da dependência afectada à higiene do corpo, a toilette é terminada sem que se tenha perdido tempo nem apanhado frio ao passar para outro lugar53. Toilette, banhos e WC formam então uma verdadeira entidade, não justificada pela aproximação de funções, mas sim, da bem mais material que diz respeito à conduta de água”54. Annals of Museu Paulista. v. 21. n.2. Jul.-Dec. 2013. Introdução Deparamo-nos com uma única toilette: “Parece, segundo os raros tratados de savoir-vivre que abordam esta questão, que a maior parte utiliza o gabinete de toilette das suas esposas, de manhã antes delas. No entanto, cada um dos cônjuges tem o seu serviço especial sobre a mesma mesa de toilette”52. A toilette encontra-se ligada ao quarto dos donos e tem também acesso directo pelo pequeno vestíbulo iluminado pelos arcos geminados. Na viragem do séc. XIX para o séc. XX, a ligação entre quarto e casa de banho torna-se um princípio, relembrando a disposição do séc. XVIII em que em torno do quarto se reuniam as várias dependências. Contudo, estas opções decorrem cada vez mais do desejo de conforto e funcionalidade e menos da vontade de exibir simplesmente um estatuto social, associado à quantidade destes compartimentos complementares. A Condessa de Gencé reconhece que a sala de banho ligada ao gabinete de toilette 53. “(…) facilite le service et présente d’inappréciables commodités puisque, lorsque l’on sort de la pièce affectée à tous les soins du corps, la toilette est terminée sans qu’il y ait à perdre de temps ni à prendre froid pour passer dans un autre local” ; cf. Comtesse de Gence (1909, p.25). A Condessa de Gencé também escreveria a obra Tratado de Civilidade e Etiqueta (1909). 54. “Toilette, bains, w.c. et cuisine forment alors souvent une véritable entité, non justifiée par le rapprochement des fonctions, mais par celui, plus matériel, des conduites d’eau” ; cf. Monique Eleb e Anne Debarre (1995, p.417). 54. “Toilette, bains, w.c. et cuisine forment alors souvent une véritable entité, non justifiée par le rapprochement des fonctions, mais par celui, plus matériel, des conduites d’eau” ; cf. Monique Eleb e Anne Debarre (1995, p.417). 113 No WC, destacam-se os azulejos de linguagem neoclássica, que apresentam uma composição policroma formada por medalhões, festões, rosas e malmequeres e rematada superiormente por um friso idêntico ao do WC social, com dois motivos dominantes, a roseta e as folhas enroladas, mas neste caso em azul claro. O banho apresenta um nicho original de forma semi-circular que se destinava a receber a plataforma circular do duche (Figuras 29 a 31). De acordo com os actuais proprietários, as loiças sanitárias apresentariam ornamentos florais relevados. Ao fundo do compartimento, localiza-se a banheira. Um sofá e uma mesa de encosto completavam o conjunto de mobiliário. Introdução Quer a azulejaria do banho, com as suas glicínias, quer os puxadores utilizados em todo o piso são Arte Nova, à semelhança dos das portas envidraçadas do piso inferior, obedecendo também a um processo de sintetização profunda que os aproxima dos modelos difundidos na Alemanha. O modelo de toalheiro de cristal com ferragens em níquel seria, do mesmo modo, aplicado nas toilettes dos quartos e nas casas de banho do palacete. 55. Em 1897 casou-se com Henriette Lecloux, também belga. Joseph e Henriette tiveram dois filhos na Covilhã: Emma (1900-1989) e Camilo (1902-1980). Henriette faleceu e Joseph voltou a casar em 1905 com uma prima, Virginie Marguerite Lemouche e deste casamento nasceriam Susana e Augusto. Informação gentilmente cedida pelo Eng. Miguel Ritto, bisneto de Korrodi e de Joseph Bouhon. 56. Encontrámos motivos similares numa pintura de Van den Abeele que retrata o interior da Villa Paolina em Viareggio, propriedade que Paolina Borghese deixou em testamento ao seu sobrinho Luís Napoleão (mais tarde Napoleão III) que a cedeu à sua irmã Zenaïde; ver Mario Praz (2008, p.215). O quarto da filha, Susana Bouhon, embora seja o menos espaçoso de todos, surge complementado por um boudoir (Figura 32). O quarto integraria a cama e respectiva mesa-de-cabeceira, um sofá, um guarda-fato e, por fim, uma cómoda ou toucador. A meia-irmã, Emma (1900-1989), casara-se em 1919, o que explica a ausência do seu quarto55. Em obra, o quarto acabaria por ser estreitado, dando origem a um recesso revestido a azulejaria com um lavatório incorporado (sendo suprimido o previsto para o boudoir), à semelhança do que acontece com os outros quartos. A linha formal dos azulejos do friso, pela sua abstracção e pelo recurso ao dourado sobre branco, leva-nos a acreditar que advêm novamente da fábrica belga referida anteriormente. No tecto, Korrodi recorre às mesmas grinaldas que encontrámos no tecto do quarto dos donos, colocadas simetricamente e enquadradas em círculos definidos por contornos lineares ou frisos com óvulos e dardos. Dada a omissão do dourado e a simplicidade dos frisos nervurados assim como o motivo central do tecto, de influência oriental (chinoiserie), em forma de leque, constatamos que este tecto evoca o estilo Império56. O boudoir, lugar de socialização feminina e qualificado de frívolo ao longo do século XIX, reaparece na viragem do séc. XX, mas unicamente na habitação de luxo57. 57. Ver Monique Eleb e Anne Debarre (1995, p.315). Anais do Museu Paulista. v. 21. n.2. Jul.- Dez. 2013. Annals of Museu Paulista. v. 21. n.2. Jul.-Dec. 2013. Introdução Neste caso, assume uma localização privilegiada no ângulo do edifício e integra uma secretária destinada à escrita, a respectiva cadeira, o toucador e uma poltrona ou uma bérgere. Na varanda, encontramos painéis de azulejaria Arte Nova, de forte verticalidade, reforçada pela representação de fitas, gerbérias, folhas e caules invertidos. O fundo apresenta uma gradação tonal, que varia desde o azul-escuro ao verde de água, em que as mesmas flores, dispostas de modo profuso no topo dos painéis, se vão libertando e caindo em direcção à tonalidade mais clara. No quarto dos filhos, Camilo e Augusto, repete-se a solução do tecto em caixotões de madeira mais clara, adoptada no escritório do piso inferior. O papel de parede apresenta uma composição mais sintetizada do que o papel que cobre o hall, mais próximo a um estilo vitoriano e eventualmente aplicado posteriormente, já no tempo da família Carneiro. Neste caso, o desenho centra-se numa sucessão de rosas 114 Anais do Museu Paulista. v. 21. n.2. Jul.- Dez. 2013. Annals of Museu Paulista. v. 21. n.2. Jul.-Dec. 2013. Figura 29 – Entrada da casa de banho completa. Fotografia de Mónica Romãozinho. Figura 29 – Entrada da casa de banho completa. Fotografia de Mónica Romãozinho. 115 Annals of Museu Paulista. v. 21. n.2. Jul.-Dec. 2013. Figura 30 – Pormenor da azulejaria da casa de banho. Fotografia de Mónica Romãozinho. Figura 30 – Pormenor da azulejaria da casa de banho. Fotografia de Mónica Romãozinho. Figura 31 – Exemplo de loiça sanitária com pormenores decorativos. Fonte: A Construcção Moderna, Lisboa, Anno VII, n.211, p.505, 20 jan. 1907. À direita: manípulo Arte Nova na Casa Bouhon. Fotografia de Mónica Romãozinho. Figura 30 – Pormenor da azulejaria da casa de banho. Fotografia de Mónica Romãozinho. Figura 30 – Pormenor da azulejaria da casa de banho. Fotografia de Mónica Romãozinho. Figura 30 – Pormenor da azulejaria da casa de banho. Fotografia de Mónica Romãozinho. Figura 31 – Exemplo de loiça sanitária com pormenores decorativos. Fonte: A Construcção Moderna, Lisboa, Anno VII, n.211, p.505, 20 jan. 1907. À direita: manípulo Arte Nova na Casa Bouhon. Fotografia de Mónica Romãozinho. Figura 31 – Exemplo de loiça sanitária com pormenores decorativos. Fonte: A Construcção Moderna, Lisboa, Anno VII, n.211, p.505, 20 jan. 1907. À direita: manípulo Arte Nova na Casa Bouhon. Fotografia de Mónica Romãozinho. Figura 31 – Exemplo de loiça sanitária com pormenores decorativos. Anais do Museu Paulista. v. 21. n.2. Jul.- Dez. 2013. 58. “Un bon lit, une table de toilette formant commode, un secrétaire, une table, deux fauteuils, plusieurs chaises, un tapis, une garniture de cheminée, voilà son ameublement complet.” ; cf. Ris-Paquot (1894, p.211). 58. “Un bon lit, une table de toilette formant commode, un secrétaire, une table, deux fauteuils, plusieurs chaises, un tapis, une garniture de cheminée, voilà son ameublement complet.” ; cf. Ris-Paquot (1894, p.211). Introdução Fonte: A Construcção Moderna, Lisboa, Anno VII, n.211, p.505, 20 jan. 1907. À direita: manípulo Arte Nova na Casa Bouhon. Fotografia de Mónica Romãozinho. 116 Anais do Museu Paulista. v. 21. n.2. Jul.- Dez. 2013. Anais do Museu Paulista. v. 21. n.2. Jul.- Dez. 2013. e lírios azuis enquadrados numa malha de fitas entrelaçadas. Korrodi criou novamente uma reentrância, localizada entre as portas, que se destina a receber as duas camas simples. O canto residual resultante de um dos chanfros da dependência seria equipado com um lavatório e um armário que ocupa o espaço sobrante inferior à escada, revelando certas preocupações higienistas transversais a todos os aposentos (Figura 33). O lavatório enquadra-se no estilo Arte Nova, devido aos seus suportes em ferro que apresentam movimentos ondulantes (Figura 34). l d h d l d O último quarto, o de hóspedes, apresenta-se iluminado por mais um vão tripartido e revestido a papel cinzento no caso do lambril e florido ao nível da restante parede. O desenho do tecto aproxima-se da linguagem dos da escadaria e do hall, mas aqui o ornamento floral é totalmente abolido. Korrodi parece ter seguido, mais uma vez, as tendências que se faziam sentir desde os finais do séc. XIX, prevendo a colocação de duas camas, mesa de cabeceira, cadeiras ou fauteuils, uma cómoda, uma secretária com o respectivo assento, um guarda-fato e uma mesa (simples ou de toilette)58. Integra também uma zona com lavatório que, Annals of Museu Paulista. v. 21. n.2. Jul.-Dec. 2013. Figura 32 – Quarto da filha. Vê-se o acesso ao boudoir e a zona de lavatório. Fotografia de Mónica Romãozinho. Figura 32 – Quarto da filha. Vê-se o acesso ao boudoir e a zona de lavatório. Fotografia de Mónica Romãozinho. 117 Annals of Museu Paulista. v. 21. n.2. Jul.-Dec. 2013. à semelhança dos outros, corresponde ao modelo de origem e surge sustentado em dois perfis de ferro. Do hall octogonal, parte a escada de serviço em direcção ao sótão, onde se localizam as dependências de apoio ao pessoal doméstico da casa. A dependência mais valorizada ao nível do sótão corresponde ao quarto da professora, localizado acima da escadaria e iluminado por um lanternim que garante um pé-direito mais elevado. O quarto, anteriormente referido, integra um vão tripartido que dá acesso a uma pequena varanda orientada a tardoz. Introdução Esta dependência é visível na secção transversal A-B que nos mostra inclusivamente a reentrância chanfrada onde se localizaria a cama (Figura 35). No lado oposto, encontra-se o quarto das criadas que surge iluminado por um grande óculo. Outra dependência, sobreposta em relação às zonas húmidas dos pisos inferiores, acolheria o depósito de abastecimento de água e uma latrina em madeira que se destinava às criadas. Por fim, costurava-se num último compartimento e por vezes no patamar de chegada ao sótão. Figura 33 – Vista da toilette ligada ao quarto dos filhos. Fotografia de Mónica Romãozinho. Figura 33 – Vista da toilette ligada ao quarto dos filhos. Fotografia de Mónica Romãozinho. 118 Anais do Museu Paulista. v. 21. n.2. Jul.- Dez. 2013. Anais do Museu Paulista. v. 21. n.2. Jul.- Dez. 2013. Figura 34 – Lavatório integrado nas toilettes dos quartos, com suportes em ferro trabalhado. Toalheiro em cristal que surge aplicado nas mesmas dependências e no wc. Fotografias de Mónica Romãozinho. Figura 34 – Lavatório integrado nas toilettes dos quartos, com suportes em ferro trabalhado. Toalheiro em cristal que surge aplicado nas mesmas dependências e no wc. Fotografias de Mónica Romãozinho. Figura 34 – Lavatório integrado nas toilettes dos quartos, com suportes em ferro trabalhado. Toalheiro em cristal que surge aplicado nas mesmas dependências e no wc. Fotografias de Mónica Romãozinho. Annals of Museu Paulista. v. 21. n.2. Jul.-Dec. 2013. Conclusões O processo de obra, consultado no Arquivo Distrital de Leiria, é vasto e inclui plantas dos pisos principais já com a localização específica de todo o mobiliário, não apenas das peças fixas, e que corresponde, neste sentido, a uma verdadeira planta de design de interiores na medida em que Korrodi procedia à cotagem de todo o mobiliário, não só da cozinha ou do WC, mas também mesas ou armários de diversas tipologias. Os próprios desenhos evidenciavam já uma definição rigorosa de todos os espaços interiores e respectiva colocação de mobiliário, revelando uma preocupação com a funcionalidade desses mesmos, com a exploração de novas relações espaciais, conforto, iluminação ou ventilação e com a própria estandardização de materiais e acabamentos escolhidos. Korrodi dedicou uma especial atenção à pormenorização dos respectivos interiores através da execução de cortes detalhada mapas de vãos e desenho de mobiliário. 119 Figura 35 – Corte transversal A-B, à escala 1:50. Acervo do Arquivo Distrital de Leiria. Figura 35 – Corte transversal A-B, à escala 1:50. Acervo do Arquivo Distrital de Leiria. No âmbito dos espaços interiores, é sobretudo assinalável o modo como o autor aborda as várias soluções decorativas e a paleta cromática, atribuindo tratamentos diferenciados de dependência para dependência. Mesmo no contexto da Primeira República portuguesa, assistimos a uma certa persistência de determinadas dependências e hierarquias espaciais assim como à distinção dos papéis do género masculino e feminino, que condiciona a localização das dependências, a sua relação de proximidade ou contiguidade e a caracterização das mesmas. Quer a cor, mais intensa (castanho) ou mais suave (branco, marfim), quer os padrões florais mais ou menos abstractos, mais escultóricos ou planos, contribuem para a clarificação dos espaços ligados à sociabilidade e aos negócios (no caso do género masculino) ou a uma sociabilidade mais privada, educação dos filhos e exigências domésticas (no caso do género feminino). Quer a saleta 120 Anais do Museu Paulista. v. 21. n.2. Jul.- Dez. 2013. quer o boudoir figuram como espaços de escala mais acolhedora que se destinavam à sociabilidade no feminino. As madeiras são pintadas de branco ou os papéis oleados de casca de ovo no caso da saleta, salão e sala de almoços que correspondem a espaços do domínio feminino e de convívio familiar, mas também nos quartos dos pais e da filha Susana. Em oposição, uma paleta mais escura surge associada ao escritório, à sala de jantar e ao quarto dos rapazes. Conclusões As opções cromáticas contribuem, do mesmo modo, para a clarificação dos espaços de permanência ou de passagem: os batentes das portas apresentam-se pintados em marfim ou em madeira aparente consoante a divisão com que confinam. A organização funcional e hierárquica do projecto evoca soluções inglesas publicadas em revistas, da autoria não só de Baillie-Scott, mas também Richard Norman Shaw (1831-1912) ou do escocês Arnold Mitchell (1863-1944), entre outros, presente na valorização de compartimentos meramente funcionais como copas, engomadorias ou rouparias, cozinhas e wc’s. A exaltação das varandas abertas sobre os jardins ou do hall como espaço simultaneamente de circulação e de estar, sempre antecedido de uma antecâmara, reflecte igualmente opções seguidas nos cottages ou country houses publicadas pela imprensa internacional. Soluções como bay windows ou bow windows aproximam, do mesmo modo, os interiores dos modelos ingleses, assim como o forte sentido de individualidade e intimidade associado às distintas espacialidade. Entradas e escadas denunciam uma separação entre áreas de circulação destinadas à família ou visitas e pessoal doméstico que resulta da mesma vontade de salvaguardar a privacidade da própria família. Contudo a Arte Nova impulsionou mudanças significativas ao nível dos seguintes pressupostos: relação de maior fluidez entre as dependências (entre as da esfera da sociabilidade, em particular) ou separação estabelecida através de vastos guardaventos em arco abatido; aproximação entre dependências até então dissociadas (entre a cozinha e a sala de jantar, por exemplo); integração e valorização de compartimentos funcionais como as copas de apoio, as casas de banho ou a própria cozinha. No presente caso, assistimos a uma forte interligação entre salas de refeições, à substituição de corredores por amplos vestíbulos e observam-se compartimentos mais amplos resultantes do recurso a uma estrutura mista, composta por vigamentos de betão. Os próprios gabinetes de toilette dos donos da casa fundem-se num só compartimento, denunciando uma mentalidade mais aberta por parte do cliente e constrastando com a divisão segundo o género habitualmente presente em outras encomendas, inclusivamente no contexto da habitação colectiva de que é exemplo o prédio de rendimento de Mendonça & Costa, situado na Rua Braancamp em Lisboa (1914). Alguns destes interiores testemunham não só programas de interiores e opções construtivas e estéticas que acompanham os padrões de gosto internacional, como incorporam componentes tecnológicas inovadoras que davam resposta ao desejo de conforto, designadamente os elevadores. Annals of Museu Paulista. v. 21. n.2. Jul.-Dec. 2013. Conclusões Na Casa da família Bouhon, multiplicam-se os compartimentos destinados aos serviços domésticos e mecanismos 121 Annals of Museu Paulista. v. 21. n.2. Jul.-Dec. 2013. que visam uma maior eficácia do serviço doméstico, como o monta-pratos que facilita a circulação dos alimentos ou as campainhas eléctricas. Ao nível do aquecimento das dependências, este era sobretudo providenciado por lareiras ou fogões, solução que contrasta com a modernidade das anteriores soluções. 59. Com 18 anos, Korrodi partiu para Itália numa visita de estudo com um colega de curso. De acordo com um diário de viagem da sua autoria, patente no Arquivo Distrital de Leiria, no Verão de 1939, dá-se a última grande viagem de Ernesto e de Quitéria, que partem para Itália, a fim de se encontrar com a sua família em S. Giulio de Orta. Passam por S. Moritz até chegarem a Zurique e pelas Baleares e Tanger, onde Ernesto participa numa excursão organizada na véspera. Por último, a Arte Nova faz-se sentir ao nível do detalhe e continuamos a vê-la relacionada com outros modelos franceses e sobretudo ingleses, em elementos isolados ou composições que exprimem a natureza verdadeiramente ecléctica da obra de Ernesto Korrodi: pormenores escultóricos, padrões da azulejaria e de papéis de parede presentes nas paredes, tectos ou lambris, nos desenhos dos tectos em caixotões ou estucados, nas curvas e contracurvas dos vãos, nas cores presentes nas molduras ou vidros texturados das portas e janelas que suscitam diferentes ambiências. Assiste- se à divisão da parede em várias secções horizontais e introdução de frisos lineares que contornam os planos superiores das paredes e que formam autênticas molduras desenhadas a stencil, constituídas por motivos fitomórficos mais abstractos Há que salientar o papel dos materiais ou soluções, produzidas pelas manufacturas alemãs, inglesas ou belgas, designadamente papéis de parede simples ou oleados, faianças, ladrilhos, vidros, ferragens, iluminação, técnicas de aplicação de soalhos de madeira ou loiças sanitárias cerâmicas. Algumas destas fórmulas industriais assumiam não só um papel estético mas também funcional no sentido de dar resposta a uma higienização progressiva da habitação. p g A atitude de Korrodi consiste em projectar em função do seu tempo e do perfil de cada cliente, retirando do passado e do presente todas as soluções que possam dar resposta às funções pretendidas. Anais do Museu Paulista. v. 21. n.2. Jul.- Dez. 2013. Conclusões Desconhecemos quais seriam os seus livros de cabeceira, mas sabemos que se cruzou com Raul Lino e que assinava algumas revistas internacionais, responsáveis pela publicação de projectos de autores com obras literárias determinantes, nomeadamente Baillie Scott. Revistas portuguesas de especialidade, designadamente A Architectura Portugueza (1908-1958) e A Construcção moderna (1900-1919), publicavam também projectos, entre eles alguns da autoria do próprio Korrodi, contribuindo ao mesmo tempo para a divulgação de concursos públicos, Prémios Valmor, Regulamentos, dando a conhecer aos profissionais o que se passava lá fora, por meio de artigos críticos e actas de congressos internacionais. As deslocações ao estrangeiro foram também certamente determinantes para as suas opções projectuais59. A sua obra reflecte a influência exercida por uma Art Nouveau que é cruzada com reinterpretações de fórmulas decorativas e espaciais do Renascimento, Barroco, Rococó ou medievalizantes, adivinhando-se, contudo, preocupações como a racionalidade, funcionalidade, padronização e integração de produtos industriais. Por outro lado, os pressupostos subjacentes a este e a outros projectos da autoria de Korrodi, ainda hoje estão na base da concepção na prática do design de interiores, concluindo-se que a Art Nouveau contribuiu, de modo incontornável, para a afirmação desta área, provocando mudanças no espaço que ultrapassaram a escala do elemento decorativo. 122 FONTES IMPRESSAS GENCÉ, Comtesse de. Le Cabinet de toilette d’une honnête femme. Paris: P.Pancier, 1909. GENCÉ, Condessa de. Tratado de civilidade e etiqueta. Tradução de Luiz Cardoso. Lisboa: Guimarães & C.a, 1909. KORRODI, Ernesto. 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